US 2010O285468A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0285468 A1 Xin (43) Pub. Date: Nov. 11, 2010

(54) DETECTION AND/OR QUANTIFICATION OF (86). PCT No.: PCT/US08/77582 NUCLECACDS S371 (c)(1), (2), (4) Date: Jul. 21, 2010 (75) Inventor: Xing Xin, Foster City, CA (US) Related U.S. ApplicationO O Data Correspondence Address: (60) Provisional application No. 60/994,969, filed on Sep. WILSON, SONSINI, GOODRICH & ROSATI 24, 2007. 650 PAGE MILL ROAD Publication Classification PALO ALTO, CA 94304-1050 (US) (51) Int. Cl. CI2O I/68 (2006.01) (73) Assignee: Allelogic Biosciences (52) U.S. Cl...... 435/6 Corporation, Hayward, CA (US) (57) ABSTRACT The present invention provides compositions, methods, and (21) Appl. No.: 12/679,534 kits for nucleic acids analyses. In particular, melting analyses are used to detect the presence or absence and to quantify (22) PCT Filed: Sep. 24, 2008 nucleic acids. Patent Application Publication Nov. 11, 2010 Sheet 1 of 7 US 2010/0285468 A1

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DETECTION AND/OR QUANTIFICATION OF 0006. In some embodiments, the invention provides a NUCLECACDS method for determining the amount of a target nucleic acid in a sample. In some embodiments, the invention provides a CROSS-REFERENCE method for determining the amount of a target nucleic acid in a sample containing a nucleic acid binding agent, a target 0001. This application claims the benefit of U.S. Provi nucleic acid and a reference nucleic acid; where the target sional Application No. 60/994.969, filed Sep. 24, 2007, which nucleic and the reference nucleic acid exhibit distinct melting application is incorporated herein by reference in its entirety. profiles and where the binding agent yields a detectable signal when bound to the target nucleic acid and/or the reference BACKGROUND OF THE INVENTION nucleic acid. The melting profiles of the target nucleic and the 0002 Nucleic acid analysis is becoming an important tool reference nucleic acid are determined by detecting the signal for the diagnosis and prognosis of infectious as well as of the binding agent at a plurality of temperatures. The genetic diseases. Genetic modifications including variation in amount of target nucleic acid is determined by comparing the gene copy number can lead to profound abnormalities at the melting profile of the target nucleic acid with the melting cellular and organismal level. Changes in gene copy number profile of the reference nucleic acid. In some embodiments, may lead to under- or overexpression of genes responsible for the melting profiles from the target and references nucleic a disease phenotype. Other genetic modifications such as acid are compared by determining the relative amount of chromosomal changes, including allelic loss, , rear signal attributable to the target nucleic acid or to the reference rangement, point , , gene amplifications and nucleic acid based on their melting profile. In some embodi acquisition of viral genomes have been identified as the hall ments, the melting profiles from the target and references mark of neoplasia. These changes can result in the loss of nucleic acid are compared by comparing the relative amount tumor Suppressor genes and the turning of a cellular proto of the signal attributable to the target nucleic acid and the oncogene to an actual oncogene. Single copy changes in reference nucleic acid. In some embodiments, the melting specific or Smaller regions can result in a num profiles from the target and references nucleic acid are com ber of developmental disorders, including Down, Prader pared by calculating the ratio between the amount of signal Willi, Angelman and Cri du chat syndromes. The extra attributable to the target nucleic acid and the amount of signal genetic material in these patients causes the phatogenic phe attributable to the reference nucleic acid. The signal can be a notype associated with these syndromes. fluorescent signal, magnetic signal, radioactive signal, 0003 Moreover, variations in the DNA sequences of Raman signal or an electrochemical signal. humans take many forms and can be correlated with how 0007. The target nucleic acid and/or the reference nucleic humans develop diseases and respond to pathogens, chemi can be amplified products of the target and/or the reference cals, drugs, vaccines, and other agents. For instance, the nucleic acids. In some embodiments, the target nucleic acid inheritance of a Substantial number of disease traits can be and/or the reference nucleic are amplified by a PCR reaction. predicted by genetic analysis. Recently, multiple studies have In some embodiments, the amplified products of the target discovered an abundance of Submicroscopic copy number nucleic acid and the amplified products of the reference variation of DNA segments ranging to kilobases (kb) to nucleic are generated by using a distinct set of primers spe megabases (Mb) in size. Deletions, insertions, duplications cific for the target nucleic acid and the reference nucleic acid and complex multi-site variations collectively known as copy respectively. number variation regions (CNVR) or copy number polymor 0008. In some embodiments, the target nucleic acid is a phism (CNP). CNVRs can affect gene expression, pheno genomic DNA region. The genomic DNA region can contain typic variation and adaptation by disrupting genes and alter one or more polymorphisms. In some embodiments, the ing gene dosage. In addition, CNVRS can cause disease, as in genomic DNA region contains one or more copy number microdeletion or microduplication disorders, or conferrisk to variable regions (CNVR). In some embodiments, the disease traits such as HIV infection and glomerulonephritis. genomic DNA region contains STRs or SNPs. In some Some CNVRs have been associated with specific diseases embodiments, the target nucleic acid and the reference such as CHARGE syndrome, Parkinson's and Alzheimer dis nucleic acid are double Stranded. In some embodiments, the CaSC. size of the target nucleic acid is about 100 bp to about 1 0004 Current methods for the analysis of cellular genetic kilobase. In some embodiments, the target nucleic acid and content include comparative genomic hybridization (CGH), the reference nucleic acid are comparable in length. spectral karyotyping (SKY), fluorescence in situ hybridiza tion (FISH), molecular subtraction methods, such as RDA, 0009. In some embodiments, the reference nucleic acid is digital karyotyping, array based approaches, MLPA and real a genomic DNA region. In some embodiments, the reference time PCR. However, these methods are of limited mapping nucleic acid is a cDNA transcript, or an oligonucleotide. In resolution, time consuming and labor intensive. In addition Some embodiments, the copy number of the reference nucleic expensive reagents and/or instruments may be required for acid is known. performing these conventional methods. 0010. In some embodiments, the target nucleic acid is associated with condition. Examples of conditions include, but are not limited to, 13, trisomy 18, trisomy 21, SUMMARY OF THE INVENTION , dup (17) (p 11.2p 11.2) syndrome, 0005. The invention relates to methods, compositions and , Pre-eclampsia, Pre-term labor, Edometrio devices, e.g., for detecting a target nucleic acid in a sample. sis, Pelizaeus-Merzbacher disease, dup (22)(d.11.2q11.2) The methods of the present invention allows for a rapid cost syndrome, , Cri-du-chat syndrome, Wolf effective single assay format to determine the presence or Hirschhorn syndrome, Williams-Beuren syndrome, Charcot absence and/or amount of nucleic acid sequences in a poly Marie-Tooth disease, neuropathy with liability to pressure nucleotide sample. palsies, Smith-Magenis syndrome, neurofibromatosis, US 2010/0285468 A1 Nov. 11, 2010

Alagille syndrome, Velocardiofacial syndrome, DiGeorge nucleic acid are compared by determining the relative amount syndrome, steroid Sulfatase deficiency, Kallmann syndrome, of signal attributable to the target nucleic acid or to the refer microphthalmia with linear skin defects, Adrenal hypoplasia, ence nucleic acid based on their melting profile. In some Glycerol kinase deficiency, Pelizaeus-Merzbacher disease, embodiments, the melting profiles from the target and refer testis-determining factor on Y. AZospermia (factor a). ences nucleic acid are compared by comparing the relative AZospermia (factorb), AZospermia (factor c), 1 p 36 deletion, amount of the signal attributable to the target nucleic acid and acute lymphoblastic , acute or chronic lymphocyctic the reference nucleic acid. In some embodiments, the melting or granulocytic tumor, acute myeloid leukemia, acute promy profiles from the target and references nucleic acid are com elocytic leukemia, adenocarcinoma, adenoma, adrenal can pared by calculating the ratio between the amount of signal cer, basal cell carcinoma, bone cancer, brain cancer, breast cancer, bronchi cancer, cervical dysplasia, chronic myelog attributable to the target nucleic acid and the amount of signal enous leukemia, colon cancer, epidermoid carcinoma, attributable to the reference nucleic acid. The signal can be a Ewing's sarcoma, gallbladder cancer, gallstone tumor, giant fluorescent signal, magnetic signal, radioactive signal, cell tumor, glioblastoma multiforma, hairy-cell tumor, head Raman signal or an electrochemical signal. cancer, hyperplasia, hyperplastic corneal nerve tumor, in situ 00.15 Examples of conditions include, but are not limited carcinoma, intestinal ganglioneuroma, islet cell tumor, Kapo to, trisomy 13, trisomy 18, trisomy 21, Klinefelter Syndrome, si's sarcoma, kidney cancer, larynx cancer, leiomyomater dup (17)(p 11.2 p 11.2) syndrome, Down syndrome, Pre tumor, liver cancer, lung cancer, , malignant car eclampsia, Pre-term labor, Edometriosis, Pelizaeus-Merz cinoid, malignant hypercalcemia, malignant melanomas, bacher disease, dup (22)(q11.2d 11.2) syndrome, Cat eye syn marfanoid habitus tumor, medullary carcinoma, metastatic drome, Cri-du-chat syndrome, Wolf-Hirschhorn syndrome, skin carcinoma, mucosal neuromas, mycosis fungoide, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, myelodysplastic syndrome, myeloma, neck cancer, neural neuropathy with liability to pressure palsies, Smith-Magenis tissue cancer, neuroblastoma, osteogenic sarcoma, osteosar syndrome, neurofibromatosis, Alagille Syndrome, Velocar coma, ovarian tumor, pancreas cancer, parathyroid cancer, diofacial syndrome, DiGeorge syndrome, steroid Sulfatase pheochromocytoma, polycythemia Vera, primary brain deficiency, Kallmann syndrome, microphthalmia with linear tumor, prostate cancer, rectum cancer, renal cell tumor, ret skin defects, Adrenal hypoplasia, Glycerol kinase deficiency, inoblastoma, rhabdomyosarcoma, seminoma, skin cancer, Pelizaeus-Merzbacher disease, testis-determining factor on Small-cell lung tumor, Soft tissue sarcoma, squamous cell Y. AZospermia (factora), AZospermia (factor b), AZospermia carcinoma, stomach cancer, thyroid cancer, topical skin (factor c), 1 p 36 deletion, acute lymphoblastic leukemia, lesion, veticulum cell sarcoma, and Wilm's tumor. acute or chronic lymphocyctic or granulocytic tumor, acute 0011. In some embodiments, the target nucleic acid is myeloid leukemia, acute promyelocytic leukemia, adenocar associated with a risk to develop a disease. Examples of cinoma, adenoma, adrenal cancer, basal cell carcinoma, bone diseases include, but are not limited to, HIV infection, glom cancer, brain cancer, breast cancer, bronchi cancer, cervical erulonephritis, CHARGE syndrome, Parkinson's disease and dysplasia, chronic myelogenous leukemia, colon cancer, epi Alzheimer's disease. dermoid carcinoma, Ewing's sarcoma, gallbladder cancer, 0012. In some embodiments, the copy number of the target gallstone tumor, giant cell tumor, glioblastoma multiforma, nucleic acid is determined. hairy-cell tumor, head cancer, hyperplasia, hyperplastic cor neal nerve tumor, in situ carcinoma, intestinal ganglion 0013. In some embodiments, the nucleic acid binding euroma, islet cell tumor, Kaposi's sarcoma, kidney cancer, agent is a DNA binding agent. In some embodiments, the larynx cancer, leiomyomater tumor, liver cancer, lung cancer, DNA binding agent is a DNA intercalator. Examples of DNA lymphomas, malignant carcinoid, malignant hypercalcemia, binding agents that can be used in the methods described malignant melanomas, marfanoid habitus tumor, medullary herein include, but are not limited to, EvaGreen, SYBR Green carcinoma, metastatic skin carcinoma, mucosal neuromas, I, PicoGreen, Cyto 9, LC Green, SYBR GreenER, Ethidium mycosis fungoide, myelodysplastic syndrome, myeloma, bromide, TOTO, YOYO, Bebo, SYTO Green and bexto. In neck cancer, neural tissue cancer, neuroblastoma, osteogenic some embodiments, the DNA binding agent is EvaGreen. sarcoma, osteosarcoma, ovarian tumor, pancreas cancer, par 0014. In some embodiments the invention provides a athyroid cancer, pheochromocytoma, polycythemia Vera, pri method of determining a genetic condition in a patient or a mary brain tumor, prostate cancer, rectum cancer, renal cell fetus by analyzing a sample. In some embodiments, the tumor, retinoblastoma, rhabdomyosarcoma, seminoma, skin invention provides a method of determining a genetic condi cancer, Small-cell lung tumor, soft tissue sarcoma, squamous tion in a patient or a fetus by analyzing a sample Suspected to cell carcinoma, stomach cancer, thyroid cancer, topical skin contain a target nucleic acid, a reference nucleic acid and a nucleic acid binding agent. The target nucleic and the refer lesion, veticulum cell sarcoma, and Wilm's tumor. ence nucleic acid exhibit distinct melting profiles. The target 0016. In some embodiments, the condition is a risk to nucleic acid and the reference nucleic acid are amplified develop a disease. Examples of diseases include, but are not using a first set of primers specific for the target nucleic acid limited to, HIV infection, glomerulonephritis, CHARGE and a second set of primers specific from the reference nucleic syndrome, Parkinson's disease and Alzheimer's disease. acid. The presence or absence of the genetic condition is 0017. In some embodiments, the target nucleic acid is a determined by comparing the melting profile of the amplified genomic DNA region. The genomic DNA region can contain target nucleic acid to the melting profile of the amplified one or more polymorphisms. In some embodiments, the reference nucleic acid, where the melting profiles of the genomic DNA region contains one or more CNVR. amplified target nucleic acid and the amplified reference 0018. In some embodiments, the genomic DNA region nucleic acid are determined by monitoring a signal from the contains STRs or SNPs. In some embodiments, the target binding agent at a plurality oftemperatures. In some embodi nucleic acid and the reference nucleic acid are double ments, the melting profiles from the target and references Stranded. In some embodiments, the size of the target nucleic US 2010/0285468 A1 Nov. 11, 2010

