USOO5654155A United States Patent (19) 11 Patent Number: 5,654,155 Murphy et al. 45 Date of Patent: Aug. 5, 1997

54 CONSENSUS SEQUENCE OF THE HUMAN Szabo, Csilla I., et al., Inherited Breast and Ovarian Cancer, BRCA1 GENE Human Molecular Genetics 4:1811-1817, 1995. Plummer, S.J., et al., Detection of BRAC1 Mutations by the 75) Inventors: Patricia D. Murphy, Slingerland, N.Y.; Protein Truncation Test 4:1989-1991, 1995. Antonette C. Allen, Millersville, Md., Chen, Y, et al., Aberrant Subcellular Localization of Christopher P. Alvares, Potomac, Md.; BRCA1 in Breast Cancer. Science 270: 789-791, Nov. 3, Brenda S. Critz, Frederick, Md. Sheri 1995. J. Olson, Arlington, Va.; Denise B. Gayther, Simon A., et al., Germline Mutations of the Schelter, Burtonsville; Bin Zeng, BRCA1 Gene in Breast and Ovarian Cancer Families Pro Rockville, both of Md. vide Evidence for A Genotype-phenotype Correlation. Nature Genetics 11: 428-433, Dec. 1995. 73 Assignee: OncorMed, Inc., Gaithersburg, Md. Durocher, F, et al., Comparison of BRCA1 Polymorphisms, Rare Sequence Variants and/or Missense Mutations in Unaf 21 Appl. No.: 598591 fected and Breast/Ovarian Cancer Populations 5: 835-842, 1996. 22 Filed: Feb. 12, 1996 Berman, D.B., et al., Two District Origins of a Common (51] Int. Cl...... C12Q 1/68; C12P 19/34; BRCA1. Mutation in Breast-Ovarian Cancer Families: A C07H 21/04: CO7H 21/02 Genetic Study of 15 185delAG-Mutation Kindreds. Am. J. 52 U.S. Cl...... 435/6: 435/91.2: 536/23.1; Hum. Genet, 58:1166-1176, 1996. Schultz, D.C., et al., Identification of Two Candidate Tumor 536/24.3: 536/24.33 Suppressor Genes on Chromosome 17p13.3. Cancer 58 Field of Search ...... 435/6, 91.2:536/23.1, Research 56:1997-2002, May 1, 1996. 536/24.3, 24.33 Couch, Fergus J., et al., Mutations and Polymorphisms in the 56 References Cited Familial Early-Onset Breast Cancer (BRCA1) Gene. Human Mutation 8:8-18, 1996. U.S. PATENT DOCUMENTS Sanger, F, et al., J. Mol. Biol. 42:1617. (1980). Beaucage, et al., Tetrahedron Letter. 22:1859-1862. 4,458,066 7/1984 Carothers et al...... 536/27 Maniatis, et al., In Molecular Cloning: A Laboratory FOREIGN PATENT DOCUMENTS Manual, Cold Spring, NY, pp. 280-281, (1982). O6997.54A1 3/1996 European Pat. Off. . Conner, et al., Proc. Natl. Acad. Sci. U.S.A. 80:278, (1983). 705903A 4/1996 European Pat. Off.. Saiki, et al., Bio/Technology 3: 1088–1012 (1985). O705903A1 4/1996 European Pat. Off.. Landegren, et al., Science 241:1007, (1988). O705902A1 4/1996 European Pat. Off.. Landgren, et al., Science 242:229-237, (1988). McPherson, M.J., et al., PCR. A Practical Approach. ILR OTHER PUBLICATIONS Press, Eds. (1992). Miki et al. Science 266: 66-71. 1994. Easton, et al., American Journal of Human Genetics 52: Friedman et al. Nature Genetics 8:399-404 Dec. 1994. 678-701, (1993). Scriver et al. In Variation in the Human Genome, Ciba Primary Examiner W. Gary Jones Foundation Symposium 197, John Wiley & Sons eds, New Assistant Examiner-Dianne Rees York, pp. 73-96. 1996. Attorney, Agent, or Firm-R. Thomas Gallegos; Albert P. Roswell, S., et al., American Journal of Human Genetics Halluin 55:861-865, (1994). Miki, Y., et al., Science 266:66–71, (1994). 57 ABSTRACT Friend, S., et al., Nature Genetics 11:238, (1995). Beaudet, Arthur L., et al.: A Suggested Nomenclature for A consensus DNA sequence has been determined for the Designating Mutations. Human Mutation 2:245–248, 1993. BRCA1 gene. As has been seven polymorphic sites and their Friedman, Lori S., et al.: Confirmation of BRCA1 by rates of occurrence in normal BRCA1 genes. The consensus Analysis of Germline Mutations Linked to Breast and Ova gene BRCA1" and the seven polymorphic sites will rian Cancer. Nature Genetics 8:399-404, Dec. 1994. provide greater accuracy and reliability for genetic testing. Shattuck-Eidens, Donna, et al.: A Collaborative Survey of One skilled in the art will be better able to avoid misinter 80 Mutations in the BRCA1. Breast and Ovarian Cancer pretations of changes in the gene, determine the presence of Susceptibility Gene. JAMA 273:535-541, Feb. 15, 1995. a normal gene, and of mutations, and to classify tumors. Ford, D., et al.: The Genetics of Breast and Ovarian Cancer. Genetics Heterogeneity 5:805-811. 4 Claims, No Drawings 5,654,155 1. 2 CONSENSUS SEQUENCE OF THE HUMAN "reduced penetrance” because they never develop cancer). BRCA1 GENE Pedigree analysis and genetic counseling is absolutely essential to the proper workup of a family prior to any lab FIELD OF THE INVENTON work. 5 Until now, only a single normal sequence for BRCA1 has This invention relates to a gene which has been associated been available for comparison. That sequence is available as with breast and ovarian cancer where the gene is found to be GenBankAccession Number U15595. There is a need in the mutated. More specifically, this invention relates to the most art, therefore, to have available a normal sequence which likely sequence (i.e. "Consensus Normal DNA sequence”) represents most likely BRCA1 sequence to be found in the for the BRCA1 gene (BRCA1". SEQ. ID. NO: 1) in O majority of the normal population, the (i.e. "Consensus normal individuals. Normal DNA sequence”). A Consensus Normal DNA BACKGROUND OF THE INVENTION sequence will make it possible for true mutations to be easily identified or differentiated from polymorphisms. Identifica It has been estimated that about 5-10% of breast cancer tion of mutations of the BRCA1 gene and protein would is inherited Rowell, S., et al., American Journal of Human 15 allow more widespread diagnostic screening for hereditary Genetics 55:861-865 (1994). Located on chromosome 17, breast and ovarian cancer than is currently possible. BRCA1 is the first geneidentified to be conferring increased A consensus normal gene sequence of the BRCA1 is risk for breast and ovarian cancer. Miki et al., Science provided which more accurately reflects the most likely 266:66-71 (1994). Mutations in this "tumor suppressor” sequence to be found in a subject. Use of the consensus gene are thought to account for roughly 45% of inherited 20 normal gene sequence (BRCA1"SEQID. NO: 1), rather breast cancer and 80-90% of families with increased risk of than the previously published BRCA1 sequence, will reduce early onset breast and ovarian cancer. Easton et al., Ameri the likelihood of misinterpreting a "sequence variation” can Journal of Human Genetics 52:678-701 (1993). found in the normal population with a pathologic"mutation” Locating one or more mutations in the BRCA1 region of (i.e. causes disease in the individual or puts the individual at chromosome 17 provides a promising approach to reducing 25 a high risk of developing the disease). With large interest in the high incidence and mortality associated with breast and breast cancer