DOCSLIB.ORG

(51) International Patent Classification: C12P21/06 (2006.01) (21

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(51) International Patent Classification: C12P21/06 (2006.01) (21 ( 2 (51) International Patent Classification: C12P21/06 (2006.01) (21) International Application Number: PCT/US20 19/032701 (22) International Filing Date: 16 May 2019 (16.05.2019) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 62/672,480 16 May 2018 (16.05.2018) US 62/779,371 13 December 2018 (13. 12.2018) US (71) Applicant: BIO-RAD LABORATORIES, INC. [US/US]; 1000 Alfred Nobel Drive, Hercules, CA 94547 (US). (72) Inventors: BIBILLO, Arkadiusz; 10100 Torre Ave. #144, Cupertino, CA 95014 (US). PATEL, Pranav; 34201 Oneil Terrace, Freemont, CA 94555 (US). (74) Agent: MEYERS, Thomas C. et al.; Brown Rudnick LLP, One Financial Center, Boston, MA 021 11 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available) : AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available) : ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: — without international search report and to be republished upon receipt of that report (Rule 48.2(g)) (54) Title: METHODS FOR PROCESSING NUCLEIC ACID SAMPLES (57) Abstract: The present disclosure provides methods and systems for amplifying and analyzing nucleic acid samples. The present disclosure provides methods for preparing cDNA and/or DNA molecules ad cDNA and/or DNA libraries using modified reverse tran¬ scriptases. METHODS FOR PROCESSING NUCLEIC ACID SAMPLES RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 62/672,480, filed on May 16, 2018, and to U.S. Provisional Application No. 62/779,371 filed on December 13, 2018, the contents of each of which are hereby incorporated by reference in their entirety. BACKGROUND [0002] A common technique used to study gene expression in living cells is to produce complementary deoxyribonucleic acid (cDNA) from a ribonucleic acid (RNA) molecule. This technique provides a means to study RNA from living cells which avoids the direct analysis of inherently unstable RNA. As a first step in cDNA synthesis, the RNA molecules from an organism are isolated from an extract of cells or tissues of the organism. After messenger RNA (mRNA) isolation, using methods such as affinity chromatography utilizing oligo dT (a short sequence of deoxy-thymidine nucleotides), oligonucleotide sequences are annealed to the isolated mRNA molecules and enzymes with reverse transcriptase activity can be utilized to produce cDNA copies of the RNA sequence, utilizing the RNA/DNA primer as a template. Thus, reverse transcription of mRNA is a key step in many forms of gene expression analyses. Generally, mRNA is reverse transcribed into cDNA for subsequent analysis by primer extension or polymerase chain reaction. [0003] Reverse transcriptase has both an RNA-directed DNA polymerase activity and a DNA-directed DNA polymerase activity. The reverse transcription of RNA templates may require a primer sequence which is annealed to an RNA template in order for DNA synthesis to be initiated from the 3 ’ OH of the primer. At room temperature, reverse transcriptase enzymes may allow formation of both perfectly matched as well as mismatched DNA/RNA hybrids. In some instances, a reverse transcriptase enzyme can produce large amounts of non-specific cDNA products as a result of such non-specific priming events. The products of non-specific reverse transcription can interfere with subsequent cDNA analyses, such as cDNA sequencing, real-time polymerase chain reaction (PCR), and alkaline agarose gel electrophoresis, among others. Non-specific cDNA templates produced by non-specific reverse transcriptase activity can present particular difficulties in applications such as real-time PCR. In particular, such non specific cDNA products can give rise to false signals which can complicate the analysis of real time PCR signals and products. Thus, the reduction of non-specific reverse transcriptase activity may result in greater specificity of cDNA synthesis. Currently, there are no reliable and easy to use methods for improving the specificity of reverse transcription. The present disclosure satisfies these and other needs. [0004] Several approaches may be used for obtaining transcriptome data from single cells. A pioneer approach used reverse transcriptase and oligo-dT primers with a T7 phage RNA polymerase promoter sequence attached to the 5’ end of the oligo-dT run. The resulting cDNA was transcribed into multiple copies of RNA which were then converted back to cDNA (Phillips, et a , Methods 10(3):283-288 (1996)). This often truncates the cDNA molecule, losing 5 ’ sequences of the original RNA, especially for relatively long transcripts, and requires multiple rounds of processing when starting with low quantity (LQ) of cells, further exacerbating cDNA truncation. A recent modification (Hashimshony, et a , Cell Rep. 2(3):666-673 (2012)) enables multiplex analyses, but this is still 3 ’ end sequence biased. Other methods are based on PCR amplification of cDNA (Liu, et ak, Methods Enzymol. 