acid is about 100 by to about 1 kilobase. In some embodi DNA binding agent is a DNA intercalator. Examples of DNA ments, the target nucleic acid and the reference nucleic acid binding agents that can be used in the methods described are comparable in length. herein include, but are not limited to, EvaGreen, SYBR Green 0019. In some embodiments, the reference nucleic acid is I, PicoGreen, Cyto 9, LC Green, SYBR GreenER, Ethidium a genomic DNA region. In some embodiments, the reference bromide, TOTO, YOYO, Bebo, SYTO Green and bexto. In nucleic acid is a cDNA transcript, or an oligonucleotide. In some embodiments, the DNA binding agent is EvaGreen. Some embodiments, the copy number of the reference nucleic 0027. In some embodiments, the invention provides a kit. acid is known. In some embodiments, the kit contains a nucleic acid binding 0020. In some embodiments, the copy number of the target agent, a set of primers specific for a reference nucleic acid or nucleic acid is determined. alternatively a reference nucleic acid; and instructions for the 0021. In some embodiments, the nucleic acid binding use of the nucleic acid binding agent, and the set of primers or agent is a DNA binding agent. In some embodiments, the the reference nucleic acid to perform any of the methods DNA binding agent is a DNA intercalator. Examples of DNA described herein. In some embodiments, the kit further con binding agents that can be used in the methods described tains a set of primers specific for a target nucleic acid. In some herein include, but are not limited to, EvaGreen, SYBR Green embodiments, the kit contains a polymerase. In some I, PicoGreen, Cyto 9, LC Green, SYBR GreenER, Ethidium embodiments, the kit contains instructions on how to perform bromide, TOTO, YOYO, Bebo, SYTO Green and bexto. In amplification of a target nucleic acid and optionally the ref some embodiments, the DNA binding agent is EvaGreen. erence nucleic acid using the polymerase. In some embodi 0022. In some embodiments, the invention provides a ments, the kit contains one or more buffers. method of determining copy number variation of a target 0028. In some embodiments, the reference nucleic acid is genomic DNA sequence in a sample. In some embodiments, a genomic DNA region, a cDNA transcript, or an oligonucle the invention provides a method of determining copy number otide. In some embodiments, the nucleic acid binding agent is variation of a target genomic DNA sequence in a sample a DNA binding agent. In some embodiments, the DNA bind potentially containing a target genomic DNA sequence, a ing agent is a DNA intercalator. Examples of DNA binding reference nucleic acid and a nucleic acid binding agent, where agents that can be used in the methods described herein the target genomic DNA sequence and the reference nucleic include, but are not limited to, EvaGreen, SYBR Green I, acid exhibit distinct melting profiles. The target DNA PicoGreen, Cyto 9, LC Green, SYBR GreenER, Ethidium sequence and the reference nucleic acid are amplified using a bromide, TOTO, YOYO, Bebo, SYTO Green and bexto. In first set of primers specific for the genomic DNA sequence some embodiments, the DNA binding agent is EvaGreen. and a second set of primes specific for the reference nucleic acid sequence. The copy number of the target genomic DNA INCORPORATION BY REFERENCE sequence is determined by comparing the melting profile of the target nucleic acid to the melting profile of the reference 0029 All publications, patents, and patent applications nucleic acid, where the melting profiles of the target and mentioned in this specification are herein incorporated by reference nucleic acid are determined by monitoring the sig reference to the same extent as if each individual publication, nal of said binding agent at a plurality of temperatures. patent, or patent application was specifically and individually 0023. In some embodiments, the melting profiles from the indicated to be incorporated by reference. target and references nucleic acid are compared by determin ing the relative amount of signal attributable to the target BRIEF DESCRIPTION OF THE DRAWINGS nucleic acid or to the reference nucleic acid based on their melting profile. In some embodiments, the melting profiles 0030. The novel features of the invention are set forth with from the target and references nucleic acid are compared by particularity in the appended claims. A better understanding comparing the relative amount of the signal attributable to the of the features and advantages of the present invention will be target nucleic acid and the reference nucleic acid. In some obtained by reference to the following detailed description embodiments, the melting profiles from the target and refer that sets forth illustrative embodiments, in which the prin ences nucleic acid are compared by calculating the ratio ciples of the invention are utilized, and the accompanying between the amount of signal attributable to said target drawings of which: nucleic acid and the amount of signal attributable to said 0031 FIG. 1 shows an example of a melting curve for a reference nucleic acid. The signal can be a fluorescent signal, reference (peak 1) and a target DNA (peak 2) in which the magnetic signal, radioactive signal, Raman signal or an elec x-axis is the temperature (T) and the y-axis is -dF/dT, where trochemical signal. F is the fluorescence intensity recorded. Various methods of 0024. In some embodiments, the target nucleic acid and determining the peak ratio for calculation of the amount of the the reference nucleic acid are double stranded. In some test DNA are also shown. embodiments, the size of the target nucleic acid is about 100 0032 FIG. 2 shows control results from the copy number by to about 1 kilobase. In some embodiments, the target determination using the method of AACt from a real-time nucleic acid and the reference nucleic acid are comparable in PCR assay. The control results are in agreement with previ length. ously reported copy numbers. 0025. In some embodiments, the reference nucleic acid is 0033 FIG.3 shows melting curves obtained from melting a genomic DNA region. In some embodiments, the reference analyses of various DNA mix ratios ranging from 1:1 to 1:10 nucleic acid is a cDNA transcript, or an oligonucleotide. In of reference DNA to target DNA. Some embodiments, the copy number of the reference nucleic 0034 FIG. 4 shows a standard response curve plotting the acid is known. peak-height ratio against the input DNA mix ratio in a double 0026. In some embodiments, the nucleic acid binding log plot. The peak-height ratio is linearly correlated with the agent is a DNA binding agent. In some embodiments, the input DNA mix ratio. US 2010/0285468 A1 Nov. 11, 2010

0035 FIG. 5 shows a standard response curve and dem 0042. An individual is not limited to a human being but onstrates that a different annealing time during amplification may also be other organisms including but not limited to affects the value of the peak-height ratio but does not affect mammals, plants, bacteria, or cells derived from any of the the linear correlation between the peak-height ratio and the above. input DNA mix ratio. 0043. Nucleic acids, including target nucleic acids and 0036 FIG. 6 shows that the total starting DNA quantity reference nucleic acids, of the present invention may include during amplification affects the peak-height ratio but does not any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and affect the linear correlation between the peak-height ratio and guanine, either deoxyribonucleotides or ribonucleotides, or the input DNA mix ratio. analogs thereof. The polymers or oligomers may be hetero 0037 FIG. 7 shows a table illustrating how the copy num geneous or homogeneous in composition, and may be iso ber of a target region was determined. lated from naturally occurring Sources or may be artificially or synthetically produced. In addition, the nucleic acids may DETAILED DESCRIPTION OF THE INVENTION be DNA or RNA, or a mixture thereof, and may exist perma nently or transitionally in single-stranded or double-stranded 0038. Unless defined otherwise, all technical and scien form, including homoduplex, heteroduplex, and hybrid tific terms used herein have the same meaning as is commonly states. Nucleic acids may have any three-dimensional struc ture, and may performany function, known or unknown. The understood by one of skill in the art to which this invention following are non-limiting examples of nucleic acids: coding belongs. All patents and publications referred to herein are or non-coding regions of a gene or gene fragment, loci (locus) incorporated by reference in their entirety. defined from linkage analysis, exons, introns, messenger 0039. As used in this application, the singular form “a, RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, “an and “the include plural references unless the context cDNA, recombinant polynucleotides, branched polynucle clearly dictates otherwise. For example, the term “an agent' otides, plasmids, vectors, isolated DNA of any sequence, includes a plurality of agents, including mixtures thereof. isolated RNA of any sequence, nucleic acid probes, and prim 0040. Throughout this disclosure, various aspects of this ers. A nucleic acid may comprise modified nucleotides. Such invention can be presented in a range format. It should be as methylated nucleotides and nucleotide analogs. If present, understood that the description in range format is merely for modifications to the nucleotide structure may be imparted convenience and brevity and should not be construed as an before or after assembly of the polymer. The sequence of inflexible limitation on the scope of the invention. Accord nucleotides may be interrupted by non-nucleotide compo ingly, the description of a range should be considered to have nents. A nucleic acid may be further modified after polymer specifically disclosed all the possible Subranges as well as ization, Such as by conjugation with a labeling component. individual numerical values within that range. For example, 0044 An oligonucleotide or polynucleotide is a nucleic description of a range such as from 1 to 6 should be consid acid ranging from about at least 3, 5, 10, or 20 nucleotides in ered to have specifically disclosed Subranges Such as from 1 length, but may be up to 100, 1000, or 10,000 nucleotides to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 long or even longer. Polynucleotides of the present invention to 6 etc., as well as individual numbers within that range, for include sequences of deoxyribonucleic acid (DNA) or ribo example, 1, 2, 3, 4, 5, and 6. This applies regardless of the nucleic acid (RNA) or mimetics thereof which may be iso breadth of the range. All references to the function log default lated from natural sources, recombinantly produced or artifi to 10 as the base. cially synthesized. The invention also encompasses situations 0041. The assay of the present invention is particularly in which there is a nontraditional base pairing such as Hoogs useful for analyzing nucleic acids (e.g., DNA, RNA, or teen base pairing which has been identified in certain trNA hybrids thereby). The methods and compositions described molecules and postulated to existina triple helix. “Polynucle herein provide a sensitive, cost and labor effective assay for otide' and "oligonucleotide' are used interchangeably in this determining the presence or absence and/or amount of a target application. nucleic acid, e.g., the presence or absence and or copy number 0045 “Genome' designates or denotes the complete, of a CNVR. The methods described herein typically involve a single-copy set of genetic material for an organism. It can be melting analysis to detect and quantify a target nucleic acid. either DNA or RNA. A genome may be multi-chromosomal In some embodiments, a target nucleic acid and a reference such that the DNA is cellularly distributed among a plurality nucleic acid that exhibit different melting profiles are ampli of individual chromosomes. For example, in human there are fied using a different set of primers. That is, a first set of 22 pairs of chromosomes plus a gender associated XX or XY primer specific for the target nucleic acid and a second set of pair. primers specific for a reference nucleic acid are used for the 0046. The term "chromosome' refers to the heredity-bear amplification. The reference nucleic acid can come from the ing gene carrier of a living cell which is derived from chro same or a different Source as the target nucleic acid. The matin and which comprises DNA and protein components melting profiles of the target nucleic acid and reference (e.g., histones). The conventional internationally recognized nucleic acid are determined and compared to deduce the individual human genome chromosome numbering system is amount of said target nucleic acid. Thus, in Some embodi employed herein. The size of an individual chromosome can ments, melting analysis is used to quantify at least two ampli vary from one type to another with a given multi-chromo cons that are co-amplified in an amplification reaction. In Somal genome and from one genome to another. In the case of some cases, the methods described herein are used for the the human genome, the entire DNA mass of a given chromo determination of nucleic acid copy number. Methods of using some is usually greater than about 100,000,000 bp. For Algorithms and computer software programs that perform the example, the size of the entire human genome is about 3x10 methods described herein are also disclosed. bp. The largest chromosome, chromosome no. 1, contains US 2010/0285468 A1 Nov. 11, 2010 about 2.4x10 by while the smallest chromosome, chromo totic reagent, an anti-coagulation reagent, an anti-thrombotic some no. 22, contains about 5.3x107 bp. reagent, magnetic property regulating reagent, a buffering 0047. A “chromosomal region' is a portion of a chromo reagent, an osmolality regulating reagent, a pH regulating some. A "genomic region' is a portion of a genome. The reagent, and/or a cross-linking reagent. actual physical size or extent of any individual chromosomal 0052. When a blood sample is obtained, a preservative or genomic region can vary greatly. The term “region' is not Such an anti-coagulation agent and/or a stabilizer can be necessarily definitive of any particular one or more genes added to the sample prior to enrichment. This allows for because a region need not take into specific account the par extended time for analysis/detection. Thus, a sample, Such as ticular coding segments (exons) of an individual gene. a blood sample, can be analyzed under any of the methods and 0.048. In practicing aspects of the present invention (e.g., systems herein within 1 week, 6 days, 5 days, 4 days, 3 days, performing amplification reactions, etc.), many conventional 2 days, 1 day, 12 hrs, 6 hrs, 3 hrs, 2 hrs, or 1 hr from the time techniques in molecular biology and recombinant DNA are the sample is obtained. optionally utilized. These techniques are well known and are 0053. In some embodiments, a blood sample can be com explained in, for example, Current Protocols in Molecular bined with an agent that selectively lyses one or more cells or Biology, Volumes I, II, and III, 1997 (F. M. Ausubel ed.); components in a blood sample. For example, fetal cells can be Sambrook et al., 2001, Molecular Cloning: A Laboratory selectively lysed releasing their nuclei when a blood sample Manual. Third Edition, Cold Spring Harbor Laboratory Press, including fetal cells is combined with deionized water. Such Cold Spring Harbor, N.Y.; Berger and Kimmel, Guide to selective lysis allows for the subsequent enrichment of fetal Molecular Cloning Techniques Methods in Enzymology Vol nuclei using, e.g., size or affinity based separation. In another ume 152 Academic Press, Inc., San Diego, Calif. (Berger), example platelets and/or enucleated red blood cells are selec DNA Cloning: A Practical Approach, Volumes I and II, 1985 tively lysed to generate a sample enriched in nucleated cells, (D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. such as fetal nucleated red blood cells (fnRBC) and maternal Gait ed.); Nucleic Acid Hybridization, 1985, (Hames and nucleated blood cells (mnBC). The finRBC's can subse Higgins); Transcription and Translation, 1984 (Hames and quently be separated from the mnBC's using, e.g., affinity to Higgins eds.); Animal Cell Culture, 1986 (R.I. Freshney ed.); antigen-i or magnetism differences in fetal and adult hemo Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, globin. 1984, A Practical Guide to Molecular Cloning; the series, Methods in Enzymology (Academic Press, Inc.); Gene Trans 0054 When obtaining a sample from an animal (e.g., fer Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P. blood sample), the amount can vary depending upon animal Calos eds., Cold Spring Harbor Laboratory); Methods in size, its gestation period, and the condition being screened. In Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and some embodiments, up to 50, 40, 30, 20, 10,9,8,7,6, 5, 4, 3, Wu, eds., respectively): PCR 2: A PRACTICAL APPROACH 2, or 1 mL of a sample is obtained. In some embodiments, (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. 1-50, 2-40, 3-30, or 4-20 mL of sample is obtained. In some (1995)). embodiments, more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 0049. In some embodiments target nucleic acids are from 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 mL of a sample is a sample obtained from an animal. Such animal can be a obtained. human, a domesticated or a laboratory model animal such as 0055 Nucleic acids from samples that can be analyzed by a mouse, cow, chicken, drosophila, pig, horse, rabbit, Zebra the methods herein include: double-stranded DNA, single fish, dog, cat, or goat. In some embodiments target nucleic stranded DNA, single-stranded DNA hairpins, DNA/RNA acids are from a sample obtained from a bacteria or virus. hybrids, RNA (e.g. mRNA or miRNA) and RNA hairpins. Samples derived from an animal, e.g., human, can include, for 0056. Where desired, nucleic acid contained in the afore example whole blood, Sweat, tears, ear flow, sputum, lymph, mentioned samples can be first extracted according to stan bone marrow Suspension, lymph, urine, saliva, semen, vagi dard methods in the art. For instance, in DNA or RNA can be nal flow, cerebrospinal fluid, brain fluid, ascites, milk, secre isolated using various lytic enzymes or chemical Solutions tions of the respiratory, intestinal or genitourinary tracts fluid. according to the procedures set forth in Sambrook et al. In some embodiments the sample is a cell sample, which can (1989), supra or extracted by nucleic-acid-binding resins fol be a primary cell sample or cultivated cell sample or a prog lowing the accompanying instructions provided by manufac eny thereof. Cell samples can be obtained from a variety of tures. tissues depending on the age and condition of the animal. Cell 0057. In some cases, sample analyses involves performing samples can be obtained from peripheral blood using well one or more genetic analyses or detection steps on nucleic known techniques. In fetal testing, a sample can be obtained acids from the enriched product (e.g., enriched cells or by amniocentesis, chorionic Villi Sampling or by isolating nuclei). Nucleic acids from enriched cells or enriched nuclei fetal cells from the blood of a pregnant individual. Other that can be analyzed by the methods herein include: double Sources of nucleic acids include blood, semen, buccal cells, or stranded DNA, single-stranded DNA, single-stranded DNA the like. Nucleic acids can be obtained from any tissue or hairpins, DNA/RNA hybrids, RNA (e.g. mRNA) and RNA organ by methods well known in the art. hairpins Examples of genetic analyses that can be performed 0050. In any of the embodiments herein, target nucleic on enriched cells or nucleic acids include, e.g., SNP detec acids are obtained from a single cell. tion, STR detection, and RNA expression analysis. 0051) To obtain a blood sample, any technique known in 0058. In some embodiments, less than 1 pg. 5 pg. 10 pg. 20 the art may be used, e.g. a syringe or other vacuum Suction pg. 30 pg. 40 pg. 50 pg. 100 pg. 200 pg. 500 pg. 1 ng, 5 ng, 10 device. A blood sample can be optionally pre-treated or pro ng, 20 ng, 30 ng, 40 ng, 50 ng, 100 ng, 200 ng, 500 ng, 1 ug, cessed prior to enrichment. Examples of pre-treatment steps 5ug. 10 ug. 20 ug. 30 ug, 40 ug, 50 ug, 100 ug, 200 ug. 500 include the addition of a reagent Such as a stabilizer, a pre ug or 1 mg of nucleic acids are obtained from the sample for servative, a fixant, a lysing reagent, a diluent, an anti-apop further genetic analysis. In some cases, about 1-5pg, 5-10 pg. US 2010/0285468 A1 Nov. 11, 2010