predisposition testing, misinterpretation is ovarian cancer through the early detection of women at high particularly worrisome. People who already have breast risk. These women, once identified, can be targeted for more cancer are asking the clinical question: "is my disease aggressive prevention programs. Screening is carried out by caused by a heritable genetic mutation?" The relatives of the a variety of methods which include karyotyping, probe 30 those with breast cancer are asking the question: "Am I also binding and DNA sequencing. a carrier of the mutation my relative has? Thus, is my risk In DNA sequencing technology, genomic DNA is increased, and should I undergo a more aggressive surveil extracted from whole blood and the coding regions of the lance program.” BRCA1 gene are amplified. Each of the coding regions is SUMMARY OF THE INVENTION sequenced completely and the results are compared to the 35 normal DNA sequence of the gene. The present invention is based on the discovery of the The BRCA1 gene is divided into 24 separate exons. most likely sequence to be found in normal human indi Exons 1 and 4 are noncoding, in that they are not part of the viduals for the BRCA1 gene. final functional BRCA1 protein product. The BRCA1 cod It is an object of the invention to provide a consensus ing region spans roughly 5600 base pairs (bp). Each exon sequence for the normal BRCA1 gene, i.e. the consensus consists of 200-400 bp, except for exon 11 which contains Sequence having the more commonly occurring nucleotides about 3600 bp. To sequence the coding region of the BRCA1 where normal polymorphisms occur. gene, each exon is amplified separately and the resulting It is another object of the invention to provide a consensus normal protein sequence of the BRCA1 protein PCR products are sequenced in the forward and reverse 45 directions. Because exon 11 is so large, we have divided it It is another object of the invention to provide a list of the into twelve overlapping PCR fragments of roughly 350 bp codon pairs which occur at each of seven polymorphic each (segments “A” through "L' of BRCA1 exon 11). points on the normal BRCA1 gene. Many mutations and normal polymorphisms have already It is another object of the invention to provide the rates of occurrence for the codons. been reported in the BRCA1 gene. A world wide web site 50 has been built to facilitate the detection and characterization It is another object of the invention to provide a method of alterations in breast cancer susceptibility genes. Such wherein BRCA1, or parts thereof, is amplified with one or mutations in BRCA1 can be accessed through the Breast more oligonucleotide primers. Cancer Information Core at: http://www.nchgr.nih.gov/dir/ It is another object of this invention to provide a method lab transfer/bic. This data site became publicly available on 55 of avoiding misinterpretation of changes which a laboratory Nov. 1, 1995. Friend, S. et al. Nature Genetics 11:238, may find in the BRCA1 gene. (1995). It is another object of this invention to provide a method The genetics of Breast/Ovarian Cancer Syndrome is auto of identifying individuals who carry no mutation(s) of the Somal dominant with reduced penetrance. In simple terms, BRCA1 gene and are therefore at no increased risk or this means that the syndrome runs through families such that susceptibility to breast or ovarian cancer based on a finding both sexes can be carriers (only women get the disease but that the individual does not carry an abnormal BRCA1 gene. men can pass it on), all generations will likely have breast/ It is another object of this invention to provide a method ovarian or both diseases and sometimes in the same of identifying a mutation leading to predisposition or higher individual, occasionally women carriers either die young susceptibility to breast or ovarian cancer. before they have the time to manifest disease (and yet 65 It is another object of the invention to provide a method offspring get it) or they never develop breast or ovarian of classifying a tumor for diagnostic and prognostic pur cancer and die of old age (the latter people are said to have poses. 5,654,155 3 4 There is a need in the art for a consensus normal sequence The term "substantially complementary to” or “substan of the BRCA1 gene and for the consensus normal protein tially the sequence” refers to primer sequences which sequence of BRCA1 as well as for an accurate list of codons hybridize to the sequences provided under stringent condi which occur at polymorphic points on a normal sequence. A tions and/or sequences having sufficient homology with person skilled in the art of genetic susceptibility testing will 5 BRCA1 sequences, such that the allele specific oligonucle find the present invention useful for: otide primers hybridize to the BRCA1 sequences to which a) identifying individuals having a normal BRCA1 gene, they are complimentary. who are therefore not at risk or have no increased The term "isolated” as used herein includes oligonucle susceptibility to breast or ovarian cancer from a otides substantially free of other nucleic acids, proteins, BRCA1 mutation; 10 lipids, carbohydrates or other materials with which they may b) avoiding misinterpretation of normal polymorphisms be associated. Such association is typically either in cellular found in the normal BRCA1 gene; material or in a synthesis medium. c) determining the presence of a previously unknown Sequencing mutation in the BRCA1 gene. 15 d) identifying a mutation which indicates a predisposition Any nucleic acid specimen, in purified or non-purified or higher susceptibility to breast or ovarian cancer; or form, can be utilized as the starting nucleic acid or acids, for providing it contains, or is suspected of containing, the specific nucleic acid sequence containing a polymorphic e) classifying a tumor for diagnostic and prognostic locus. Thus, the process may amplify, for example, DNA or purposes. 