303:45- 55 (1999), Ozsolak, et ak, Genome Res. 20(4):5 19-525 (2010), Gonzalez, et ak, PLoS ONE. 5(l2):el44l8 (2010), Kanamori, et ak, Genome Res. 21(7): 1150-1 159 (2011), Islam, et ak, Genome Res. 21(7): 1160-1 167 (2011), Tang, et ak, Nat. Methods. 6(5):377-382 (2009), Kurimoto, et ak, Nucleic Acids Res. 34(5):e42 (2006), Qiu S, et ak, Front Genet. 3:124 (2012)). [0005] These approaches, however, may yield biased representations of sequences along the mRNA, and fail to give complete sequences for mRNAs (e.g., long mRNAs) because DNA templates (e.g., long DNA templates) are discriminated against even when a long PCR reaction is used. SUMMARY [0006] In some aspects, the disclosure provides a method for generating a non-naturally occurring enzyme comprising: a) expressing a heterologous sequence encoding said non- naturally occurring enzyme in a host, wherein said non-naturally occurring enzyme comprises: a first domain, such as a finger domain, derived from an R2 retrotransposon; a second domain, such as a thumb domain, derived from an R2 retrotransposon; a third domain, such as a palm domain, derived from an R2 retrotransposon; and an endonuclease domain derived from an R2 retrotransposon; b) purifying said non-naturally occurring enzyme from said host, thereby generating said non-naturally occurring enzyme. In some instances, said non-naturally occurring enzyme further comprises a fusion-tag molecule. In other instances, said fusion tag-molecule stabilizes said non-naturally occurring enzyme and said fusion-tag molecule is selected from the group consisting of: Fh8, MBP, NusA, Trx, SUMO, GST, SET, GB1, ZZ, HaloTag, SNUT, Skp, T7PK, EspA, Mocr, Ecotin, CaBP, ArsC, IF2-domain I, an IF2-domain I derived tag, RpoA, SlyD, Tsf, RpoS, PotD, Crr , msyB, yjgD, rpoD, and His6. In other cases, said fusion-tag molecule is selected from the group consisting of: His-tag, His6-tag, Calmodulin-tag, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softag-l, Softag-3, V5-tag, Xpress-tag, Isopeptag, SpyTag, B, HPC (heavy chain of protein C) peptide tags, GST, MBP, biotin, biotin carboxyl carrier protein, glutathione-S- transferase-tag, green fluorescent protein-tag, maltose binding protein-tag, Nus-tag, Strep-tag, and thioredoxin-tag. In some instances, at least one of said first domain, said second domain, said third domain, or said endonuclease domain, is derived from an arthropod. In some instances at least one of said first domain, said second domain, said third domain, or said endonuclease is derived from a vertebrate, an echinoderm, a flatworm, a hydra, or silkmoth. In some instances, said non-naturally occurring enzyme has at least 90% identity to SEQ ID NOs: 1-20. In some aspects, said host is selected from bacteria, yeast, algae, cyanobacteria, fungi, a plant cell, E. coli, or any combination thereof. In some instances, said non-naturally occurring enzyme comprises a mutagenized motif- 1 sequence. In some instances, said mutagenized motif - 1sequence has an improved jumping activity as compared to a wild-type sequence. In some instances, said non-naturally occurring enzyme comprises a mutagenized motif 0 sequence. In some instances, said mutagenized motif 0 sequence has an improved jumping activity as compared to a wild-type sequence. In some instances, said non-naturally occurring enzyme comprises a mutagenized thumb sequence. In some instances, said mutagenized second domain sequence has an improved single-stranded priming efficiency or an improved processivity. [0007] In some instances, the disclosure provides a non-naturally occurring enzyme, comprising (i) a first domain, such as a finger domain, from an R2 retrotransposon; (ii) a second domain, such as a thumb domain, derived from an R2 retrotransposon; (iii) a third domain, such as a palm domain, derived from an R2 retrotransposon; and (iv) an endonuclease domain derived from an R2 retrotransposon.