10-100 pg. 100 pg-1 ng, 1-5 ng, 5-10 ng, 10-100 ng, 100 ng-1 plastic and/or cancer cell. In some embodiments, the methods ug of nucleic acids are obtained from the sample for further described herein are used to detect and/or quantify target genetic analysis. nucleic acids to diagnose cancer and/or a neoplastic condi 0059. In some embodiments, the methods described tion. In some embodiments, the methods described herein are herein are used to detect and/or quantified a target nucleic used to detect and/or quantify target nucleic acids to detect acid molecule. In some embodiments, the methods described biomarkers in a neoplastic and/or cancer cell. In some herein are used to detect and/or quantified multiple target embodiments the methods described herein are used to deter nucleic acid molecules. The methods described herein can mine the copy number of a target nucleic acid in a neoplastic analyzed at least 1,2,3,4, 5, 10, 20, 50, 100, 200, 500, 1,000, and/or cancer cell. 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, different tar 0065. As used herein the term "diagnose' or “diagnosis' get nucleic acids. of a condition includes predicting or diagnosing the condi 0060. In some embodiments, the methods described tion, determining predisposition to the condition, monitoring herein are used to detect and/or quantified a target nucleic treatment of the condition, diagnosing a therapeutic response acid molecule which is about 10, 20, 30, 40, 50, 60, 70, 80,90, of the disease, and prognosis of the condition, condition pro 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, gression, and response to particular treatment of the condi 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, tion. 340, 350, 360, 370, 380,390, 400, 400, 410, 420, 430, 440, 0.066 Conditions in a patient that can be detected using the 450, 460, 470,480,490, 500,510,520, 530, 540, 550, 560, systems and methods herein include, but are not limited to, 570, 580, 590, 600, 600, 610, 620, 630, 640, 650, 660, 670, infection (e.g., bacterial, viral, or fungal infection), neoplastic 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or cancer conditions (e.g., acute lymphoblastic leukemia, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, acute or chronic lymphocyctic or granulocytic tumor, acute 920,930, 940,950, 960,970,980, 990, 1000 by in length. In myeloid leukemia, acute promyelocytic leukemia, adenocar some embodiments, the methods described herein are used to cinoma, adenoma, adrenal cancer, basal cell carcinoma, bone detect and/or quantified a target nucleic acid molecule which cancer, brain cancer, breast cancer, bronchi cancer, cervical larger that 1 kb in length. In some embodiments, the methods dysplasia, chronic myelogenous leukemia, colon cancer, epi described herein are used to detect and/or quantified a target dermoid carcinoma, Ewing's sarcoma, gallbladder cancer, nucleic acid molecule which is about 1,2,3,4,5,6,7,8,9, 10 gallstone tumor, giant cell tumor, glioblastoma multiforma, Kb in length. hairy-cell tumor, head cancer, hyperplasia, hyperplastic cor 0061. In some embodiments, the methods described neal nerve tumor, in situ carcinoma, intestinal ganglion herein are used to detect allelic loss, point mutations, dele euroma, islet cell tumor, Kaposi's sarcoma, kidney cancer, tions, amplifications, or rearrangement in cells from an indi larynx cancer, leiomyomater tumor, liver cancer, lung cancer, vidual. Normal cells that are heterozygous at one or more loci lymphomas, malignant carcinoid, malignant hypercalcemia, may give rise to tumor cells that are homozygous at those loci. malignant melanomas, marfanoid habitus tumor, medullary This loss of heterozygosity may result from structural dele carcinoma, metastatic skin carcinoma, mucosal neuromas, tion of normal genes or loss of the chromosome carrying the mycosis fungoide, myelodysplastic syndrome, myeloma, normal gene, mitotic recombination between normal and neck cancer, neural tissue cancer, neuroblastoma, osteogenic mutant genes, followed by formation of daughter cells sarcoma, osteosarcoma, ovarian tumor, pancreas cancer, par homozygous for deleted or inactivated (mutant) genes; or loss athyroid cancer, pheochromocytoma, polycythemia Vera, pri of the chromosome with the normal gene and duplication of mary brain tumor, prostate cancer, rectum cancer, renal cell the chromosome with the deleted or inactivated (mutant) tumor, retinoblastoma, rhabdomyosarcoma, seminoma, skin gene. cancer, Small-cell lung tumor, soft tissue sarcoma, squamous 0062. A homozygous deletion is a deletion of both copies cell carcinoma, stomach cancer, thyroid cancer, topical skin of a gene or of a genomic region. Diploid organisms generally lesion, Veticulum cell sarcoma, or Wilm's tumor), inflamma have two copies of each autosomal chromosome and there tion, etc. fore have two copies of any selected genomic region. If both 0067. In some embodiments, the methods described copies of a genomic region are absent the cell or sample has herein are used to distinguish between target nucleic acids a homozygous deletion of that region. Similarly, a hemizy that differ from another nucleic acid by a single nucleotide. In gous deletion is a deletion of one copy of a gene or of a some embodiments, the methods described herein are used to genomic region. distinguish between target nucleic acids that differ from 0063 Genetic rearrangement occurs when errors occur in another nucleic acid by 1 nucleotide (nt) or more than 1, 2, 3, DNA replication and cross over occurs between nonhomolo 5, 10, 15, 20, 21, 22, 24, 25, 30 or more nucleotides. gous regions resulting in genetic material moving from one 0068. In some embodiments, the methods described chromosomal location to another. Rearrangement may result herein are used to detect and/or quantify target nucleic acids in altered expression of the genes near the rearrangement. without the need of target nucleic acid isolation. In some 0064. In some embodiments, the methods described embodiments, the methods described hereinareused to detect herein are used to detect and/or quantify target nucleic acids and/or quantify a target nucleic acid directly from a nucleic to profile a specific tissue or a specific condition. In some acid sample comprising DNA and RNA molecules. embodiments, the methods described hereinareused to detect 0069. The methods described herein can be used to detect and/or quantify target nucleic acids to detect biomarkers for and/or quantify genomic DNA regions. The method specific tissue or condition. In some embodiments the meth described herein can be used to detect the copy number of one ods described herein are used to determine the copy number or more genomic DNA regions. In some embodiments, the of a target nucleic acid for specific tissue or condition. In methods described herein are used to diagnose a fetal abnor some embodiments, the methods described herein are used to mality. means the condition of having less than or detect and/or quantify target nucleic acids to profile a neo more than the normal diploid number of chromosomes. In US 2010/0285468 A1 Nov. 11, 2010 other words, it is any deviation from euploidy. Aneuploidy taining a DNA polymorphism. A polymorphism refers to the includes conditions such as (the presence of only occurrence of two or more genetically determined alternative one chromosome of a pairina cell's nucleus), trisomy (having sequences oralleles in a population. A polymorphic marker or three chromosomes of a particular type in a cell's nucleus), site is the locus at which divergence occurs. Preferred mark (having four chromosomes of a particular type in a ers have at least two alleles, each occurring at a frequency of cell's nucleus), pentasomy (having five chromosomes of a preferably greater than 1%, and more preferably greater than particular type in a cell's nucleus), triploidy (having three of 10% or 20% of a selected population. A polymorphism may every chromosome in a cell's nucleus), and tetraploidy (hav comprise one or more base changes, an insertion, a repeat, or ing four of every chromosome in a cell's nucleus). Birth of a live triploid is extraordinarily rare and such individuals are a deletion. A polymorphic locus may be as Small as one base quite abnormal, however triploidy occurs in about 2-3% of all pair. Polymorphic markers include single nucleotide poly human pregnancies and appears to be a factor in about 15% of morphisms (SNPs), restriction fragment length polymor all . Tetraploidy occurs in approximately 8% of phisms (RFLP's), variable number of tandem repeats (VN all miscarriages. (http://www.emedicine.com/med/ TR's), hypervariable regions, minisatellites, dinucleotide topic3241.htm). repeats, trinucleotide repeats, tetranucleotide repeats, simple 0070. In some embodiments, the methods described sequence repeats, and insertion elements such as Alu. The herein are used to detect and/or quantify genomic DNA first identified allelic form is arbitrarily designated as the regions to diagnose a fetal condition Such as aneuploidy. In reference form and otherallelic forms are designated as alter some embodiments, the methods described herein are used to native or variant alleles. The allelic form occurring most diagnose a fetal abnormality by quantifying a DNA region frequently in a selected population is sometimes referred to as chosen on a chromosome Suspected of aneuploidy and on a the wildtype form. Diploid organisms may be homozygous or control chromosome. In some embodiment aneuploidy is tri heterozygous for allelic forms. A diallelic polymorphism has Somy selected from the group consisting of trisomy 13, tri two forms. A triallelic polymorphism has three forms. A somy 18, trisomy21 (Down Syndrome), Klinefelter Syn polymorphism between two nucleic acids can occur naturally, drome (XXY), or other irregular number of sex or autosomal or be caused by exposure to or contact with chemicals, chromosomes, and a combination thereof. Examples of chro enzymes, or other agents, or exposure to agents that cause mosomes that are often trisomic include chromosomes 21, damage to nucleic acids, for example, ultraviolet radiation, 18, 13, and X. In some cases, 1 or more than 1, 2, 3, 4, 5, 6, 7, mutagens or carcinogens. In some embodiments, the methods 8, 9, 10, 15, or 20 regions are detected and quantified per described herein can discriminate and quantitate a DNA chromosome tested. In some embodiments, the methods region containing a DNA polymorphisms. The methods described herein can discriminate and quantitate genomic described herein can discriminate and quantitate DNA poly DNA regions. The methods described herein candiscriminate morphism of at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, and quantitate genomic DNA regions of at least 1, 2, 3, 4, 5, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, differ 10, 20, 50, 100, 200,500, 1,000, 2,000, 5,000, 10,000, 20,000, ent genomic DNA regions. 50,000, 100,000, different genomic DNA regions. The meth 0073. The methods described herein can be used to detect ods described hereincan discriminate and quantitate genomic and/or quantify genomic DNA regions such as copy number DNA regions varying by 1 nt or more than 1,2,3,5, 10, 15, 20, variable region (CNVR). Until recently, the importance of 21, 22, 24, 25, 30 nt. large-scale copy number changes in the genomes of humans 0071 Fetal conditions that can be determined based on the and other vertebrates has been under-appreciated. Two methods and systems herein include the presence of a fetus reports using comparative genomic hybridization with DNA and/or a condition of the fetus Such as fetal aneplouidy e.g., microarrays (array-CGH), highlighted the widespread nature trisomy 13, trisomy 18, trisomy 21 (Down Syndrome), of this normal copy number variation (Iafrate et al. 2004: Klinefelter Syndrome (XXY) and other irregular number of Sebat et al. 2004). Other studies have now confirmed and sex or autosomal chromosomes. Other fetal conditions that further detailed the extent of copy number variation (CNV) in can be detected using the methods herein include segmental human and primate genomes (Newman et al. 2005: Sharp et aneuploidy, such as 1 p 36 duplication, dup (17) (p. 11.2p11.2) al. 2005: Tuzun et al. 2005; Conrad et al. 2006: Perry et al. syndrome, Down syndrome, Pelizaeus-Merzbacher disease, 2006). CNVR are copy number variation of DNA segments dup (22)(q11.2d 11.2) syndrome, Cat eye syndrome. In some ranging to kilobases (kb) to megabases (Mb) in size, includ embodiment, the fetal abnormality to be detected is due to one ing deletions, insertions, duplications and complex multi-site or more deletions in sex or autosomal chromosomes, includ variants. CNVRS can affect gene expression, phenotypic ing Cri-du-chat syndrome, Wolf-Hirschhorn syndrome, Wil variation and adaptation by disrupting genes and altering liams-Beuren syndrome, Charcot-Marie-Tooth disease, gene dosage. In addition, CNVRS can cause disease, as in Hereditary neuropathy with liability to pressure palsies, microdeletion or microduplication disorders, or conferrisk to Smith-Magenis Syndrome, Neurofibromatosis, Alagille Syn disease traits such as HIV infection and glomerulonephritis. drome, Velocardiofacial syndrome, DiGeorge syndrome, Ste Some CNVRs have been associated with specific diseases roid sulfatase deficiency, Kallmann syndrome, such as CHARGE syndrome, Parkinson's and Alzheimer dis Microphthalmia with linear skin defects, Adrenal hypoplasia, CaSC. Glycerol kinase deficiency, Pelizaeus-Merzbacher disease, 0074. In some embodiments, the methods described testis-determining factor on Y. AZospermia (factor a). herein are used to detect and/or quantified a CNVR molecule. AZospermia (factor b), AZospermia (factor c) and 1 p 36 dele In some embodiments, the methods described herein are used tion. In some cases, the fetal abnormality is an abnormal to detect and/or quantified multiple CNVR molecules. In decrease in chromosomal number, such as XO syndrome. some embodiments, the methods described herein are use to 0072 The methods described herein can be used to detect determine the copy number of one or more CNVRs. The and/or quantify genomic DNA regions such as a region con methods described herein can analyzed at least 1, 2, 3, 4, 5, US 2010/0285468 A1 Nov. 11, 2010