20 RNA, including messenger RNA, wherein DNA or RNA f) performing gene repair. may be single stranded or double stranded. In the event that DETALED DESCRIPTION OF THE RNA is to be used as a template, enzymes, and/or conditions INVENTION optimal for reverse transcribing the template to DNA would 25 be utilized. In addition, a DNA-RNA hybrid which contains Definitions one strand of each may be utilized. A mixture of nucleic acids may also be employed, or the nucleic acids produced The following definitions are provided for the purpose of in a previous amplification reaction herein, using the same understanding this invention. or different primers may be so utilized. See TABLE II. The "Consensus Normal Sequence” refers to the nucleic acid specific nucleic acid sequence to be amplified, i.e., the or protein sequence, the nucleic or amino acids of which are 30 polymorphic locus, may be a fraction of a larger molecule or known to occur with high frequency in a population of can be present initially as a discrete molecule, so that the individuals who carry the gene which codes for a normally specific sequence constitutes the entire nucleic acid. It is not functioning protein, or which nucleic acid itself has normal necessary that the sequence to be amplified be present function. initially in a pure form; it may be a minor fraction of a "Consensus normal DNA sequence” also called "consen 35 complex mixture, such as contained in whole human DNA. sus normal gene sequence” refers to a nucleic acid sequence, DNA utilized herein may be extracted from a body the nucleic acid of which are known to occur at their sample, such as blood, tissue material and the like by a respective positions with high frequency in a population of variety of techniques such as that described by Maniatis, et. individuals who carry the gene which codes for a normally 40 al. in Molecular Cloning. A Laboratory Manual, Cold Spring functioning protein, or which itself has normal function. Harbor, N.Y., p 280–281, 1982). If the extracted sample is "Consensus Normal Protein Sequence” refers to the pro impure, it may be treated before amplification with an tein sequence, the amino acids of which are known to occur amount of areagent effective to open the cells, or animal cell with high frequency in a population of individuals who carry membranes of the sample, and to expose and/or separate the the gene which codes for a normally functioning protein. strand(s) of the nucleic acid(s). This lysing and nucleic acid “BRCA1G"(SEQ ID NO: 1)" refers to a consensus 45 denaturing step to expose and separate the strands will allow sequence for the BRCA1 gene. The consensus sequence was amplification to occur much more readily. found by end to end sequencing,of the BRCA1 gene from 5 The deoxyribonucleotide triphosphates dATP, dCTP, individuals randomly drawn from the population and found dGTP, and dTTP are added to the synthesis mixture, either to have no family history of breast or ovarian cancer. The 50 separately or together with the primers, in adequate amounts sequenced gene was found not to contain any mutations. The and the resulting solution is heated to about 90°-100° C. consensus was determined by calculating the frequency with from about 1 to 10 minutes, preferably from 1 to 4 minutes. which nucleic acids occur and inserting the nucleic acid with After this heating period, the solution is allowed to cool, highest frequency of occurrence at each polymorphic site. In which is preferable for the primer hybridization. To the some cases the insertion of a nucleic acid at a polymorphic 55 cooled mixture is added an appropriate agent for effecting site indicated a codon change and a change of amino acid the primer extension reaction (called herein "agent for from previously published information. In other cases the polymerization”), and the reaction is allowed to occur under frequency of occurrence of a nucleic acid was found to differ conditions known in the art. The agent for polymerization from the published frequency. may also be added together with the other reagents if it is The term "primer" as used herein refers to a sequence heat stable. This synthesis (or amplification) reaction may comprising about 20 or more nucleotides of the BRCA1 occur at room temperature up to a temperature above which gene. the agent for polymerization no longer functions. Thus, for A “target polynucleotide" refers to the nucleic acid example, if DNA polymerase is used as the agent, the sequence of interest e.g., the BRCA1 encoding polynucle temperature is generally no greater than about 40° C. Most otide. Other primers which can be used for primer hybrid 65 conveniently the reaction occurs at room temperature. ization will be known or readily ascertainable to those of The allele specific oligonucleotide primers are useful in skill in the art. determining whether a subject is at risk of having breast or 5,654,155 S 6 ovarian cancer, and also useful for characterizing a tumor. perform primer extension after being subjected to tempera Primers direct amplification of a target polynucleotide prior tures sufficiently elevated to cause denaturation), such as to sequencing. These unique BRCA1 oligonucleotide prim Taq polymerase. Suitable enzyme will facilitate combination ers of TABLE II were designed and produced specifically to of the nucleotides in the proper manner to form the primer optimize amplification of portions of BRCA1 which are to extension products which are complementary to each poly be sequenced. morphic locus nucleic acid strand. Generally, the synthesis The primers used to carry out this invention embrace will be initiated at the 3' end of each primer and proceed in oligonucleotides of sufficient length and appropriate the 5' direction along the template strand, until synthesis sequence to provide initiation of polyrmerization. Environ terminates, producing molecules of different lengths. mental conditions conducive to synthesis include the pres 10 The newly synthesized strand and its complementary ence of nucleoside triphosphates and an agent for nucleic acid strand will form a double-stranded molecule polymerization, such as DNA polymerase, and a suitable under hybridizing conditions described above and this temperature and pH. The primer is preferably single hybrid is used in subsequent steps of the process. In the next stranded for maximum efficiency in amplification, but may step, the newly synthesized double-stranded molecule is be double stranded. If double stranded, the primer is first 15 subjected to denaturing conditions using any of the proce treated to separate its strands before being used to prepare dures described above to provide single-stranded molecules. extension products. The primer must be sufficiently long to The steps of denaturing, annealing, and extension product prime the synthesis of extension products in the presence of synthesis can be repeated as often as needed to amplify the the inducing agent for polymerization. The exact length of target polymorphic locus nucleic acid sequence to the extent primer will depend on many factors, including temperature, 20 necessary for detection. The amount of the specific nucleic buffer, and nucleotide composition. The oligonucleotide acid sequence produced will accumulate in an exponential primer typically contains 12–20 or more nucleotides, fashion. although it may contain fewer nucleotides. Amplification is described in PCR. A Practical Approach, Primers used to carry out this invention are designed to be ILR Press, Eds. M. J. McPherson, P. Quirke, and