Load more

Recommended publications
  • Use of Vectorette and Subvectorette PCR to Isolate Transgene Flanking DNA
    Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Use of Vectorette and Subvectorette PCR to Isolate Transgene Flanking DNA Maxine J. Allen, Andrew Collick, and Alec J. Jeffreys Department of Genetics, University of Leicester, Leicester LE1 7RH, UK Vectorette PCR permits the specific PCR allows specific amplification of structures and in the recovery of un- amplification of DNA segments flank- DNA fragments between two regions of known bacterial sequences. ~4-6~ In this ing a known DNA sequence. It en- known sequence to which primers can paper we detail its use in the systematic ables the application of the PCR be directed. Often, however, molecular isolation of 5'- and 3'-flanking junction where sequence information is only biological techniques require the isola- sequences around mouse transgenes of available for one primer site. We now tion and subsequent sequence analysis the minisatellite MS32. MS32 is a hu- show that vectorette PCR can be used of unknown regions of DNA. PCR can be man minisatellite composed of a tan- for the systematic mapping and re- used to retrieve this unknown sequence dem array of 29-bp repeat-unit sequence trieval of transgene flanking DNA. if it is flanking a region of known se- variants. The interspersion of these We also show that the sequence of quence. In this case, a primer can be de- sequence variants can be assayed in large vectorette PCR fragments can signed in either orientation from the the minisatellite variant repeat (MVR)- be obtained without cloning, by the known sequence.
    [Show full text]
  • Download At: Amplification May Be Performed During Library Con- Fenyolab/Tipseqhunter (See Table 6)
    Steranka et al. Mobile DNA (2019) 10:8 https://doi.org/10.1186/s13100-019-0148-5 METHODOLOGY Open Access Transposon insertion profiling by sequencing (TIPseq) for mapping LINE-1 insertions in the human genome Jared P. Steranka1,2, Zuojian Tang3,4, Mark Grivainis3,4, Cheng Ran Lisa Huang2, Lindsay M. Payer1, Fernanda O. R. Rego5, Thiago Luiz Araujo Miller5,6, Pedro A. F. Galante5, Sitharam Ramaswami7, Adriana Heguy7, David Fenyö3,4, Jef D. Boeke4* and Kathleen H. Burns1,2* Abstract Background: Transposable elements make up a significant portion of the human genome. Accurately locating these mobile DNAs is vital to understand their role as a source of structural variation and somatic mutation. To this end, laboratories have developed strategies to selectively amplify or otherwise enrich transposable element insertion sites in genomic DNA. Results: Here we describe a technique, Transposon Insertion Profiling by sequencing (TIPseq), to map Long INterspersed Element 1 (LINE-1, L1) retrotransposon insertions in the human genome. This method uses vectorette PCR to amplify species-specific L1 (L1PA1) insertion sites followed by paired-end Illumina sequencing. In addition to providing a step-by-step molecular biology protocol, we offer users a guide to our pipeline for data analysis, TIPseqHunter. Our recent studies in pancreatic and ovarian cancer demonstrate the ability of TIPseq to identify invariant (fixed), polymorphic (inherited variants),aswellassomatically-acquiredL1insertionsthat distinguish cancer genomes from a patient’s constitutional make-up. Conclusions: TIPseq provides an approach for amplifying evolutionarily young, active transposable element insertion sites from genomic DNA. Our rationale and variations on this protocol may be useful to those mapping L1 and other mobile elements in complex genomes.