10, 20, 50, 100, 200,500, 1,000, 2,000, 5,000, 10,000, 20,000, 0078. The present invention provides a method for deter 50,000, 100,000, different CNVRs. mining methylation status of CpG dinucleotides within a 0075. The methods described herein can be used to detect target nucleic acid molecule. CpG islands (a stretch of CpGs), and/or quantify CNVRs to profile a specific tissue or a spe are typically unmethylated. Hypermethylation in CpG islands of promoter regions leads to silence the associated cific condition. In some embodiments, the methods described gene expression. Aberrant methylation has been associated to herein are used to detect and/or quantify CNVRs to detect different pathogenesis including neoplasia. In some embodi biomarkers for specific tissue or condition. In some embodi ments, the methods described herein can discriminate and ments, the methods described herein are used to detect and/or quantitate the methylation state of at least 1, 2, 3, 4, 5, 10, 20, quantify CNVRs to profile a neoplastic and/or cancer cell. In 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, some embodiments, the methods described herein are used to 50,000, 100,000, different target nucleic acids. In some detect and/or quantify CNVRs to diagnose cancer and/or a embodiments, the methods described hereinareused to detect neoplastic condition. In some embodiments, the methods and/or quantify methylation status of target nucleic acids with described herein are used to detect and/or quantify CNVRs to similar sequences. The methods described herein can dis detect biomarkers in a neoplastic and/or cancer cell. criminate and quantitate the methylation state of target 0076. In some embodiments, the methods described nucleic acids varying by 1 nt or more than 1, 2, 3, 4, 5, 10, 12. herein are used to detect and/or quantify genomic DNA 15, 20 nt. regions to diagnose a fetal condition Such as any disorder or 0079. In some embodiments, the methods described condition associated with CNVRs. Such disorders include, herein are used to detect and/or quantify gene expression. In but are not limited to, Parkinson's disease, Alzheimer's dis some embodiments, the methods described herein provide ease, dementia, an disorder, Susceptibility to high discriminative and quantitative analysis of multiples viral infection such as HIV, and CHARGE Syndrome. Autism genes. The methods described herein can discriminate and spectrum disorders include Asperger syndrome, autism, PDD quantitate the expression of at least 1, 2, 3, 4, 5, 10, 20, 50. not otherwise specified, and Rett disorder. Other known dis 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, orders related to CNVRs include, but are not limited to, 12q14 100,000, different target nucleic acids. , 15q13.3 microdeletion syndrome, 15q24 recurrent microdeletion syndrome, 16 p. 11.2-p 12.2 Amplification Procedures microdeletion syndrome, 17q21.3 microdeletion syndrome, 0080. The practice of one or more methods disclosed 1p 36 microdeletion syndrome, liq21.1 recurrent microdele herein may involve amplifying a target nucleic acid and/or a tion, 1 q21.1 recurrent microduplication, 1 q21.1 Susceptibil reference nucleic acid. In some embodiments, at least one ity locus for Thrombocytopenia-Absent Radius (TAR) syn target nucleic acid and at least one reference nucleic acid are drome, 22q11 deletion syndrome (Velocardiofacial/ amplified using a different set of primers. That is, a first set of DiGeorge syndrome), 22d 11 duplication syndrome, 22d 11.2 primer specific for the target nucleic acid and a second set of distal deletion syndrome, (Phelan primers specific for a reference nucleic acid are used for the Mcdermid syndrome), 2 p 15-16.1 microdeletion syndrome, amplification. Typically, the target nucleic acid and the refer 2q33.1 deletion syndrome, 2c37 monosomy, 3q29 microde ence nucleic acid are comparable in length. letion syndrome, 3q29 microduplication syndrome, 6p dele I0081. Usually the quantity of the reference nucleic acid is tion syndrome, 7q11.23 duplication syndrome, 8p 23.1 dele known. In some embodiments, a reference nucleic acid is tion syndrome, 9q Subtelomeric deletion syndrome, Adult added to the sample containing the target nucleic acid. The onset autosomal dominant leukodystrophy (ADLD), reference nucleic acid may be isolated from the same or (Type 1), Angelman syndrome (Type 2), different source as the target nucleic acid. In addition, the ATR-16 syndrome, AZFa, AZFb. AZFb-i-AZFc, AZFc, Cat reference nucleic acid may be recombinantly produced or Eye Syndrome (Type I), Charcot-Marie-Tooth syndrome type artificially synthesized. For example, the target nucleic acid 1 A (CMT1A), (5p deletion), Early and the reference nucleic acid can be different parts of a single onset Alzheimer disease with cerebral amyloid angiopathy, cDNA transcript, such as 5' end vs. 3' end. Alternatively, the Familial Adenomatous Polyposis, Hereditary Liability to target nucleic acid and the reference nucleic acid can be Pressure Palsies (HNPP), Leri-Weill dyschondrostosis different transcripts from a single cDNA preparation. The (LWD) -SHOX deletion, Miller-Dieker syndrome (MDS), target nucleic acid and the reference nucleic acid can be NF1-microdeletion syndrome, Pelizaeus-Merzbacher dis different parts from a single gene, or two different regions of ease, Potocki-Lupski syndrome (17p11.2 duplication syn the same chromosome, or two different regions from the same drome), Potocki-Shaffer syndrome, Prader-Willi syndrome human DNA from the same or different sample preparations. (Type 1), Prader-Willi Syndrome (Type 2), RCAD (renal Where desired, the reference nucleic acid has a length com cysts and diabetes), Rubinstein-Taybi Syndrome, Smith-Ma parable to that of the target nucleic acid. For example, a genis Syndrome, Sotos syndrome, Split hand/foot malforma reference nucleic acid differs in length as compared to the tion 1 (SHFM1), Steroid sulphatase deficiency (STS), WAGR target nucleic acid by less than about 50%, 40%, 30%, 20%, 11 p 13 deletion syndrome, Williams-Beuren Syndrome 10%, 5% or less. A reference nucleic acid typically exhibits a (WBS), Wolf-Hirschhorn Syndrome, and Xq28 (MECP2) distinct melting profile from that of the target nucleic acid. duplication. I0082 Amplification of target nucleic acids can be per 0077. The methods described herein can be used to detect formed by any means known in the art. In some cases, target and/or quantitate a DNA epigenetic change including but not nucleic acids are amplified by polymerase chain reaction limited to chemical modifications and chromatin structure. In (PCR). Examples of PCR techniques that can be used include, Some embodiments the DNA epigenetic change comprises a but are not limited to, quantitative PCR, quantitative fluores chemical modification. In some embodiments, the chemical cent PCR (QF-PCR), multiplex fluorescent PCR (MF-PCR), modification comprises DNA methylation. real time PCR(RT-PCR), single cell PCR, restriction frag US 2010/0285468 A1 Nov. 11, 2010

ment length polymorphism PCR (PCR-RFLP), PCR-RFLP/ reference nucleic acid may be isolated from the same or RT-PCR-RFLP, hot start PCR, nested PCR, in situ polonony different source as the target nucleic acid. In addition, the PCR, in situ rolling circle amplification (RCA), bridge PCR, reference nucleic acid may be recombinantly produced or picotiter PCR and emulsion PCR. Other suitable amplifica artificially synthesized. For example, the target nucleic acid tion methods include the ligase chain reaction (LCR), tran and the reference nucleic acid can be different parts of a single Scription amplification, self-sustained sequence replication, cDNA transcript, such as 5' end vs. 3' end. Alternatively, the selective amplification of target polynucleotide sequences, target nucleic acid and the reference nucleic acid can be consensus sequence primed polymerase chain reaction (CP different transcripts from a single cDNA preparation. The PCR), arbitrarily primed polymerase chain reaction (AP target nucleic acid and the reference nucleic acid can be PCR), degenerate oligonucleotide-primed PCR (DOP-PCR) different parts from a single gene, or two different regions of and nucleic acid based sequence amplification (NABSA). the same chromosome, or two different regions from the same Other amplification methods that can be used herein include human DNA from the same or different sample preparations. those described in U.S. Pat. Nos. 5,242,794; 5,494,810; Typically, the target nucleic acid and the reference nucleic 4,988,617; and 6,582,938. acid are comparable in length, e.g., when the target nucleic 0083. In any of the embodiments, amplification of target acid and reference nucleic are not known to be in similar nucleic acids and/or reference nucleic acid occurs on a Solid quantity, or when the target nucleic acid and reference nucleic Support including but not limited to beads, nanoparticles and are known to be in similar quantity. The relative length of the quantom dots. target and reference could be adjusted according to the quan 0084. In any of the embodiments herein, the nucleic acid tities of the two so that the melting areas are comparable in (s) of interest can be pre-amplified prior to the amplification melting analysis. step (e.g., PCR). In some cases, a nucleic acid sample may be 0090. In some embodiments, the target nucleic acid ana pre-amplified to increase the overall abundance of genetic lyzed by the methods described herein is a genomic DNA material to be analyzed (e.g., DNA). Pre-amplification can region. In some cases, the genomic DNA region contains one therefore include whole genome amplification Such as mul or more polymorphisms such as a CNVR or a STRs or SNPs. tiple displacement amplification (MDA) or amplifications The reference nucleic acid could also be a genomic DNA with outer primers in a nested PCR approach. region. Alternatively, the reference nucleic acid could be a 0085. In some embodiments, the target nucleic acid is cDNA transcript or an oligonucletide. The reference nucleic amplified through other isothermal amplification schemes acid may be isolated from the same or different source as the known in the art. In some embodiments of the invention, the target nucleic acid. In addition, the reference nucleic acid may target nucleic acid is quantified before the amplification steps. be recombinantly produced or artificially synthesized. For example, the target nucleic acid and the reference nucleic acid Melting Analysis can be different parts of a single cDNA transcript, such as 5' I0086. The methods describe herein generally utilize melt end vs. 3' end. Alternatively, the target nucleic acid and the ing analysis to detect and quantify target nucleic acids. reference nucleic acid can be different transcripts from a Results from melting analysis with a test sample containing a single cDNA preparation. The target nucleic acid and the target nucleic acid of interest are optionally compared with reference nucleic acid can be different parts from a single similar target nucleic acids from a control samples, e.g. con gene, or two different regions of the same chromosome, or trol cell population. Melting, also called denaturation, is the two different regions from the same human DNA from the process by which double-stranded nucleic acid unwinds and same or different sample preparations. separates into single-stranded strands through the breaking of 0091 Melting analysis can be carried out using any suit hydrogen bonding between the bases. The melting tempera able instrument known in the art such as a UV spectropho ture (Tm) is defined as the temperature at which half of the tometer. Typical melting analyses can be done on instru nucleic acid strands are in the double-helical state and halfare ments, such as ABI 7900, 7500, BioRad iQ5, Chromo4, in the “random-coil state. The melting temperature typically Corbett Rotogene 6000, Roche Lightcycler 480, Idaho tech depends on both the length of the molecule, and the GC nology Genetyper, or Stratagene MX4000. content of that molecule. Short nucleic acid fragments usually 0092. In some embodiments, melting analyses are per have only one melting unit manifested as a single melting formed by measuring the absorbance of the target nucleic acid peak, while long nucleic acid fragment may have multiple and the reference nucleic acid at a plurality of temperatures. melting units, manifested as multiple melting curves. Typically, absorbance is measured at 260. 0087 Melting analyses can be conviniently done in the 0093 Melting analysis can also be performed in the pres presence of a nucleic binding dye concurrent with or after an ence of a nucleic acid binding agent. In some embodiments, amplification reaction is completed. When thermal resolution the binding agent emits a signal when the agent is bound to a is at 0.05° C. or smaller, melting analyses called high-reso nucleic acid. The signal can be fluorescent signal, magnetic lution melting analyses (HRM) are used. HRM could provide signal, radioactive signal, Raman signal or an electrochemi very detailed Tm information, including single base changes cal signal. In some embodiments the binding agent is a such as SNPs (single nucleotide polymorphisms). nucleic acid intercalator. Examples of nucleic acid binding 0088 Nucleic acids from samples that can be analyzed by agents that can be used in the methods described herein the methods herein include: double-stranded DNA, single include, but are not limited to, EvaGreen(R) (Biotium, Hay stranded DNA, single-stranded DNA hairpins, DNA/RNA ward, Calif.), SYBR(R) Green I, PicoGreenTM, LC GreenTM, hybrids, RNA (e.g. mRNA or miRNA) and RNA hairpins. SYBR GreenERR), PO-PROR).-1, BO-PROR).-1, SYTOR 9, 0089. In some embodiments, to quantify the target nucleic SYTORTM43, SYTOR. 44, SYTOR. 45, SYTOX(R) Blue, acid content in a target sample, a reference a reference nucleic POPOTM-1, POPOTM-3, BOBOTM-1, BOBOTM-3, acid with known quantity is added. The reference and the LO-PROTM-1, JO-PROTM-1, YO-PROR)-1, TO-PROR)-1, target nucleic acid can be amplified as described above. The SYTOR 9, SYTOR11, SYTOR13, SYTOR15, SYTOR16, US 2010/0285468 A1 Nov. 11, 2010