G. R. substantially complementary to each strand of the genomic 25 Taylor, 1992. locus to be amplified. This means that the primers must be The amplification products may be detected by Southern sufficiently complementary to hybridize with their respec blots analysis, without using radioactive probes. In such a tive strands under conditions which allow the agent for process, for example, a small sample of DNA containing a polymerization to perform. In other words, the primers very low level of the nucleic acid sequence of the polymor should have sufficient complementarity with the 5' and 3' 30 phic locus is amplified, and analyzed via a Southern blotting sequences flanking the mutation to hybridize therewith and technique or similarly, using dot blot analysis. The use of permit amplification of the genomic locus. non-radioactive probes or labels is facilitated by the high Oligonucleotide primers of the invention are employed in level of the amplified signal. Alternatively, probes used to the amplification process which is an enzymatic chain detect the amplified products can be directly or indirectly reaction that produces exponential quantities of polymor 35 detectably labeled, for example, with a radioisotope, a phic locus relative to the number of reaction steps involved. fluorescent compound, a bioluminescent compound, a Typically, one primer is complementary to the negative (-) chemiluminescent compound, a metal chelator or an strand of the polymorphic locus and the other is comple enzyme. Those of ordinary skill in the art will know of other mentary to the positive (+) strand. Annealing the primers to suitable labels for binding to the probe, or will be able to denatured nucleic acid followed by extension with an 40 ascertain such, using routine experimentation. enzyme, such as the large fragment of DNA polymerase I Sequences amplified by the methods of the invention can (Klenow) and nucleotides, results in newly synthesized + be further evaluated, detected, cloned, sequenced, and the and - strands containing the target polymorphic locus like, either in solution or after binding to a solid support, by sequence. Because these newly synthesized sequences are 45 any method usually applied to the detection of a specific also templates, repeated cycles of denaturing, primer DNA sequence such as PCR, oligomer restriction (Saiki, et. annealing, and extension results in exponential production al., Bio/Technology,3:1008-1012, 1985), allele-specific oli of the region (i.e., the target polymorphic locus sequence) gonucleotide (ASO) probe analysis (Conner, et al., Proc. defined by the primers. The product of the chain reaction is Natl. Acad. Sci. U.S.A., 80:278, 1983), oligonucleotide a discreet nucleic acid duplex with termini corresponding to 50 ligation assays (OLAs) (Landgren, et al., Science,241:1007, the ends of the specific primers employed. 1988), and the like. Molecular techniques for DNA analysis The oligonucleotide primers of the invention may be have been reviewed (Landgren, et. al., Science, prepared using any suitable method, such as conventional 242:229-237, 1988). phosphotriester and phosphodiester methods or automated Preferably, the method of amplifying is by PCR, as embodiments thereof. In one such automated embodiment, 55 described herein and as is commonly used by those of diethylphosphoramidites are used as starting materials and ordinary skillin the art. Alternative methods of amplification may be synthesized as described by Beaucage, et al., Tet have been described and can also be employed as long as the rahedron Letters, 22:1859-1862, 1981. One method for BRCA1 locus amplified by PCR using primers of the synthesizing oligonucleotides on a modified solid support is invention is similarly amplified by the alternative means. described in U.S. Pat. No. 4458,066. Such alternative amplification systems include but are not The agent for polymerization may be any compound or limited to self-sustained sequence replication, which begins system which will function to accomplish the synthesis of with a short sequence of RNA of interest and a T7 promoter. primer extension products, including enzymes. Suitable Reverse transcriptase copies the RNA into cDNA and enzymes for this purpose include, for example, E. coli DNA degrades the RNA, followed by reverse transcriptase poly polymerase I, Klenow fragment of E. coli DNA polymerase, 65 merizing a second strand of DNA. Another nucleic acid polymerase muteins, reverse transcriptase, other enzymes, amplification technique is nucleic acid sequence-based including heat-stable enzymes (e.i., those enzymes which amplification (NASBA) which uses reverse transcription 5,654,155 7 8 and T7 RNA polymerase and incorporates two primers to The consensus normal BRCA1 amino acid sequence may target its cycling scheme. NASBA can begin with either be used to provide a level of protection for patients against DNA or RNA and finish with either, and amplifies to 10 risk of breast or ovarian cancer. The isolated consensus copies within 60 to 90 minutes. Alternatively, nucleic acid normal BRCA1 gene can be constructed from amplification can be amplified by ligation activated transcription (LAT). products and inserted into a vector such as the LXSN vector. LAT works from a single-stranded template with a single Fresh lymphocytes of a patient having a mutation in the primer that is partially single-stranded and partially double BRCA1 gene, are cultured. The cells are transduced with the stranded. Amplification is initiated by ligating a cDNA to the vector above, and cultured. Extracted BRCA1 protein can be promoter oligonucleotide and within a few hours, amplifi provided by injection or other known means to patients who cation is 10 to 10 fold. The QB replicase system can be 10 are at risk. utilized by attaching an RNA sequence called MDV-1 to RNA complementary to a DNA sequence of interest. Upon mixing with a sample, the hybrid RNA finds its complement EXAMPLE 1. among the specimen’s mRNAS and binds, activating the replicase to copy the tag-along sequence of interest. Another Determination of the Sequence of the BRCA1" nucleic acid amplification technique, ligase chain reaction 15 Gene From Five Normal Individuals (LCR), works by using two differently labeled halves of a Materials and Methods sequence of interest which are covalently bonded by ligase in the presence of the contiguous sequence in a sample, Approximately 150 volunteers were screened in order to forming a new target. The repair chain reaction (RCR) identify individuals with no cancer history in their imme nucleic acid amplification technique uses two complemen 20 diate family (i.e. first and second degree relatives). Each tary and target-specific oligonucleotide probe pairs, thermo