    [Show full text]
  • Loop-Mediated Isothermal Amplification (LAMP)
    cells Review Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR? Marianna Soroka, Barbara Wasowicz * and Anna Rymaszewska Department of Genetics and Genomics, Institute of Biology, University of Szczecin, 3c Felczaka St., 71-412 Szczecin, Poland; [email protected] (M.S.); [email protected] (A.R.) * Correspondence: [email protected] Abstract: In 1998, when the PCR technique was already popular, a Japanese company called Eiken Chemical Co., Ltd. designed a method known as the loop-mediated isothermal amplification of DNA (LAMP). The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets. Furthermore, the Bst DNA polymerase most used in LAMP shows a high strand displacement activity, which eliminates the DNA denaturation stage. One of the most significant advantages of LAMP is that it can be conducted at a stable temperature, for instance, in a dry block heater or an incubator. The products of LAMP can be detected much faster than in standard techniques, sometimes only requiring analysis with the naked eye. The following overview highlights the usefulness of LAMP and its effectiveness in various fields; it also considers the superiority of LAMP over PCR and presents RT-LAMP as a rapid diagnostic tool for SARS-CoV-2. Keywords: LAMP method; isothermal amplification; SARS-CoV-2 detection Citation: Soroka, M.; Wasowicz, B.; Rymaszewska, A.
    [Show full text]
  • Are Molecular Tools Solving the Challenges Posed by Detection Of
    Curr. Issues Mol. Biol. 11: 13-46. Molecular Tools for DetectionOnline journal of Plant at http://www.cimb.org Pathogens 13 Are Molecular Tools Solving the Challenges Posed by Detection of Plant Pathogenic Bacteria and Viruses? María M. López*, Pablo Llop, Antonio Olmos, Ester plant pathogens due to their low titre in the plants, their Marco-Noales, Mariano Cambra and Edson Bertolini uneven distribution, the existence of latent infections and the lack of validated sampling protocols. Research Centro de Protección Vegetal y Biotecnología, Instituto based on genomic advances and innovative detection Valenciano de Investigaciones Agrarias (IVIA). Carretera methods as well as better knowledge of the pathogens’ de Moncada a Náquera km 4.5, 46113 Moncada, lifecycle, will facilitate their early and accurate detection, Valencia, Spain thus improving the sanitary status of cultivated plants in the near future. *For correspondence: [email protected] Introduction Abstract Plant pathogenic bacteria, phytoplasmas, viruses and Plant pathogenic bacteria, phytoplasmas, viruses and viroids cause harmful, widespread and economically viroids are difficult to control, and preventive measures important diseases in a very broad range of plant are essential to minimize the losses they cause each species worldwide (Agrios, 2001; Janse, 2007). Damage year in different crops. In this context, rapid and accurate is often sufficient to cause significant yield losses in methods for detection and diagnosis of these plant cultivated plants (Schaad, 1988; Scortichini, 1995; pathogens are required to apply treatments, undertake Cambra et al., 2006b). The two main effects on agronomic measures or proceed with eradication agriculture are decreased production and, in a less direct practices, particularly for quarantine pathogens.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 9,546,389 B2 Li (45) Date of Patent: Jan
    USOO9546,389 B2 (12) United States Patent (10) Patent No.: US 9,546,389 B2 Li (45) Date of Patent: Jan. 17, 2017 (54) METHODS AND SYSTEMS FOR NUCLEIC (56) References Cited ACIDAMPLIFICATION U.S. PATENT DOCUMENTS (71) Applicant: Coyote Bioscience Co., Ltd., Beijing 5,759,821. A * 6/1998 Teasdale .............. C12O 1/6806 (CN) 435,912 2004/0209331 A1* 10, 2004 Ririe ....................... BO1L 3,505 435,912 (72) Inventor: Xiang Li, Beijing (CN) 2008/0085541 A1 4/2008 Spangler 2012/0308990 A1* 12/2012 TeMaat ..................... BO1L, 7,52 (73) Assignee: COYOTE BIOSCIENCE CO.,LTD., 435/3 Beijing (CN) 2013/0022963 A1 1/2013 Exner et al. FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 CN 101597652. A 12/2009 U.S.C. 154(b) by 0 days. CN 103074349 A 5, 2013 JP EP 1069 190 A2 * 1 2001 ............. C12O 1/686 WO WO-2012109604 A1 8, 2012 (21) Appl. No.: 14/963,986 OTHER PUBLICATIONS (22) Filed: Dec. 9, 2015 Callahan et al. Use of a portable real-time reverse transcriptase (65) Prior Publication Data polymerase chain reaction assay for rapid detection of foot-and mouth disease virus. JAVMA 220: 1636-1642 (2002).* US 2016/O115513 A1 Apr. 28, 2016 Gilbert et al. Typing of bovine viral diarrhea viruses directly from blood of persistently infected cattle by multiplex PCR. Journal of Clinical Microbiology 37:2020-2023 (1999).* International search report and written opinion dated Feb. 26, 2015 Related U.S. Application Data for PCT Application No. CN2014/094914.