SYTOR20, SYTOR23, TOTOTM3, YOYOR-3 (Molecular 0100 Another method to determine nucleic acid quantity Probes, Inc., Eugene, Oreg.), GelStar R. (Cambrex Bio Sci is by measuring the peak height. Three examples are pre ence Rockland Inc., Rockland, Me.), Ethidium Bromide, sented in FIGS. 1D, 1E and 1F. thiazole orange (Aldrich Chemical Co., Milwaukee, Wis.), BEBO, BETO, BOXTO (TATAA Biocenter AB., Goteborg, 0101. In some embodiments, the quantity of nucleic acid is Sweden). determined by comparing the results of the melting analyses 0094. The binding agent can emit a signal when bound to of the target and reference nucleic acid to a standard curve. either single or double stranded nucleic acids. Alternative, the The quantity of nucleic acid can be calculated in grams, binding agent can emit a signal when is no longer bound to Dalton, bp, or copy numbers. For instance, after determining either single or double stranded nucleic acids. In other the area under the peak or the peak height ratio of the target embodiments, the absorbance of a binding agent is measured and reference nucleic acids as described above, the quantity to perform the melting analysis described herein. All these of target nucleic acid can be determined using a standard type of binding agents can be used and are encompassed in the methods described herein. curve such as the one depicted in FIG. 6B. In Example 7 using 0095. The change in the signal from the nucleic acid bind the peak-height ratio of the standard response curve in FIG. ing agent (e.g., fluorescence change) or absorbance (e.g. 6B, the input ratio of Chrl to Chr8 was determined as shown binding agent or nucleic acid) is monitored at different tem in Row 5 of FIG. 7. peratures. For example, in Some embodiments, the signal 0102. In some embodiments, the methods described from the nucleic acid binding agent can be monitored at herein are used to detect and/or quantified a target nucleic increasing temperatures from 0°C. to 100°C. The intensities acid molecule. In some embodiments, the methods described of the signals of the binding agent are typically inversely herein are used to detect and/or quantified multiple target proportional to the degree of melting of a nucleic acid mol nucleic acid molecules. The methods described herein can ecule in double stranded or multi-stranded state. The intensity readout at various temperatures points are recorded and plot analyzed at least 1,2,3,4, 5, 10, 20, 50, 100, 200, 500, 1,000, ted to derive a melting curve or a melting profile of the target 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, different tar or reference nucleic acids. Typical melting analyses can be get nucleic acids. automatically performed on instruments, such as ABI 7900, 0103) In some embodiments, less than 1 pg. 5 pg. 10 pg. 20 7500, BioRadio5, Chromo4, Corbett Rotogene 6000, Roche pg. 30 pg. 40 pg. 50 pg. 100 pg. 200 pg. 500 pg. 1 ng, 5 ng, 10 Lightcycler 480, Idaho technology Genetyper, or Stratagene ng, 20 ng, 30 ng, 40 ng, 50 ng, 100 ng, 200 ng, 500 ng, 1 ug, MX4000. Any instrument known in the art suitable for melt ing analysis can be used with the methods described herein. 5ug. 10 ug. 20 ug. 30 ug, 40 ug, 50 ug, 100 ug, 200 ug. 500 0096. In some embodiments a melting curve is generated ug or 1 mg of nucleic acids are obtained from the sample for from the melting analysis described above. FIG. 1A shows an further genetic analysis. In some cases, about 1-5pg, 5-10 pg. example of a melting curve obtained after performing melting 10-100 pg. 100 pg-1 ng, 1-5 ng, 5-10 ng, 10-100 ng, 100 ng-1 analyses of the target and reference nucleic acid. The X-axis is ug of nucleic acids are obtained from the sample for further the temperature (T), the y-axis is the -dF/dT, where F is the genetic analysis. fluorescence intensity recorded. dF/dT is the first derivative 0104. In some embodiments, the methods described against the temperature. Two melting peaks (1 and 2) corre herein are used to detect and/or quantified a target nucleic spond to reference nucleic acid and target nucleic acid respec tively. acid molecule which is about 10, 20, 30, 40, 50, 60, 70, 80,90, 0097. There are various ways in which the nucleic acid 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, content can be quantified. In some embodiments, the melting 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, profiles from the target and references nucleic acids are com 340, 350, 360, 370, 380,390, 400, 400, 410, 420, 430, 440, pared to determine the relative amount of signal attributable 450, 460, 470, 480,490, 500,510,520, 530, 540, 550,560, to the target nucleic acid or the reference nucleic acid based 570, 580, 590, 600, 600, 610, 620, 630, 640, 650, 660, 670, on their melting profile. In some embodiments, the relative 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, amount of the signal attributable to the target nucleic acid and 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, the reference nucleic acid are compared. In some embodi 920,930,940,950, 960,970,980, 990, 1000 by in length. In ments, a ratio is calculated between the amount of signal some embodiments, the methods described herein are used to attributable to the target nucleic acid and the amount of signal detect and/or quantified a target nucleic acid molecule which attributable to the reference nucleic acid. The quantity of larger that 1 kb in length. In some embodiments, the methods nucleic acid can be calculated in grams, Dalton, bp, or copy described herein are used to detect and/or quantified a target numbers. nucleic acid molecule which is about 1,2,3,4,5,6,7,8,9, 10 0098 FIGS. 1B, 1C, 1D and 1E show various exemplary kb in length. embodiments for the quantification of nucleic acid content. 0105. The methods described herein can be employed to One method to determine nucleic acid quantity is to measure discriminate between the amount of nucleic acid (e.g. copy the area under the peak as shown in FIGS. 1B, and 1C.. In number) and nucleic acid sequences. The difference between general, the amount of target nucleic acid and the area of the the target nucleotide sequences can be, for example, a single melting peak follow the formula I as shown below: nucleic acid base difference, deletion, insertion, amplifica tion or rearrangement. As a result, the process of the present (Peak Area 1)/(Peak Area. 2)=(Amount of DNA 1). invention is able to detect infectious diseases, genetic dis (Amount of DNA 2) (I) eases, and cancer. It is also useful in environmental monitor 0099. In many cases, the area of the peak is proportional to ing, forensics, and food Science. Examples of genetic analy the peak height, the formula I can be modified to formula II as ses that can be performed on nucleic acids include e-g., shown below: CNVR detection, SNP detection, STR detection, RNA (Peak Height 1)/(Peak height 2)=(Amount of DNA expression analysis, promoter methylation, gene expression, 1)/(Amount of DNA 2) (II) virus detection, viral Subtyping and drug resistance. US 2010/0285468 A1 Nov. 11, 2010

0106 A wide variety of infectious diseases can be detected Cat eye syndrome, Cri-du-chat syndrome, Wolf-Hirschhorn by the process of the present invention. Typically, these are syndrome, Williams-Beuren syndrome, Charcot-Marie caused by bacterial, viral, parasite, and fungal infectious Tooth disease, neuropathy with liability to pressure palsies, agents. The resistance of various infectious agents to drugs Smith-Magenis syndrome, neurofibromatosis, Alagille Syn can also be determined using the present invention. drome, Velocardiofacial syndrome, DiGeorge syndrome, Ste 0107 Bacterial infectious agents which can be detected by roid Sulfatase deficiency, Kallmann syndrome, the present invention include Escherichia coli, Salmonella, microphthalmia with linear skin defects, Adrenal hypoplasia, Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes, Glycerol kinase deficiency, Pelizaeus-Merzbacher disease, Mycobacterium tuberculosis, Mycobacterium aviumintracel testis-determining factor on Y. AZospermia (factor a). lulare, Yersinia, Francisella, Pasteurella, Brucella, AZospermia (factor b), AZospermia (factor c), and 1 p 36 Clostridia, Bordetella pertussis, Bacteroides, Staphylococ deletion. cus aureus, Streptococcus pneumonia, B-Hemolytic strep., 0113 Cancers which can be detected by the process of the Corynebacteria, Legionella, Mycoplasma, Ureaplasma, present invention generally involve oncogenes, tumor Sup Chlamydia, Neisseria gonorrhea, Neisseria meningitides, pressor genes, or genes involved in DNA amplification, rep Hemophilus influenza, Enterococcus faecalis, Proteus vul lication, recombination, or repair Examples of these include: garis, Proteus mirabilis, Helicobacter pylori, Treponema BRCA1 gene, p53 gene, APC gene. Her2/Neu amplification, palladium, Borrelia burgdorferi, Borrelia recurrentis, Rick Bcr/Ab1, K-ras gene, and human papillomavirus Types 16 ettsial pathogens, Nocardia, and Acitinomycetes. and 18.Various aspects of the present invention can be used to 0108 Fungal infectious agents which can be detected by identify amplifications, large deletions as well as point muta the present invention include Cryptococcus neoformans, tions and Small deletions/insertions of the above genes in the Blastomyces dermatitidis, Histoplasma capsulatum, Coccid following common human cancers: leukemia, colon cancer, ioides immitis, Paracoccidioides brasiliensis, Candida albi breast cancer, lung cancer, prostate cancer, brain tumors, cans, Aspergillus fumigautus, Phycomycetes (Rhizopus), central nervous system tumors, bladder tumors, melanomas, Sporothrix schenckii, Chronomycosis, and Maduromycosis. liver cancer, osteosarcoma and other bone cancers, testicular 0109 Viral infectious agents which can be detected by the and ovarian carcinomas, head and neck tumors, and cervical present invention include human immunodeficiency virus, neoplasms. Examples of different cancer and/or neoplastic human T-cell lymphocytotrophic virus, hepatitis viruses (e.g., conditions include but are not limited to acute lymphoblastic Hepatitis BVirus and Hepatitis C Virus), Epstein-Barr Virus, leukemia, acute or chronic lymphocyctic or granulocytic cytomegalovirus, human papillomaviruses, orthomyxo tumor, acute myeloid leukemia, acute promyelocytic leuke viruses, paramyxo viruses, adenoviruses, corona viruses, mia, adenocarcinoma, adenoma, adrenal cancer, basal cell rhabdo viruses, polio viruses, toga viruses, bunya viruses, carcinoma, bone cancer, brain cancer, breast cancer, bronchi arena viruses, rubella viruses, and reo viruses. cancer, cervical dysplasia, chronic myelogenous leukemia, 0110 Parasitic agents which can be detected by the colon cancer, epidermoid carcinoma, Ewing's sarcoma, gall present invention include Plasmodium falciparum, Plasmo bladder cancer, gallstone tumor, giant cell tumor, glioblas dium malaria, Plasmodium vivax, Plasmodium ovale, toma multiforma, hairy-cell tumor, head cancer, hyperplasia, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schis hyperplastic corneal nerve tumor, in situ carcinoma, intestinal tosoma spp., Entamoeba histolytica, Cryptosporidum, Giar ganglioneuroma, islet cell tumor, Kaposi's sarcoma, kidney dia spp., Trichimonas spp., Balatidium coli, Wuchereria ban cancer, larynx cancer, leiomyomater tumor, liver cancer, lung crofti, Toxoplasma spp., Enterobius vermicularis, Ascaris cancer, lymphomas, malignant carcinoid, malignant hyper lumbricoides, Trichuris trichiura, Dracunculus medinesis, calcemia, malignant melanomas, marfanoid habitus tumor, trematodes, Diphyllobothrium latum, Taenia spp., Pneu medullary carcinoma, metastatic skin carcinoma, mucosal mocystis carinii, and Necator americanis. neuromas, mycosis fungoide, myelodysplastic syndrome, 0111. The present invention is also useful for detection of myeloma, neck cancer, neural tissue cancer, neuroblastoma, drug resistance by infectious agents. For example, Vancomy osteogenic sarcoma, osteosarcoma, ovarian tumor, pancreas cin-resistant Enterococcus faecium, methicillin-resistant Sta cancer, parathyroid cancer, pheochromocytoma, poly phylococcus aureus, penicillin-resistant Streptococcus pneu cythemia Vera, primary brain tumor, prostate cancer, rectum moniae, multi-drug resistant Mycobacterium tuberculosis, cancer, renal cell tumor, retinoblastoma, rhabdomyosarcoma, and AZT-resistant human immunodeficiency virus can all be seminoma, skin cancer, Small-cell lung tumor, soft tissue identified with the present invention. sarcoma, squamous cell carcinoma, stomach cancer, thyroid 0112 Genetic diseases can also be detected by the process cancer, topical skin lesion, Veticulum cell sarcoma, and of the present invention. This can be carried out by prenatal or Wilm's tumor. post-natal screening for chromosomal and genetic aberra 0114. The methods described herein can be used to deter tions or for genetic diseases. Examples of detectable genetic mine whether an individual is at risk to develop a disease. diseases include: 21 hydroxylase deficiency, cystic fibrosis, Examples of such diseases include, but are not limited to HIV , , Duchenne Muscular infection, glomerulonephritis, CHARGE syndrome, Parkin Dystrophy, Down Syndrome or other , heart disease, son's disease and Alzheimer's disease. single gene diseases, HLA typing, phenylketonuria, sickle 0.115. In the area of environmental monitoring, the present cell anemia, Tay-Sachs Disease, thalassemia, Klinefelter invention can be used for detection, identification, and moni Syndrome, Huntington Disease, autoimmune diseases, lipi toring of pathogenic and indigenous microorganisms in natu dosis, obesity defects, hemophilia, inborn errors of metabo ral and engineered ecosystems and microcosms such as in lism, diabetes, trisomy 13, trisomy 18, trisomy 21, Klinefelter municipal waste water purification systems and water reser Syndrome, dup (17) (p. 11.2p 11.2) syndrome, Down syn Voirs or in polluted areas undergoing bioremediation. It is also drome, Pre-eclampsia, Pre-term labor, Edometriosis, Peliza possible to detect plasmids containing genes that can metabo eus-Merzbacher disease, dup (22)(q11.2d 11.2) syndrome, lize Xenobiotics, to monitor specific target microorganisms in US 2010/0285468 A1 Nov. 11, 2010