person was asked to fill out a hereditary cancer prescreening stable polymerase and ligase, and DNA nucleotides to questionnaire See TABLE I below. Five of these were geometrically amplify targeted sequences. A 2-base gap randomly chosen for end-to-end sequencing of their BRCA1 separates the oligonucleotide probe pairs, and the RCR fills gene.” A first degree relative is a parent, sibling, or off and joins the gap, mimicking normal DNA repair, Nucleic 25 spring. A second degree relative is an aunt, uncle, acid amplification by strand displacement activation (SDA) grandparent, grandchild, niece, nephew, or half-sibling. utilizes a short primer containing a recognition site for hincII with short overhang on the 5' end which binds to target TABLE DNA. A DNA polymerase fills in the part of the primer opposite the overhang with Sulfur-containing adenine ana 30 Hereditary Cancer Pre-Screening Questionnaire logs. HincII is added but only cuts the unmodified DNA Part A: Answer the following questions about your family strand, ADNA polymerase that lacks 5' exonuclease activity 1. To your knowledge, has anyone in your family been diagnosed with enters at the cite of the nick and begins to polymerize, a very specific hereditary colon disease called Familial Adenomatous displacing the initial primer strand downstream and building 35 Polyposis (FAP)? a new one which serves as more primer. SDA produces 2. To your knowledge, have you or any aunt had breast cancer diagnosed before the age 35? greater than 10-fold amplification in 2 hours at 37° C. 3. Have you had inflammatory Bowel Disease, also called Crohn's Unlike PCR and LCR, SDA does not require instrumented Disease or Ulcerative Colitis, for more than 7 years? Temperature cycling. Another amplification system useful in Part B: Refer to the list of cancers below for your responses only to the method of the invention is the QB Replicase System. 40 questions in Part B Although PCR is the preferred method of amplification if the Bladder Cancer Lung Cancer Pancreatic Cancer invention, these other methods can also be used to amplify Breast Cancer Gastric Cancer Prostate Cancer the BRCA1 locus as described in the method of the inven Colon Cancer Malignant Melanoma Renal Cancer tion Endometrial Cancer Ovarian Cancer Thyroid Cancer 4. Have your mother or father, your sisters or brothers or your children The BRCA1" Consensus Normal DNA sequence was 45 had any of the listed cancers? obtained by end to end sequencing offive normal subjects in 5. Have there been diagnosed in your mother's brothers or sisters, or the manner described above followed by analysis of the data your mother's parents more than one of the cancers in the above list? obtained. The data obtained provided us with the opportu 6. Have there been diagnosed in your father's brothers or sisters, or your father's parents more than one of the cancers in the above list? nity to evaluate six previously published normal polymor Part C: Refer to the list of relatives below for responses only to phisms for correctness and frequency in the population, and 50 questions in Part C to identify an additional polymorphism not previously found. You Your mother Your sisters or brothers Your mothers's sisters or brothers The consensus normal gene sequence can be used for (maternal aunts and uncles) gene therapy. A complete description of the method is Your children Your mother's parents (maternal provided in Anderson et al. U.S. Pat. No. 5,399,346, issued 55 grandparents) 7 Have there been diagnosed in these relatives 2 or more identical Mar. 21, 1995. The isolated consensus normal BRCA1 gene types of cancer? --- can be constructed from amplification products and inserted Do not count "simple" skin cancer, also called basal cell or squamous into a vector such as the LXSN vector. Fresh lymphocytes cell skin cancer. of a patient having a mutation in the BRCA1 gene, are 8. Is there a total of 4 or more of any cancers in the list of relatives cultured. The cells are transduced with the vector above, and 60 above other than "simple" skin cancers? culturing is continued. The cultured, transformed cells are Part D: Refer to the list of relatives below for responses only to infused into the patient. questions in Part D. The consensus normal BRCA1 amino acid sequence may You Your father Your sisters or brothers Your fathers's sisters or brothers (paternal be used to make diagnostic probes. Labeled diagnostic aunts and uncles) probes may be used by any hybridization method to deter 65 Your children Your father's parents (paternal mine the level of BRCA1 protein in serum or lysed cell grandparents) suspension of a patient, or solid surface cell sample. 5,654,155 9 10 demic Press, Inc., 1993. Fluorescent dye was attached for TABLE I-continued automated sequencing using the Taq Dye Terminator Kit (PERKIN-ELMER® cath 401628). DNA sequencing was Hereditary Cancer Pre-Screening Questionnaire performed in both forward and reverse directions on an 9, Have there been diagnosed in these relatives 2 or more identical Applied Biosystems, Inc. (ABI) automated sequencer types of cancer? (Model 377). The software used for analysis of the resulting Do not count "simple" skin cancer, also called basal cell or squamous data was "Sequence Navigator" purchased through ABI. cell skin cancer. 1. Polymerase Chain Reaction (PCR) Amplification 10. Is there a total of 4 or more of any cancers in the list of relatives above other than "simple' skin cancers? Genomic DNA (100 nanograms) extracted from white O blood cells offive normal subjects. Each of the five samples G Copyright 1996, OncorMed, Inc. was sequenced end to end. Each sample was amplified in a final volume of 25 microliters containing 1 microliter (100 Genomic DNA was isolated from white blood cells offive nanograms) genomic DNA, 2.5 microliters 10XPCR buffer normal subjects selected from analysis of their answers to (100 mM Tris, pH 8.3, 500 mM KCl, 1.2 mM MgCl), 2.5 the questions above. Dideoxy sequence analysis was per microliters 10X dNTP mix (2 mM each nucleotide), 2.5 formed following polymerase chain reaction amplification. 15 microliters forward primer, 2.5 microliters reverse primer, All exons of the BRCA1 gene were subjected to direct and 1 microliterTaq polymerase (5 units), and 13 microliters dideoxy sequence analysis by asymmetric amplification of water. using the polymerase chain reaction (PCR) to generate a The primers in Table II, below were used to carry out single stranded product amplified from this DNA sample. 20 amplification of the various sections of the BRCA1 gene Shuldiner, et at., Handbook of Techniques in Endocrine samples. The primers were synthesized on an DNA/RNA Research, p. 457-486, DePablo, F, Scanes, C., eds., Aca Synthesizer Model 394(8).