    [Show full text]
  • Biochemistry 24 Polymerase Chain Reaction
    Biochemical Techniques Biochemistry 24 Polymerase Chain Reaction Description of Module Subject Name Biochemistry Paper Name 12 Biochemical Techniques Module Name/Title 24 Polymerase Chain Reaction Biochemical Techniques Biochemistry 24 Polymerase Chain Reaction 1. Objectives 1.1 To understand principle of Polymerase chain reaction (PCR) technique. 1.2 To explain how this techniques is performed. 1.3 What are helpful hints which help in successful performance of PCR technique? 1.4 What are potential applications of PCR? 1.5 What are different variants of PCR? 2.0 Introduction and principle Polymerase chain reaction (PCR) can be recognized as a revolutionary technology for which the inventor Kary B. Mullis was awarded with the prestigious Nobel Prize in 1993 for chemistry along with Michael Smith. The secret story of any living organism which gives it unique characteristics is coded within the large and complexed DNA of that individual. To understand the story, it was needed to bring the DNA outside the living cell into the laboratory, this gave birth to the concept of PCR. Kary Mullis conceptualized of a machine through which from a single molecule of DNA we can get multiples of its copy after the end, like a Xerox machine does. With this in 1983 Kary Mullis developed PCR which is now a most generalized technique used in medical or biological science research labs for a range of applications. Examples are its use in DNA cloning, phylogeny, functional analysis of genes, diagnosis of diseases, genetic fingerprints for forensic sciences as paternity test where the aim is to amplify a single or a few copies of a specific DNA segment in generating thousands to millions of copies of that specific DNA sequence.
    [Show full text]
  • M.Sc. (MICROBIOLOGY)
    SAURASHTRA UNIVERSITY RAJKOT (ACCREDITED GRADE “A” BY NAAC) FACULTY OF SCIENCE Syllabus for M. Sc. (MICROBIOLOGY) Choice Based Credit System With Effect From: 2016-17 Department of Microbiology Course Structure and Scheme of Examination For Choice Based Credit System (CBCS) (Total 96 credits) Effective from June 2016 M. Sc. Microbiology Program Outcomes (PO) PO1: Academic Competence Understanding of the subject with respect to structure, diversity, metabolisms and applications of the microorganisms is extensively developed. It also involves structure and function of biological molecules. At the end, the students gain depth of scientific knowledge regarding microorganisms. PO2: Critical thinking Students are expected to develop skills in conducting practical, designing experiments and analyzing the data reaching to conclusions. The theoretical base is developed on the various aspects of microorganisms and ability to solve the problems. PO3: Research and development Students gain ideas and competence regarding research and data analysis which will be highly useful to them in higher studies and exploring job opportunities. They acquire abilities for self direction and originality in tackling and solving problems and implementing tasks at professional levels. PO4: Personal and Behavioural Competence Professional skills concerning biochemical analysis, carrying out experiments and developing ability in specific areas of microorganisms are also developed. PO5: Effective Communication 1 During this programme, the students develop the ability for articulation of ideas, scientific writing and report reporting, conference presentations and delivering seminars. They also develop conversational competence and effective verbal and written. PO6: Social Competence The students will develop abilities to manage projects in order to achieve objectives and ability to work in team.