population dynamic studies, or either to detect, identify, or can be determine instantly as the instrument is running the monitor genetically modified microorganisms in the environ analysis or it can be calculated at a later point. ment and in industrial plants. 0116. The present invention can also be used in a variety of Kits forensic areas, including for human identification for military I0122. In an embodiment, a kit is provided for a detection personnel and criminal investigation, paternity testing and and/or quantitation of a target nucleic acid. The kit includes: family relation analysis, HLA compatibility typing, and a nucleic acid binding agents as described herein and an oligo screening blood, sperm, or transplantation organs for con mix containing the oligonucleotide probes described herein. tamination. In addition, kits are provided which comprise reagents and 0117. In the food and feed industry, the present invention instructions for performing methods of the present invention, has a wide variety of applications. For example, it can be used or for performing tests or assays utilizing any of the compo for identification and characterization of production organ sitions, or assemblies of articles of the present invention. The isms such as yeast for production of beer, wine, cheese, kits may further comprise buffers, restriction enzymes, adap yogurt, bread, etc. Another area of use is with regard to quality tors, primers, a polymerase, dNTPS, NTPs, detection control and certification of products and processes (e.g., live reagents and instructions necessary for use of the kits, option stock, pasteurization, and meat processing) for contaminants. ally including troubleshooting information. Other uses include the characterization of plants, bulbs, and seeds for breeding purposes, identification of the presence of EXAMPLES plant-specific pathogens, and detection and identification of Veterinary infections. Example 1

Instruments Preparation of a Standard of Reference DNA 0118. Also provided herein is an instrument for use in a I0123. In a 20 LL reaction volume, a 10 ng fragment of melting analysis described herein comprising multiple ther DNA, which resides in human , containing the mal cycles, comprising: an automated thermal cycler capable Sequence 5'-TGATTCTCTATACCCATTATGACCTG of alternately heating and cooling, and adapted to receive, at GATATTGGTATTATTGTGGCCATTTCTACCTCAT CACACGTTCTGGAGAATTGT-3' (SEQ ID NO: 1) was least one reaction vessel containing an reaction mixture com amplified using primers, ChrlF (5'-TTGATTCTCTATAC prising a target nucleic acid, a reference nucleic agent, and CCATT-3', SEQID NO: 2) and ChrlR (5'-AACAATTCTC nucleic acid binding agent; wherein the cycler is program CAGAACGTG-3', SEQID NO:3), in the presence of 1x of mable to control temperature. The reaction mixture may also EvaGreen(R) qPCR Basic Mix (Biotium, Hayward, Calif.) and comprise reagents to perform an amplification reaction. In 1 unit of Taq polymerase (Fermentas). The following ther Some embodiments, the instrument additionally comprises a mocyle procedure was used for amplification: 95° C. for 4 display capable of indicating the melting profile of the target minutes, 40 cycles of 95° C. for 15 second, 45° C. for 60 and reference nucleic acid. Such a display may aid the user of second, and 60°C. for 60 second. Standard agarose gel elec the instrument in performing the methods disclosed herein. trophoresis was used to confirm amplification of the DNA 0119 The instrument may further comprise a detector fragment. Using standard cloning protocols, the resulting operable to detect a fluorescence optical signal while the amplified DNA fragment was cloned into pTOPO CRII vec melting analysis is in progress. The detector is for example tor (Invitrogen, Carlsbad, Calif.) to generate the pChrl plas operable to detect a fluorescence optical signal in at least one mid. of the following wavelength regions: from about 510 to about 530 nm, from about 540 to about 550 nm, from 560 to about Example 2 580 nm, from about 585 to about 595 nm, from 590 to about 610 nm, from 660 to about 680 nm, from about 690 to about Preparation of a Standard of Target DNA 710 nm, or from 770 to about 790 nm. The instrument may also be adapted to receive a plurality of reaction vessels, each 0.124. In a 20 uL reaction volume, alOng fragment of containing an reaction mixture. DNA, which resides in human , containing the 0120. Other instruments known in the art are also suitable Sequence 5'-ATTTAAACGGATAGTTCTGCAGCCT for performing the methods of the invention. Such instru GAACTTAAATGTTTTCAGGATAAAACAGTTTCAAA ments are described, for example, in U.S. Pat. Nos. 6,814, 0.125 AATGACTTACCGAAAATCTTCAACT 934, 5,475,610, 5,928,907, 5,972,716, and 6,015,674, all of TGTGGCAATGGAATTTTGGAACCTACA which are hereby incorporated by reference. GAGCAGTGTGATTGTGGCTATAAAGA-3', SEQID NO: 0121. Also provided herein are algorithms and computer 4) was amplified using primers, Chr8F (5'-ATTTAAACG Software programs capable of analyzing melting analysis as GATAGTTCTG-3', SEQ ID NO. 5) and Chr8R (5-TCTT described herein to determine the amount of one or more TATAGCCACAATCAC-3', SEQID NO: 6) in the presence of target nucleic acids. The quantity of nucleic acid can be 1x of EvaGreen(R) qPCR Basic Mix (Biotium, Hayward, calculated in grams, Dalton, bp, or copy numbers. Such algo Calif.) and 1 unit of Taq polymerase (Fermentas). The fol rithms and computer Software programs may aid the user of lowing thermocyle procedure was used for amplification: 95° the instrument in performing the methods disclosed herein. C. for 4 minutes, 40 cycles of 95°C. for 15 second, 45° C. for This algorithms and computer software programs can be 60 second, and 60° C. for 60 second. Standard agarose gel attached, incorporated or separate to the instrument running electrophoresis was used to confirm amplification of the DNA the melting analysis. The quantity of the target nucleic acid fragment. Using standard cloning protocols, the resulting US 2010/0285468 A1 Nov. 11, 2010

amplified DNA fragment was cloned into pTOPO CRII vec I0134. The relative Ct difference between the fragment tor (Invitrogen, Carlsbad, Calif.) to generate the pChr8 plas from chromosome 8 and the Amel fragment was obtained mid using Equation 4: Example 3 AC Chris, x-CIAnel. x-Clich-8. x (IV) I0135) The smallest number of ACts, of the six DNA Copy Number Determination of Target DNA Using samples is used as Min(ACts, x). Real-Time PCR 10136. AACt, is obtained using Equation 5: 0126 DNA samples from two sets of family trios were purchased from Coriell Cell Repositories (Salt lake city, AACicii. xACtchi, x-Min (ACtchi, x (V): Utah): Ref NA10846, Ref NA12144, Ref NA 12145, Ref 10137 AACts, is obtained using Equation 6: NA06994, Ref NA07000, Ref NA07029 (see Redon et al, AACics, x-ACics, x-Min (ACtcs, x (VI); Nature 444:444-454, 2006) and herein denoted as A1, A2, A3, B1, B2 and B3, respectively. Redon et al determined that 0.138 FIG. 2 is a chart plotting copy number determination these sample DNA. A1, A2, A3, B1, B2, and B3 respectively using AACt from the real-time PCR assay as described above. have 3, 2, 3, 3, 4, and 4 copies in a region of Chromosome 8. A1, A2, A3, B1, B2 and B3 are DNA samples from two family Target DNA (SEQ ID NO: 4) from example 2 is within the trios as described. According to Redon et al., each DNA region of chromosome 8 that exhibits copy number variation. sample have exactly 2 copies in a region of chromosome 1, 0127. Each of the six DNA samples (A1, A2, A3, B1, B2, and each respectively have 3, 2, 3, 3, 4, and 4 copies in a B3) were amplified using 3 sets of primers in 3 separate region of chromosome 8. amplication reactions. All reactions were performed in dupli (0.139. The chart shows AACt. (labeled as Delta Delta cate. In a 20 Jul, reaction volume, along concentration of each Ct (Chrl) and represented by the square columns), and DNA were amplified using 10 uI of 2x 1x of EvaGreen(R) AACt. (labeled as Delta Delta Ct (Chr8) and represented qPCR Basic Mix HS (Biotium, Hayward, Calif.). For each by the round columns) are plotted following mathematical reaction, 500 nM of one of the following 3 primer pairs were manipulation as described above. Assuming that each indi used: (1) ChrlF and ChrlR; (2) Chr8F and Chr8R; and (3) vidual has one pair of sex chromosome, each individual must AmelF (5'-CCTGGGCTCTGTAAAGAATAGT-3', SEQ ID have two copies of the Amel gene. The relative Ct difference NO: 7) and AmelR (5'-CAGAGCTTAAACTGGGAAGCT between the fragment from chromosome 1 and the Amel 3', SEQID NO:8). Primers ChrlF and ChrlR were used for Ct fragment and between the fragment from chromosome 8 and determination of chromosome 1. Primers Chr8F and Chr8R Amel fragment were obtained using Equations 3 and 4. were used for Ct determination of chromosome 8. Primers respectively and were used to calculate copies of chromo AmelF and AmelR were used for Ct determination of sex some 1 and 8. After rounding to the nearest digit, DNA chromosomes. The following thermocyle procedure was used samples A1, A2, A3, B1, B2, and B3 were determined to each for amplification of each reaction: 95°C. for 4 minutes, 30 have 2 copies of chromosome 1 (SEQID NO: 1) and 3, 2, 3, cycles of 95°C. for 15 second, 45° C. for 60 second, and 60° 3, 4, and 4 copies of chromosome 8 (SEQID NO: 4), respec C. for 60 second. tively. These copy numbers are in agreement with previously I0128. Ct. is the Ct generated using the primer pair, published results by Redon et al. AmelF and AmelR, with sample X wherein X is A1, A2, A3, B1, B2, or B3. Example 4 I0129. Ct. is the Ct generated using the primer pair, Standard Response Curve for Calculation of DNA ChrlF and ChrlR, with sample X whereinX is A1, A2, A3, B1, Mix Ratios Using Peak-Height Ratio B2, or B3. I0130 Ctes, is the Ct generated using the primer pair 0140. In separate reaction tubes, pChrl and pChr8 were Chr8F and Chr8R with sample X wherein X is A1, A2, A3, mixed in various quantities according to the chart shown B1, B2, or B3. below:

Mix ratio of pChrl to pChr

1O TO 1 8 TO 1 6TO 1 4TO 1 2 TO 1 1 TO 1 1 TO 2 1 TO 4 1 TO 6 1 TO 8 1 TO 10 Units of pChr1 10 8 6 4 2 1 1 1 1 1 1 Units of pChr8 1 1 1 1 1 1 2 4 6 8 10

0131 AACt were generated using the following equations (0.141. Each unit of pChrl or pChr8 has about 3000 copies III-VI. of the plasmid per 1 uL. Each reaction containing one of the 0132) The relative Ct difference between the fragment above DNA mix ratio were amplified in a 20 L reaction from chromosome 1 and the Amel fragment was obtained volume using 1 tit of the DNA mix and lx of EvaGreen(R) using Equation 3: qPCR Basic Mix HS (Biotium, Hayward, Calif.) master mix with 2 sets of primers ChrlF, Chrl R. Chr8F and Chr8R in one AClchri, x-CIAnel. x-Clichrix (III) reaction. The following thermocyle procedure was used for I0133) The smallest number of ACt, of the six DNA amplification of each reaction: 95°C. for 4 minutes, 30 cycles samples is used as Min(ACtc.). of 95°C. for 15 second, 45° C. for 60 second, and 60° C. for US 2010/0285468 A1 Nov. 11, 2010