TABLE II BRCA PRIMERS AND SEQUENCING DATA SEQ. ID EXON SEQUENCE NO. MER MG + 1 SEZE EXON 2 2F 5 GAA GT, GTCATTTTAAAACCTTT-3' 3 24 1.6 -275 2R 5 TFT. CTTTCTTC CCTAGTATG 5.3' 4. 22 EXON 3 3F 5TCCTGACACAGC AGA CATTTA-3' 5 21 1.4 -375 3R 5 TTG GATT CGTCT CACTTA-3' 6 21 EXON 5 SF 5 CTCTA AGG GCA GT, GOGAG-3' 7 20 1.2 -275 15R 5 TTC CACGTGGG CTTCC 8 20 EXON 6 6F 5 CTTATTTIA GTGTCCTTAAAAGG-3' 9 23 1.6 -250 6R STT CATGGA CAG CACTTGAGTG-3' 10 22 EXON T TF 5' CAC AACAAGAG. CATACAAG GG-3' 11 23 15 -275 6FTR SCGGGTTCACTCTGTAGAAG-3' 12 20 EXON 8, 8F 5 TTCCTTCAGGAGGAAAAGCA-3' 13 2. 1.2 -270 8R1 5 GCT GCC TAC CACAAATACAAA-3' 14 21 EXON 9 9F 5' CCA CAG TAGATG CTCAGTAAATA-3' 15 23 1.2 -250 9R 5'TAG GAAAATACCAGCTTCATAGA-3' 16 23 EXOMO 10F 5 TGGTCA, GCTTTC TGTAATCG-3 17 20 1.6 -250 10R 5 GTATCTACC CACTCTCTT CTT CAG-3 18 24 EXON 1A 11AF 5 CCACCTCCAAGGTGTATCA-3' 19 19 1.2 372 11AR 5"TGTAT GTTGGCTCCTTG CT3' 20 20 EXON 1B 11BF1 5 CACTAAAGACAGAA GAATCTA-3; 21 2. 1.2 -400 11BR1 5 GAAGAA CCA GAATATTCATCTA-3' 22 2. EXON 11C 11CF1 5'TGATGG GGAGCTGAATCAA-3' 23 20 1.2 -400 11CR1 5TCT GCTTCTGATAAAATCC T3' 24 22 EXON 1D DF1 SAGCGTCCCCTCA CAAATAAA-3' 25 20 1.2 -400 11DR1 5 TCAAGC GCATGAATATGC CT3 26 20 EXON 11E 1EF 5 GTATAAGCAATATGGAACTCGA-3 27 22 1.2 388 1ER 5 TTAAGTTCACTG GTATTGAA CA-3' 28 23 EXON 11F 1FF 5 GACAGC GATACTTTC CCAGA-3' 29 20 1.2 382 1FR STGGAACAAC CAT GAATTAGTC-3' 30 21 EXON 11G 1GF 5 GGAAGTTAG CACTCTAGGGA-3' 31 20 1.2 423 11GR 5 GCA GTGATATTAACT GTCTGTA-3' 32 22 EXON 11H 11HF 5 TGG GTC CTTAAAGAAACAAAGT3' 33 22 1.2 366 11HR 5. CAGGTGACATTGAATCTTCC-3' 34 21. EXON 11 11F SCCA CTTTTTCCC ATCAAGTCA-3' 35 21 1.2 377 1R 5''CAGGATGCTTACAATTACT C-3 36 2. EXON 11 11F 5 CAAAATTGAATG CTATGCTAAGA-3' 37 23 1.2 377 11R STCG GTAACC CTGAGCCAAAT3' 38 20 EXON11K 11KF 5 GCAAAAGCGTCCAGAAAG GA-3' 39 20 1.2 396 11KR-1 STATTTG CAGTCAAGT CTTCCAA-3 40 22 EXON 1.1L 1.1LF-1 5 GTAAATG GCAAAG GCATCT3' 41 22 1.2 360 1LR 5'TAAAAT GTG CCCCCAAAAGCA-3' 42 22 EXON 2 .2F 5 GTC CTG CCAATGAGAAGAAA-3 43 20 1.2 -300 12R 5 TGT CAG CAAACC TAAGAATGT3' 44 21 EXON 13 3F SAATGGAAAG CTTCTCAAAGA-3' 45 2 1.2 -325 13R 5ATGTG GAGCCA GGT CCTTAC-3' 46 21 EXON 14 14F 5 CTAACCTGAATTACACTATCA-3' 47 22 1.2 -310 14R 5 GTGTATAAATGC CTGTATGCA-3 48 21. 5,654,155 11 12

TABLE II-continued BRCA1 PRIMERS AND SEQUENCING DATA SEQ, ID EXON SEQUENCE NO. MER MG + 1 SEZE EXON 5 15F 5' TGG CTG CCCAGGAAG TAT G-3 49 19 1.2 375 SR 5'AAC CAGAATATC TTTATG TAGGA-3' 50 23 EXON 16 6F 5'AATTCTTAACAGAGA CCA GAA C-3' 51 22 1.6 -550 16R 5'AAAACT CTTTCCAGAATGTTG 5-3 52 22 EXON 17 17F 5 GTG TAGAAC GTG CAG GATTG-3' 53 20 1.2 -275 1TR 5 TCG CCT CAT GTGGTTTTA-3' 54 18 EXON 18, 18F 5 GGCCTTTAGCTTCTTAGGAC-3 55 21 1.2 -350 18R 5 GAGACCATTTTC CCAGCATC-3' 56 20 EXON 19 19F 5 CTG CATTCTTC CTG TGCTC-3' 57 20 i.2 -250 19R 5' CATTGTTAAGGAAAGTGGTGC-3' 58 21 EXON 20 20F 5ATATGA CGT GTCTGCTCCAC-3' 59 20 1.2 -425 2OR 5' GGGAATCCAAATTACACA GC-3' 60 20 EXON 21 21F 5' AAG CTCTTCCTTTTT GAAAGT C-3' 61 22 1.6 -300 21R 5 GTA GAGAAA TAGAAT AGC CTCT-3' 62 22 EXON 22 22F 5 TCC CATTGA GAG GCTG CT3' 63 20 16 -300 22R 5 GAGAAG ACTTCT GAG GCTAC-3' 64 2O EXON 23 23F-1 5 TGAAGTGACAGTTCCAGTAGT3' 65 2 1.2 -250 23R-1 5' CATTTTAGC CATTCATTC AACAA-3' 66 23 EXON 24 24F 5' ATGAATTGACACTAATCT CTG C-3 67 22 1.4 -285 24R 5 GTAGCCAGGACA GTA. GAA. GGA-3' 68 2.1

25 Thirty-five cycles were performed, each consisting of denaturing (95° C.; 30 seconds), annealing (55° C.; 1 TABLE I minute), and extension (72° C.; 90 seconds), except during the first cycle in which the denaturing time was increased to NORMAL PANEL TYPING 5 minutes, and during the last cycle in which the extension 30 AMNo time was increased to 5 minutes, ACD CHANGE EXON 1 2 3 4. 5 FREQUENCY PCR products were purified4. using QLA-QUICKGE PCR SER(SERE. ) 11E CFC CFT CIT TIT TFT 0.4O.6T C purification kits (QIAGEN, catt 28104; Chatsworth, Calif.). 35 LEUCLEU) 11F TT CIT CIT CIC CIC 0.4T Yield and purity of the PCR product determined spectro- (771) 0.6C photometrically at OD2 on a Beckman DU 650 spectro- Rep. El) 11G CIT Ci Cf. TT TT 3. photometer. GLU(GLY) 11E AIA AG AIG GIG GIG 0.4A (1038) 0.6 G. 40 LYSCARG) 11J AJA A/G AIG GIG GIG 0.4A 2. Dideideoxy Sequence Analysis (1183)SER(SER) 13 TFT T/T T/C CfC CFC 0.6O5T G. (1436) O.5T SER(GLY) 6 AIA AIG AIG GIG GIG 0.4A Fluorescent dye was attached to PCR products for auto- (1613) 0.6 G. mated sequencing using the Taq Dye Terminator Kit 45 (PERKIN-ELMER(8) cati401628). DNA sequencing was performed in both forward and reverse directions on an A consensus normal sequence of the BRCA1 gene was Applied Biosystems, Inc. (ABI) Foster City, Calif., auto- determined by calculating the percentage of occurrence of mated sequencer (Model 377). The software used for analy- each polymorphism and inserting the more frequently occur sis of the resulting data was "SEQUENCENAVIGATORCE" 50 ring polymorphism into the published BRCA1 (Genbank purchased through ABI. Accession Number U15595). The normal consensus BRCA1" is shown as SEQ. ID NO. 1 at page 29. 3. Results The data show that for each of the samples. The BRCA1 gene is identical except in the region of seven polymor 55 phisms. These polymorphic regions, together with their Differences in the nucleic acids of the five normal indi locations, the amino acid groups of each codon, the fre viduals were found in seven locations on the gene. The quency of their occurence and the amino acid coded for by changes and their positions are found on TABLE III, below. each codon are found in TABLE IV below, 5,654,155 13 14

TABLE TV COEDON AND BASE CHANGESIN SEVEN POLYMORPHC SITES OF BRCA NORMAL, GENE

SAMPLE BASE CODON AA PUBLISHED NUMBER CHANGE POSITION EXON CHANGE CHANGE FREQUENCY REFERENCES 2, 3, 4, 5 C-T 22O1 11E AGCCAGT) SER-SER C UNPUBLISHED 2, 3, 4, 5 T-C 2430 11F TTG(CTG) Sieu T 67% 13 1, 2, 3, 4, 5 C-T 2731 11G CCG(CTG) E. LEU T= 66% 12 2, 3, 4, 5 A-G 3232 11 GAAGGA) SEgy At 67% 13 2, 3, 4, 5 A-G 3667 11J AAA(AGA) SPARg A = 68% 12 3, 4, 5 TC 4427 13 TCT(TCC) SE'sER Ta 67% 12 2, 3, 4, 5 A-G 4956 16 AGT(GGT) Sicily A 6.7% 12 (1613) Reference numbers correspond To The Table Of References on Page 28.