    [Show full text]
  • Lecture 37: Polymerase Chain Reaction
    Proteomics & Genomics Dr. Vikash Kumar Dubey Lecture 37: Polymerase Chain Reaction We have already studied basics of DNA/RNA structure and recombinant DNA technology in previous classes. Polymerase Chain Reaction (PCR) is another revolutionary method developed by Kary Mullis and Michael Smith. Both shared Nobel Prize in Chemistry for the work in 1993. PCR is based on ability of DNA polymerase to synthesize complementary strand to the template strand. As DNA polymerase can add a nucleotide only onto a 3'-OH group, it needs an artificial DNA strand (called DNA primer) of about 18 to 25 nucleotides complementary to 3’ end of the DNA template. As shown below, each polynucleotide has a free 3’ –OH group and 5’ phosphate group. Moreover, a DNA strand has complimentary sequence, already paired by hydrogen IIT Guwahati Page 1 of 9 Proteomics & Genomics Dr. Vikash Kumar Dubey bonding. Thus, primer can bind only when DNA strands are separated. This is generally done by heating. The primers anneal to the single-stranded DNA template at specific temperature (depends on primer sequence) and then DNA-Polymerase binds to this double stranded DNA produced. The again reaction mixture is heated to 72°C (extension); a temperature optimum for DNA- polymerase functions. This starts synthesis of the new DNA strand. Than reaction mixture is cooled to lower temperature for short term storage, if required. This completes one cycle. After first cycle, one DNA molecule has become two. After multiple cycle of the PCR reaction, the specific sequence will be accumulated in billions of copies. The PCR reaction requires the following components: DNA template: DNA template is DNA target sequence.
    [Show full text]
  • Polymerase Chain Reaction. Wikipedia. Last Modified on 13
    Polymerase chain reaction - Wikipedia, the free encyclopedia Create account Log in Article Talk Read Edit View Polymerase chain reaction From Wikipedia, the free encyclopedia Main page Contents "PCR" redirects here. For other uses, see PCR (disambiguation). Featured content It has been suggested that Applications of PCR be merged into Current events this article. (Discuss) Proposed since June 2013. Random article Donate to Wikipedia The polymerase chain reaction (PCR) is a biochemical technology in molecular Interaction biology to amplify a single or a few copies Help of a piece of DNA across several orders About Wikipedia of magnitude, generating thousands to Community portal millions of copies of a particular DNA Recent changes sequence. Contact page Developed in 1983 by Kary Mullis,[1][2] Tools PCR is now a common and often Print/export indispensable technique used in medical and biological research labs for a variety Languages of applications.[3][4] These include DNA Afrikaans cloning for sequencing, DNA-based phylogeny, or functional analysis of A strip of eight PCR tubes, each containing a 100 μl Azərbaycanca genes; the diagnosis of hereditary reaction mixture Български diseases; the identification of genetic Català fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of Česky infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Dansk Smith for his work on PCR.[5] Deutsch The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the Eesti reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) Ελληνικά containing sequences complementary to the target region along with a DNA polymerase (after Español which the method is named) are key components to enable selective and repeated amplification.
    [Show full text]
  • (PCR-RFLP) and Gel Electrophoresis – Valuable Tool for Genotyping and Genetic Fingerprinting
    18 Restriction Fragment Length Polymorphism Analysis of PCR-Amplified Fragments (PCR-RFLP) and Gel Electrophoresis – Valuable Tool for Genotyping and Genetic Fingerprinting Henrik Berg Rasmussen Institute of Biological Psychiatry, Mental Health Centre Sct. Hans, Copenhagen University Hospitals, Roskilde, Denmark 1. Introrduction PCR-restriction fragment length polymorphism (RFLP)-based analysis, also known as cleaved amplified polymorphic sequence (CAPS), is a popular technique for genetic analysis. It has been applied for the detection of intraspecies as well as interspecies variation. There exist several techniques that are related with PCR-RFLP and also involve gel electrophoresis including techniques for DNA fingerprinting and expression profiling. This chapter describes PCR-RFLP and related techniques. 1.1 Genetic variation There are different types of genetic variations. The so-called small-scale genetic variation includes single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and microindels. MNPs are multiple, consecutive nucleotide variations of a single common length such as double nucleotide polymorphisms (DNPs) and triple nucleotide polymorphisms (TNPs) with two and three variable nucleotides, respectively (Figure 1). Microindels are deletions, duplications and combinations thereof involving the gain or loss of 1 to 50 nucleotides (Gonzalez et al., 2007). The human genome contains more than 3 million SNPs located with an average distance of approximately 1000 bp (International HapMap Consortium 2005, Levy et al., 2007). The frequency of DNPs and TNPs, the most common forms of MNPs, amounts to ∼1% of the total number of SNPs (Rosenfeld et al., 2010). Most likely, the genome-wide occurrence of small insertions and deletions has been underestimated, probably reflecting an inaccuracy of the current sequencing techniques (Krawitz et al., 2010).