60 second. Following amplification, melting analysis was each DNA mix ratio in a 20 LL reaction Volume, 1 uL., 2 LL. performed in the presence of a DNA binding dye, EvaGreen, and 4 ul, (labeled as 1x, 2x, and 4x, respectively, in FIG. 6A) from a temperature of 60° C. to 95°C., with increment of 0.2° of DNA mix ratio were separately amplified using 1x of C. per step and exciting at wavelength of around 488 nm and EvaGreen(R) qPCR Basic Mix HS (Biotium, Hayward, Calif.) colleting around 510 nm on a BioRad’s iCycler IQ. Other with 2 sets of primers ChrlF/ChrlR and Chr8F/Chr8R in one non-limiting examples of DNA binding dyes include SYBR reaction. The following thermocyle procedure was used for Green I, PicoGreen, Cyto 9, LC Green, SYBR GreenER, amplification of each reaction: 95°C. for 4 minutes, 30 cycles Ethidium Bromide, TOTO, YOYO, Bebo, and Bexto. Non of 95°C. for 15 second, 45° C. for 90 second, and 60° C. for limiting examples of various instruments that can be used to 60 second. Following amplification, melting analysis was perform melting analyses are ABI 7900, 7500, BioRad iOS, performed and the melting curves were generated as Chromo4, Corbett Rotogene 6000, Roche Lightcycler 480, described (not shown). The peak-height ratio was calculated Idaho technology Genetyper, or Stratagene MX4000. FIG. 3 using panel 5 of FIG. 1 and plotted against the starting quan shows the melting curves generated from the melting analy tity of input DNA (1x, 2x, 4x) for each reaction in the above ses. The peak-height ratio was calculated using panel 5 of chart (FIG. 6A). Results from FIG. 6A indicate that a different FIG. 1 and plotted against the DNA mix ratio for each reac starting quantity of input DNA affects the peak-height ratios. tion in the above chart (FIG. 4). Results from FIG. 4 indicate As the starting quantity of input DNA increased, the peak that the peak-height ratio is linearly correlated to the input height ratio decreased. In addition, the extent to which the DNA mix ratio. The linear correlation demonstrates that cal peak-height ratio was affected is a function of the input DNA culation of the peak-height ratio allows the input DNA mix mix ratio; the degree of decrease in the peak-height ratio ratio to be determined. became less noticeable as the relative amount of pChrl to pChr8 decreased. Example 5 0144. Although the starting quantity of input DNA affects Annealing Time Affects the Peak-Height Ratios but the peak-height ration, the input DNA amount can be calcu does not Affect not the Linearity of the Peak-Height lated by the Ct and therefore the ratio corresponding to a fixed Ratios to the Input DNA Mix Ratios amount of input DNA can be corrected. FIG. 6B shows a 0142 Five DNA mix ratios were prepared (units of pChrl representative standard response curve with peak-height ratio to units of pChr8): 4 to 1, 2 to 1, 1 to 1, 1 to 2, and 1 to 4. Each VS. input DNA mix ratio using the means of peak-height ratios unit of pChrl or pChr8 has about 3000 copies of the plasmid of lx, 2x, and 4x starting amounts. Results show a linear per 1 uL. Each reaction containing one of the above DNA mix correlation and that each point is well separated. A plot of the ratio were amplified in a 20LL reaction Volume using 1 uL of Ct corrected ratio vs. input DNA mix ratio showed that the the DNA mix and 1x of EvaGreen R qPCR Basic Mix HS error bars of the points are small for the various input (Biotium, Hayward, Calif.) with 2 sets of primers ChrlF, amounts. This illustrates that the reaction tolerates at least 4 ChrlR, Chr8F and Chr8R in one reaction. The following fold fluctuations in the starting quantity of input DNA. thermocyle procedure was used for amplification of each reaction: 95° C. for 4 minutes, 30 cycles of 95° C. for 15 Example 7 second, 45° C. for 90 second, and 60° C. for 60 second. Following amplification, melting analysis was performed and Determination of Copy Number of Genomic DNA the melting curves were generated as described (not shown). (0145 Each of the six DNA samples (A1, A2, A3, B1, B2, The peak-height ratio was calculated using panel 5 of FIG. 1 B3) were amplified using both sets of ChrlF/ChrlR and and plotted against the DNA mix ratio for each reaction in the Chr8F/Chr8R primers together. All reactions were performed above chart (FIG. 5). Results from FIG. 5 indicate that a in duplicate. In a 20 LL reaction Volume, a 10 ng concentra different annealing time (90s) from the annealing time used tion of each DNA were amplified using 1x of EvaGreen(R) in Example 4 (60s) during amplification does not affect the qPCR Basic Mix HS (Biotium, Hayward, Calif.), Taq poly linearity of the peak-height ratios to the input DNA mix merase, and both sets of primers. The following thermocyle ratios. However the different annealing time used resulted in procedure was used for amplification of each reaction: 95°C. differences of the absolute numbers of the peak-height ratio. for 4 minutes, 30 cycles of 95°C. for 15 second, 45° C. for 60 Results show that a strict thermocycle procedure should be second, and 60° C. for 60 second. followed during amplification. In addition, we observed dif 0146 Following amplification, melting analysis was per ferent brand of instrument may yield different absolute melt formed and the melting curves were generated as described ing profiles. Different manufactures use different temperature (not shown). The peak-height ratio of Chrl and Chr8 were increment to collect melting fluorescence, and may use dif calculated using panel 5 of FIG. 1 and are shown in FIG. 7, ferent mathematic algorithm for data treatment as their rows 2 and 3, respectively. Row 4 of FIG. 7 shows the peak default settings. The absolute melting profiles of the same height ratio of Chrl to Chr8. Using the peak-height ratio of the sample may look different from instruments of different standard response curve in FIG. 6B, the input ratio of Chrl to manufacturers; however, results are quite repeatable when the Chr8 was determined and shown in Row 5 of FIG.7. The copy melting was performed on the same instrument. numbers of Chrl (reference DNA) for A1, A2, A3, B1, B2, B3 Example 6 were previously determined to all be 2 (see Example 3). Using the ratio of Chrl to Chr8 and the copy number of Chrl, the Starting Quantity of Input DNA Mix Amount Affects copy number of Chr8 was determined for A1, A2, A3, B1, B2, the Peak Ratio but does not Affect not the Linearity B3 and are shown in Row 6 to be 3, 2, 3, 3, 4, 4, respectively. of the Peak-Height Ratios to the Input DNA Mix These copy number results indicate that the methods dis Ratios closed herein are in agreement with the copy number results 0143 Five DNA mix ratios were prepared (units of pChrl obtained by the conventional AACt method (see Example 3) to units of pChr8): 4 to 1, 2 to 1, 1 to 1, 1 to 2, and 1 to 4. For and with the reported numbers of Redon et al. US 2010/0285468 A1 Nov. 11, 2010

Example 8 chromosome 20q13 and CD24 gene is located on human chromosome 6q21. Genomic DNAs are isolated from colon Detection of Gene Duplication in Charcot-Marie cancer, breast cancer, or ovarian cancer samples using stan Tooth Disease Type 1 dard protocols known in the art. The Amel gene can be used 0147 Charcot-Marie-Tooth disease type 1A (CMT1A) as the reference gene. Using known protocols in the art, and hereditary neuropathy with liability to pressure palsies amplification primers directed to the CTSZ or CD24 gene can (HNPP) are autosomal dominant disorders associated with be designed and generated. DNA duplication or deletion of a specific 1.5-Mb genomic I0152 A DNA sample from each type of cancer is ampli fragment of the peripheral myelin protein 22 (PMP22) gene fied using two sets of primer pairs, one directed to amplifying located at chromosome 17p11.2-p 12. DNA from individuals the target gene (CTSZ or CD24) and another directed to with CMT1A or HNPP are extracted from peripheral blood amplifying a reference region (e.g. Amel). All reactions are leukocytes using standard methods (see e.g. Sambrook, J. and performed in duplicate. In a 20 Jull it reaction volume, a 10 ng D. W. Russell (2001). Control DNA samples are obtained concentration of each DNA is amplified using 1x of from individuals without CMT1A and HNPP to ascertain the EvaGreen(R) qPCR Basic Mix HS (Biotium, Hayward, Calif.), absence of a duplication or deletion at 17p11.2-p 12. Taq polymerase, and both sets of primers. The following 0148 Oligonucleotide primers are designed to amplify thermocyle procedure is used for amplification of each reac part of the PMP22 target sequence that lie within the poten tion: 95°C. for 5 minutes, 26 cycles of 94° C. for 30 second, tially duplicated or deleted target region and have a different 58°C. for 30 second, and 72° C. for 30 second. The PCR cycle melting profile than a reference region. The Amel gene can be ends with 72°C. for 10 min. It is understood that variations in used as the reference gene. the thermocycle procedure used will depend on various fac 0149 Each DNA sample from CMT1A, HNPP, and unaf tors such as the size and nucleotide content of the template fected individuals is amplified using both sets of primer pairs, DNA and primers. to amplify the target region (PMP22) and reference region, I0153. Following amplification, melting analysis is per respectively. All reactions are performed in duplicate. In a 20 formed and the melting curves are generated using methods uL reaction volume, a 10 ng concentration of each DNA is described. The peak-height ratio of reference region and amplified using 1x of EvaGreen(R) qPCR Basic Mix HS (Bi either CTSZ or CD24 are calculated using the method shown otium, Hayward, Calif.), Taq polymerase, and both sets of in panel 5 of FIG.1. A standard response curve is generated primers. The following thermocyle procedure is used for using similar procedures as those used to generate FIG. 6B. amplification of each reaction: 95°C. for 5 minutes, 26 cycles Using the peak-height ratio of the standard response curve, of 94° C. for 30 second, 58° C. for 30 second, and 72° C. for the input ratio of reference region to either CTSZ or CD24 is 30 second. The PCR cycle ends with 72° C. for 10 min. determined and allows the amount of product amplified for 0150. Following amplification, melting analysis is per either the CTSZ or CD24 sequence to be compared with the formed and the melting curves are generated using methods amount of PCR product generated from reference region described. The peak-height ratio of the reference region and sequence. The copy number of the reference region is usually PMP22 are calculated using the method shown in panel 5 of known. The copy number of the reference Amel is 2. Using FIG.1. A standard response curve is generated using similar the input ratio of reference region to either CTSZ or CD24 procedures as those used to generate FIG. 6B. Using the and the known copy number of Amel, the copy number of peak-height ratio of the standard response curve, the input either CTSZ or CD24 is determined. Comparison of the num ratio of reference region to PMP22 is determined and allows ber of CTSZ or CD24 DNA copies detected in the sample to the amount of product amplified for the PMP22 sequence to a control or a known value, allows determination of whether be compared with the amount of PCR product generated from the CTSZ or CD24 gene is amplified in the biological test the reference region The copy number of the reference is subject. Amplification of the CTSZ or CD24 gene indicates a usually known. The copy number of the reference Amel is 2. cancer in the tissue. Using the input ratio of reference region to PMP22 and the 0154) While preferred embodiments of the present inven known copy number of the reference region, the copy number tion have been shown and described herein, it will be obvious of PMP22 is determined. The copy number of PMP22 from to those skilled in the art that such embodiments are provided unaffected individuals is expected to be 2. by way of example only. Numerous variations, changes, and Substitutions will now occur to those skilled in the art without Example 9 departing from the invention. It should be understood that various alternatives to the embodiments of the invention Detection of Amplification of the CTSZ and CD24 described herein may be employed in practicing the inven Genes in Human Cancers tion. It is intended that the following claims define the scope 0151. The Cathepsin Z (CTSZ) and small cell lung carci of the invention and that methods and structures within the noma cluster 4 antigen (CD24) genes are frequently amplified Scope of these claims and their equivalents be covered in tumor tissue and cell lines. The human CTSZ gene maps to thereby.

SEQUENCE LISTING

<16 O NUMBER OF SEQ ID NOS: 8

<21 Os SEQ ID NO 1 US 2010/0285468 A1 Nov. 11, 2010 16

- Continued

&211s LENGTH: 81 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OOs, SEQUENCE: 1 tgattct cita tacccattat gacctggata ttgg tatt at tdtggccatt totacct cat 6 O cacacgttct ggagaattgt t 81

<210s, SEQ ID NO 2 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 2 ttgatt ct ct at acccatt 19

<210s, SEQ ID NO 3 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 3 aacaattctic cagaacgtg 19

<210s, SEQ ID NO 4 &211s LENGTH: 138 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide

<4 OOs, SEQUENCE: 4 atttaaacgg at agttctgc agcctgaact taaatgttitt caggataaaa cagtttcaaa 6 O aatgacttac Caaaatctt Caacttgttgg caatggaatt ttggalacct a cagagcagtg 12 O tgattgttggc tataaaga 138

<210s, SEQ ID NO 5 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 5 atttaaacgg at agttctg 19

<210s, SEQ ID NO 6 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic US 2010/0285468 A1 Nov. 11, 2010 17

- Continued primer

<4 OOs, SEQUENCE: 6 t ctittatago cacaat cac 19

SEO ID NO 7 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OO > SEQUENCE: 7

Cctgggct ct gtaaagaata git 22

SEQ ID NO 8 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 8 cagagcttaa actgggaagc t 21