EXAMPLE 2 The software used for analysis of the resulting data is “SEQUENCE NAVIGATOR(8” purchased through ABI. Determination of a Normal Individual Using 1. Polymer Chain Reaction (PCR) Amplification BRCA1" and the Seven Polymorphisms for 25 Genomic DNA (100 nanograms) extracted from white Reference blood cells of the subject is amplified in a final volume of 25 Aperson skilled in the art of genetic susceptibility testing microliters containing 1 microliter (100 nanograms) genomic DNA, 2.5 microliters 10X PCR buffer (100 mM will find the present invention useful for: Tris, pH 8.3, 500 mM KCl, 1.2 mMMgCl), 2.5 microliters a) identifying individuals having a normal BRCA1 gene, 30 10X dNTP mix (2 mM each nucleotide), 2.5 microliters who are therefore not at risk or have no increased forward primer (BRCA1-11K-F, 10 micromolar solution), susceptibility to breast or ovarian cancer from a 2.5 microliters reverse primer (BRCA1-11K-R, 10 micro BRCA1 mutation; molar solution),and 1 microliter Taq polymerase (5 units), b) avoiding misinterpretation of normal polymorphisms and 13 microliters of water. The PCR primers used to found in the normal BRCA1 gene; 35 amplify a patient's sample BRCA1 gene are listed in Table Sequencing is carried out as in EXAMPLE 1 using a blood II. The primers were synthesized on an DNA/RNA Synthe sample from the patient in question. However, the BRCA1 sizer Model 394(8). Thirty-five cycles are of amplification are "sequence is used for reference and polymorphic sites performed, each consisting of denaturing (95° C.; 30 are compared to the nucleic acid sequences listed above for seconds), annealing (55° C.; 1 minute), and extension (72° normal codons at each polymophic site. A normal sample is C.; 90 seconds), except during the first cycle in which the one which compares to the BRCA1" sequence and con denaturing time is increased to 5 minutes, and during the last tains one of the normal base variations which occur at each cycle in which the extension time is increased to 5 minutes. of the polymorphic sites. The codons which occur at each of PCR products are purified using QIA-QUICKGE PCR the polymorphic sites are paired below. purification kits (QLAGENGE), cati 28104; Chatsworth, AGC and AGT at position 2201, 45 Calif.). Yield and purity of the PCR product determined spectrophotometrically at ODo on a Beckman DU 650 TTG and CTG at position 2430, spectrophotometer. CCG and CTG at position 2731, 2. Dideoxy Sequence Analysis GAA and GGA at position 3232, Fluorescent dye is attached to PCR products for auto AAA and AGA at position 3667, 50 mated sequencing using the Taq Dye Terminator Kit TCT and TCC at position 4427, and (PERKIN-ELMER(8) cath: 401628). DNA sequencing is per AGT and GGT at position 4956. formed in both forward and reverse directions on an Applied The availability of these polymorphic pairs provides added Biosystems, Inc. (ABI) Foster City, Calif., automated assurance that one skilled in the art can correctly interpret sequencer (Model 377). The software used for analysis of the polymorphic variations without mistaking a normal 55 the resulting data is “SEQUENCE NAVIGATOR6)” pur variation for a mutation. chased through ABI. The BRCA16" sequence is entered Exon 11 of the BRCA1 gene is subjected to direct dideoxy into the Sequence Navigator software as the Standard for sequence analysis by asymmetric amplification using the comparison. The Sequence Navigator software compares the polymerase chain reaction (PCR) to generate a single sample sequence to the BRCA1" standard, base by base. stranded product amplified from this DNA sample. The Sequence Navigator highlights all differences between Shuldiner, et al., Handbook of Techniques in Endocrine the BRCA1" (SEQID. NO: 1) consensus normal DNA Research, p. 457-486, DePablo, F. Scanes, C., eds., Aca sequence and the patient's sample sequence. demic Press, Inc., 1993. Fluorescent dye is attached for A first technologist checks the computerized results by automated sequencing using the Taq Dye Terminator Kit comparing visually the BRCA1" standard against the (PERKIN-ELMER® cat 401628). DNA sequencing is per 65 patient's sample, and again highlights any differences formed in both forward and reverse directions on an Applied between the standard and the sample. The first primary Biosystems, Inc. (ABI) automated Sequencer (Model 377). technologist then interprets the sequence variations at each 5,654,155 15 16 position along the sequence. Chromatograms from each at position 4956, AGT and GGT occuratfrequencies from sequence variation are generated by the Sequence Navigator about 35-45%, and from about 55-65%, respectively. and printed on a color printer. The peaks are interpreted by Exon 11 of the BRCA1 gene is subjected to direct dideoxy the first primary technologist and a second primary tech sequence analysis by asymmetric amplification using the nologist. A secondary technologist then reviews the chro 5 polymerase chain reaction (PCR) to generate a single matograms. The results are finally interpreted by a geneti stranded product amplified from this DNA sample. cist. In each instance, a variation is compared to known Shuldiner, et al., Handbook of Techniques in Endocrine normal polymorphisms for position and base change. If the Research, p. 457-486, DePablo, F, Scanes, C., eds., Aca sample BRCA1 sequence matches the BRCA1("standard, demic Press, Inc., 1993. Fluorescent dye is attached for with only variations within the known list of O automated sequencing using the Taq Dye Terminator Kit polymorphisms, it is interpreted as a normal gene sequence. (PERKIN-ELMER(8) catf 401628). DNA sequencing is per formed in both forward