    [Show full text]
  • Magdalena Sztyler Phd 2014
    MOLECULAR ANALYSIS OF MICROBIAL COMMUNITIES FROM OIL INDUSTRY ENVIRONMENTS Magdalena K. Sztyler A thesis submitted in partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy of the University of Portsmouth Institute of Biomedical and Biomolecular Sciences School of Pharmacy and Biomedical Sciences May 2014 “Więcej się można nauczyć podróżując...” Olga A. Jackowska Rodzicom AUTHORS’ DECLARATION Whilst registered as a candidate for the above degree, I have not been registered for any other research award. The results and conclusions embodied in this thesis are the work of the named candidate and have not been submitted for any other academic award. Magdalena K. Sztyler I ABSTRACT The effects of microbiologically influenced corrosion (MIC) can be very expensive to correct, dangerous to workers and its mechanisms are poorly understood. Understanding these processes is important so that they can be monitored and mitigated (Koch et al., 2001). It is now accepted that for the assessment of biocorrosion risks, the most powerful approach is to detect functional genes encoding the enzymes that play an important part in material deterioration (Schadt et al., 2004). The main aim of this study was to identify the microbial community present in corroded and non-corroded systems, and to detect genes that might be implicated in corrosion processes, particularly iron corrosion, so that a biochip could be designed for risk assessment of oil environments. In this thesis the microbial populations and their actives were assessed using sequencing and hybridisation techniques for three oil field sites, generating information that can help identify MIC risk. The final section of the thesis describes the development and design of functional gene probes, identified from hybridisation studies that might be included in a biochip for risk assessment in oil field environments.
    [Show full text]
  • Review Article Loop Mediated Isothermal Amplification (LAMP) Test– a Novel Nucleic Acid Based Assay for Disease Diagnosis
    Advances in Animal and Veterinary Sciences 2 (6): 344 – 350 http://dx.doi.org/10.14737/journal.aavs/2014/2.6.344.350 Review Article Loop Mediated Isothermal Amplification (LAMP) Test– a Novel Nucleic Acid Based Assay for Disease Diagnosis Valsala Rekha 1*, Rajneesh Rana1, Thachappully Remesh Arun1, Plantharayil Bharathan Aswathi2, Jeny Kalluvila John1, Devi Gopinath1, Gurupriya Vijayasaraswathy Sadanandan1, Aron Jacob 1 1Indian Veterinary Research Institute, Izatnagar, Bareilly, UP; 2Central Avian Research Institute, Izatnagar, Bareilly, UP; *Corresponding author: [email protected] ARTICLE HISTORY ABSTRACT Received: 2014–05–26 Disease diagnosis is of crucial importance in making appropriate therapeutic decisions. Revised: 2014–07–14 Conventionally, diagnosis was based on isolation and identification of the causative agent. Accepted: 2014–07–15 This was followed in timeline by the development of serological tests. Nowadays, molecular tests credited with high sensitivity like polymerase chain reaction and its variants are replacing the older diagnostic techniques. Major obstacle in adoption of PCR based Key Words: LAMP, PCR, technique is the need for thermocycler, which precludes its use in low resource setting areas. Isothermal amplification, Isothermal amplification system like Loop mediated isothermal amplification (LAMP) test is Visual detection a good alternative. It is a novel nucleic acid amplification assay developed in 2000. Amplification of nucleic acid is carried out under isothermal condition, usually at temperature ranging from 60–65℃. Comparatively higher sensitivity, visual detection, lower reaction time, and dispensable use of thermocycler make LAMP an attractive option for field diagnosis. As the test is not susceptible to inhibitory effect of biological substances, it can be very well applied for direct detection from clinical samples without processing.
    [Show full text]