What is claimed is: 8. The method of claim 6 wherein said polymorphisms are 1. A method for determining the amount of a target nucleic STRS or SNPS. acid comprising: 9. The method of claim 1 wherein said target nucleic acid providing a sample comprising a nucleic acid binding and said reference nucleic acid are double stranded. agent, a target nucleic acid and a reference nucleic acid; 10. The method of claim 1 wherein the size of said target wherein said target nucleic and said reference nucleic nucleic acid is about 100 by to about 1 kilobase. acid exhibit distinct melting profiles and wherein said 11. The method of claim 10 wherein said target nucleic acid binding agent yields a detectable signal when bound to and said reference nucleic acid are comparable in length. the target nucleic acid and/or the reference nucleic acid; 12. The method of claim 1 wherein said reference nucleic determining the melting profiles of said target nucleic and acid comprises a genomic DNA region. said reference nucleic acid by detecting the signal of said 13. The method of claim 1 wherein said reference nucleic binding agent at a plurality of temperatures; acid is a cDNA transcript, or an oligonucleotide. comparing the melting profile of said target nucleic acid to 14. The method of claim 12 wherein the copy number of the melting profile of said reference nucleic acid; and said reference nucleic acid is known. determining the amount of said target nucleic acid based on 15. The method of claim 1 wherein said target nucleic acid said comparison. is associated with condition. 2. The method of claim 1 wherein said target nucleic acid 16. The method of claim 15 wherein said condition is and/or said reference nucleic are amplified products of said condition is selected from the group consisting of trisomy 13, trisomy 18, trisomy 21, Klinefelter Syndrome, dup (17) (p target and said reference nucleic acids. 11.2p11.2) syndrome, Down syndrome, Pre-eclampsia, Pre 3. The method of claim 2 wherein said amplified products term labor, Edometriosis, Pelizaeus-Merzbacher disease, dup of said target nucleic acid and/or said reference nucleic are (22)(q11.2d 11.2) Syndrome, Cat eye syndrome, Cri-du-chat amplified by a PCR reaction. syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren 4. The method of claim 2 wherein said amplified products syndrome, Charcot-Marie-Tooth disease, neuropathy with of the target nucleic acid amplified product and said amplified liability to pressure palsies, Smith-Magenis syndrome, neu products of the reference nucleic are generated with the use of rofibromatosis, Alagille syndrome, Velocardiofacial Syn distinct sets of primers. drome, DiGeorge syndrome, Steroid Sulfatase deficiency, 5. The method of claim 1 wherein said target nucleic acid Kallmann syndrome, microphthalmia with linear skin comprises a genomic DNA region. defects, Adrenal hypoplasia, Glycerol kinase deficiency, Pel 6. The method of claim 5 wherein said region of genomic izaeus-Merzbacher disease, testis-determining factor on Y. DNA comprises one or more polymorphisms. AZospermia (factor a), AZospermia (factor b), AZospermia 7. The method of claim 5 wherein said region of genomic (factor c), 1 p 36 deletion, and a combination thereof. DNA comprises one or more copy number variable region 17. The method of claim 15 wherein said condition is (CNVR). selected from the group consisting of condition is acute lym US 2010/0285468 A1 Nov. 11, 2010

phoblastic leukemia, acute or chronic lymphocyctic or granu providing a reference nucleic acid and a nucleic acid bind locytic tumor, acute myeloid leukemia, acute promyelocytic ing agent, wherein said target nucleic and said reference leukemia, adenocarcinoma, adenoma, adrenal cancer, basal nucleic acid exhibit distinct melting profiles; cell carcinoma, bone cancer, brain cancer, breast cancer, amplifying said target nucleic acid and said reference bronchi cancer, cervical dysplasia, chronic myelogenous leu nucleic acid using a first set of primers specific for said kemia, colon cancer, epidermoid carcinoma, Ewing's sar target nucleic acid and a second set of primes specific coma, gallbladder cancer, gallstone tumor, giant cell tumor, from said reference nucleic acid; glioblastoma multiforma, hairy-cell tumor, head cancer, determining the melting profiles of said amplified target hyperplasia, hyperplastic corneal nerve tumor, in situ carci nucleic acid and said amplified reference nucleic acid by noma, intestinal ganglioneuroma, islet cell tumor, Kaposi's monitoring a signal from said binding agentata plurality sarcoma, kidney cancer, larynx cancer, leiomyomater tumor, of temperatures; liver cancer, lung cancer, lymphomas, malignant carcinoid, malignant hypercalcemia, malignant melanomas, marfanoid comparing the melting profile of said target nucleic acid to habitus tumor, medullary carcinoma, metastatic skin carci the melting profile of said reference nucleic acid; and noma, mucosal neuromas, mycosis fungoide, myelodysplas determining the presence of absence of said genetic con tic syndrome, myeloma, neck cancer, neural tissue cancer, dition based on said comparison. neuroblastoma, osteogenic sarcoma, osteosarcoma, ovarian 30. The method of claim 29 wherein said condition is tumor, pancreas cancer, parathyroid cancer, pheochromocy condition is selected from the group consisting of trisomy 13, toma, polycythemia Vera, primary brain tumor, prostate can trisomy 18, trisomy 21, Klinefelter Syndrome, dup (17) (p cer, rectum cancer, renal cell tumor, retinoblastoma, rhab 11.2p11.2) syndrome, Down syndrome, Pre-eclampsia, Pre domyosarcoma, seminoma, skin cancer, Small-cell lung term labor, Edometriosis, Pelizaeus-Merzbacher disease, dup tumor, Soft tissue sarcoma, squamous cell carcinoma, stom (22)(q11.2d 11.2) Syndrome, Cat eye syndrome, Cri-du-chat ach cancer, thyroid cancer, topical skin lesion, Veticulum cell syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren sarcoma, and Wilm's tumor. syndrome, Charcot-Marie-Tooth disease, neuropathy with 18. The method of claim 15 wherein said condition is a risk liability to pressure palsies, Smith-Magenis syndrome, neu to develop a disease. rofibromatosis, Alagille syndrome, Velocardiofacial Syn 19. The method of claim 18 wherein said disease is selected drome, DiGeorge syndrome, Steroid Sulfatase deficiency, from the group consisting of HIV infection, glomerulone Kallmann syndrome, microphthalmia with linear skin phritis, CHARGE syndrome, Parkinson's disease and Alzhe defects, Adrenal hypoplasia, Glycerol kinase deficiency, Pel imer's disease. izaeus-Merzbacher disease, testis-determining factor on Y. 20. The method of claim 1 wherein determining the amount AZospermia (factor a), AZospermia (factor b), AZospermia of said target nucleic acid further comprises determining the (factor c), 1 p 36 deletion, and a combination thereof. copy number of said target nucleic acid. 31. The method of claim 29 wherein said condition is 21. The method of claim 1 wherein said nucleic acid bind selected from the group consisting of condition is acute lym ing agent is a DNA binding agent. phoblastic leukemia, acute or chronic lymphocyctic or granu 22. The method of claim 21 wherein said DNA binding locytic tumor, acute myeloid leukemia, acute promyelocytic agent is a DNA intercalator. leukemia, adenocarcinoma, adenoma, adrenal cancer, basal cell carcinoma, bone cancer, brain cancer, breast cancer, 23. The method of claim 21 wherein said DNA binding bronchi cancer, cervical dysplasia, chronic myelogenous leu agent is selected from the group consisting of EvaGreen, kemia, colon cancer, epidermoid carcinoma, Ewing's Sar SYBR Green I, PicoGreen, Cyto 9, LC Green, SYBR coma, gallbladder cancer, gallstone tumor, giant cell tumor, Green ER, Ethidium bromide, TOTO, YOYO, Bebo, SYTO glioblastoma multiforma, hairy-cell tumor, head cancer, Green and bexto. hyperplasia, hyperplastic corneal nerve tumor, in situ carci 24. The method of claim 23 wherein said DNA binding noma, intestinal ganglioneuroma, islet cell tumor, Kaposi's agent is EvaGreen. sarcoma, kidney cancer, larynx cancer, leiomyomater tumor, 25. The method of claim 1 wherein comparing the melting liver cancer, lung cancer, lymphomas, malignant carcinoid, profiles from said target and references nucleic acid com malignant hypercalcemia, malignant melanomas, marfanoid prises determining the relative amount of signal attributable habitus tumor, medullary carcinoma, metastatic skin carci to said target nucleic acid or to said reference nucleic acid noma, mucosal neuromas, mycosis fungoide, myelodysplas based on their melting profile. tic syndrome, myeloma, neck cancer, neural tissue cancer, 26. The method of claim 25 further comprising comparing neuroblastoma, osteogenic sarcoma, osteosarcoma, ovarian the relative amount of the signal attributable to said target tumor, pancreas cancer, parathyroid cancer, pheochromocy nucleic acid and said reference nucleic acid. toma, polycythemia Vera, primary brain tumor, prostate can 27. The method of claim 25 further comprising calculating cer, rectum cancer, renal cell tumor, retinoblastoma, rhab the ratio between the amount of signal attributable to said domyosarcoma, seminoma, skin cancer, Small-cell lung target nucleic acid and the amount of signal attributable to tumor, soft tissue sarcoma, squamous cell carcinoma, stom said reference nucleic acid. ach cancer, thyroid cancer, topical skin lesion, Veticulum cell 28. The method of claim 1 wherein said detectable signal is sarcoma, and Wilm's tumor. a fluorescent signal, magnetic signal, radioactive signal, 32. The method of claim 29 wherein said condition is a risk Raman signal or an electrochemical signal. to develop a disease. 29. A method of determining a genetic condition in a 33. The method of claim32 wherein said disease is selected patient or a fetus comprising: from the group consisting of HIV infection, glomerulone providing a sample Suspected to contain a target nucleic phritis, CHARGE syndrome, Parkinson's disease and Alzhe acid imer's disease. US 2010/0285468 A1 Nov. 11, 2010

34. The method of claim 29 wherein said target nucleic acid determining the copy number of said target genomic DNA comprises a genomic DNA region. sequence based on said comparison. 35. The method of claim 34 wherein said region of genomic 52. The method of claim 51 wherein the size of said target DNA comprises one or more polymorphisms. nucleic acid is about 100 bp to about 1 kilobase. 36. The method of claim 34 wherein said region of genomic 53. The method of claim 52 wherein said target nucleic acid DNA comprises one or more CNVR. and said reference nucleic acid are comparable in length. 37. The method of claim 35 wherein said polymorphisms 54. The method of claim 51 wherein said reference nucleic are STRS or SNPS. acid comprises a genomic DNA region. 38. The method of claim 29 wherein the size of said target 55. The method of claim 51 wherein said reference nucleic nucleic acid is about 100 bp to about 1 kilobase. acid is a cDNA transcript, or an oligonucleotide. 39. The method of claim38 wherein said target nucleic acid and said reference nucleic acid are comparable in length. 56. The method of claim 51 wherein determining the pres 40. The method of claim 29 wherein said reference nucleic ence or absence of said condition further comprises determin acid comprises a genomic DNA region. ing the copy number of said target nucleic acid. 41. The method of claim 29 wherein said reference nucleic 57. The method of claim 51 wherein said nucleic acid acid is a cDNA transcript, or an oligonucleotide. binding agent is a DNA binding agent. 42. The method of claim 29 wherein determining the pres 58. The method of claim 57 wherein said DNA binding ence or absence of said condition further comprises determin agent is a DNA intercalator. ing the copy number of said target nucleic acid. 59. The method of claim 57 wherein said DNA binding 43. The method of claim 29 wherein said nucleic acid agent is selected from the group consisting of EvaGreen, binding agent is a DNA binding agent. SYBR Green I, PicoGreen, Cyto 9, LC Green, SYBR 44. The method of claim 43 wherein said DNA binding GreenER, Ethidium bromide, TOTO, YOYO, Bebo, SYTO agent is a DNA intercalator. Green and bexto. 45. The method of claim 43 wherein said DNA binding 60. The method of claim 59 wherein said DNA binding agent is selected from the group consisting of EvaGreen, agent is EvaGreen. SYBR Green I, PicoGreen, Cyto 9, LC Green, SYBR 61. The method of claim 51 wherein comparing the melting Green ER, Ethidium bromide, TOTO, YOYO, Bebo, SYTO profiles from said target and references nucleic acid com Green and bexto. prises determining the relative amount of signal attributable 46. The method of claim 45 wherein said DNA binding to said target nucleic acid or to said reference nucleic acid agent is EvaGreen. based on their melting profile. 47. The method of claim 29 wherein comparing the melting 62. The method of claim 61 further comprising comparing profiles from said target and references nucleic acid com the relative amount of the signal attributable to said target prises determining the relative amount of signal attributable nucleic acid and said reference nucleic acid. to said target nucleic acid or to said reference nucleic acid 63. The method of claim 61 further comprising calculating based on their melting profile. the ratio between the amount of signal attributable to said 48. The method of claim 47 further comprising comparing target nucleic acid and the amount of signal attributable to the relative amount of the signal attributable to said target said reference nucleic acid. nucleic acid and said reference nucleic acid. 64. The method of claim 51 wherein said signal is a fluo 49. The method of claim 47 further comprising calculating rescent signal, magnetic signal, radioactive signal, Raman the ratio between the amount of signal attributable to said signal or an electrochemical signal. target nucleic acid and the amount of signal attributable to 65. A kit comprising: said reference nucleic acid. a nucleic acid binding agent, 50. The method of claim 29 wherein said signal is a fluo rescent signal, magnetic signal, radioactive signal, Raman a set of primers specific for a reference nucleic acid or signal or an electrochemical signal. alternatively a reference nucleic acid; and 51. A method of determining copy number variation of a instructions for use of said nucleic acid binding agent, and target genomic DNA sequence comprising: said set of primers specific or said reference nucleic acid providing a sample potentially containing a target genomic to perform the method describe in claim 1. 66. The kit of claim 65 further comprising a set of primers DNA sequence specific for a target nucleic acid. providing a reference nucleic acid and a nucleic acid bind ing agent, wherein said target genomic DNA sequence 67. The kit of claim 66 further compromising a poly said reference nucleic acid exhibit distinct melting pro CaS. files; 68. The kit of claim 67 further comprising instruction on amplifying the target genomic DNA sequence and the ref how to perform amplification of said target nucleic acid and erence nucleic acid using a first set of primers specific optionally of said reference nucleic acid using said poly for said genomic DNA sequence and a second set of CaS. primes specific for said reference nucleic acid sequence; 69. The kit claim 65 further comprising a buffer. determining the melting profiles of said amplified target 70. The method of claim 65 wherein said reference nucleic genomic DNA sequence and said amplified reference acid comprises a genomic DNA region. nucleic acid by monitoring the signal of said binding 71. The method of claim 65 wherein said reference nucleic agent at a plurality of temperatures; acid is a cDNA transcript, or an oligonucleotide. comparing the melting profile of said target nucleic acid to 72. The method of claim 65 wherein said nucleic acid the melting profile of said reference nucleic acid; and binding agent is a DNA binding agent. US 2010/0285468 A1 Nov. 11, 2010 20

73. The method of claim 72 wherein said DNA binding GreenER, Ethidium bromide, TOTO, YOYO, Bebo, SYTO agent is a DNA intercalator. Green and bexto. 74. The method of claim 65 wherein said DNA binding agent75. isThe EvaGreen. method of claim 74 wherein said DNA binding agent is selected from the group consisting of EvaGreen, SYBR Green I, PicoGreen, Cyto 9, LC Green, SYBR ck