and reverse directions on an Applied EXAMPLE 3 Biosystems, Inc. (ABI) automated sequencer (Model 377) The software used for analysis of the resulting data is Determining the Presence of a Mutation in the 15 “SEQUENCE NAVIGATOR6” purchased through ABI. BRCA1 Gene Using BRCA1" and Seven 1. Polymerase Chain Reaction (PCR) Amplification Polymorphisms for Reference Genomic DNA (100 nanograms) extracted from white A person skilled in the art of genetic susceptibility testing blood cells of the subject is amplified in a final volume of 25 will find the present invention useful for determining the microliters containing 1 microliter (100 nanograms) presence of a known or previously unknown mutation in the genomic DNA, 2.5 microliters 10X PCR buffer (100 mM BRCA1 gene. A list of mutations of BRCA1 is publicly Tris, pH 8.3, mMKCl, 1.2 mMMgCl), 2.5 microliters 10X available in the Breast Cancer Information Core at: http:// dNTP mix (2 mM each nucleotide), 2.5 microliters forward www.nchgr.nih.gov/dir/lab transfer/bic. This data site primer (BRCA1-11K-F, 10 micromolar solution), 2.5 micro became publicly available on Nov. 1, 1995. Friend, S. et al. liters reverse primer (BRCA1-11K-R, 10 micromolar Nature Genetics 11:238, (1995). Sequencing is carried out as 25 solution),and 1 microliter Taq polymerase (5 units), and 13 in EXAMPLE 1 using a blood sample from the patient in microliters of water. The PCR primers used to amplify a question. However, the BRCA (?" (SEQ ID NO: 1) patient's sample BRCA1 gene are listed in Table II. The sequence is used for reference and polymorphic sites are primers were synthesized on an DNA/RNA Synthesizer compared to the nucleic acid sequences listed above for Model 394(8). Thirty-five cycles are of amplification are normal codons at each polymophic site. A normal sample is 30 performed, each consisting of denaturing (95° C. 30 one which compares to the BRCA1" sequence and con Seconds), annealing (55° C. 1 minute), and extension (72 tains one of the normal base variations which occur at each C.; 90 seconds), except during the first cycle in which the of the polymorphic sites. The codons which occur at each of denaturing time is increased to 5 minutes, and during the last the polymorphic sites are paired below. cycle in which the extension time is increased to 5 minutes. 35 PCR products are purified using QIA-QUICKGE PCR AGC and AGT at position 2201, purification kits (QIAGEN(8), catt 28104; Chatsworth, TTG and CTG at position 2430, Calif.). Yield and purity of the PCR product determined CCG and CTG at position 2731, spectrophotometrically at ODo on a Beckman DU 650 GAA and GGA at position 3232, spectrophotometer. AAA and AGA at position 3667, 40 2. Dideoxy Sequence Analysis Fluorescent dye is attached to PCR products for auto TCT and TCC at position 4427, and mated sequencing using the Taq Dye Terminator Kit AGT and GGT at position 4956. (PERKIN-ELMER® cath: 401628). DNA sequencing is per The availability of these polymorphic pairs provides added formed in both forward and reverse directions on an Applied assurance that one skilled in the art can correctly interpret 45 Biosystems, Inc. (ABI) Foster City, Calif., automated the polymorphic variations without mistaking a normal sequencer (Model 377). The software used for analysis of variation for a mutation. the resulting data is “SEQUENCE NAVIGATOR6)” pur As evident from the data in Table III and IV, the statistical chased through ABL. The BRCA16" sequence (SEQID. analysis (on average) shows one or more normal codon pairs NO: 1) is entered into the Sequence Navigator software as wherein the codons occur in the following frequencies in the 50 the Standard for comparison. The Sequence Navigator soft normal population, respectively: ware compares the sample sequence to the BRCA1" at position 2201, AGC and AGT occur at frequencies of standard (SEQ ID NO: 1), base by base. The Sequence about 40%, and from about 55-65%, respectively; Navigator highlights all differences between the BRCA1 at position 2430, TTG and CTG occuratfrequencies from " consensus normal DNA sequence and the patient's about 35-45%, and from about 55-65%, respectively; 55 Sample sequence. A first technologist checks the computerized results by at position 2731, CCG and CTG occuratfrequencies from comparing visually the BRCA1" standard (SEQID. NO: about 25-35%, and from about 65–75%, respectively; 1) against the patient's sample, and again highlights any at position 3232, GAA and GGA occur at frequencies differences between the standard and the sample. The first from about 35-45%, and from about 55-65%, respec 60 primary technologist then interprets the sequence variations tively; at each position along the sequence. Chromatograms from at position 3667, AAA and AGA occur at frequencies each sequence variation are generated by the Sequence from about 35-45%, and from about 55-65%, respec Navigator and printed on a color printer. The peaks are tively; interpreted by the first primary technologist and also by a at position 4427, TCT and TCC occur at frequencies from 65 second primary technologist. A secondary technologist then about 45-55%, and from about 45–55%, respectively; reviews the chromatograms. The results are finally inter and preted by a geneticist. In each instance, a variation is 5,654,155 17 18 compared to known normal polymorphisms for position and Fluorescent dye is attached to PCR products for auto base change. If the sample BRCA1 sequence matches the mated sequencing using the Taq Dye Terminator Kit BRCA1"standard, with only variations within the known (PERKIN-ELMER(8) Cat # 401628). DNA sequencing is list of polymorphisms, it is interpreted as a normal gene performed in both forward and reverse directions on an sequence. Mutations are noted by the length of non Applied Biosystems, Inc. (ABI) Foster City, Calif., auto matching variation. Such a lengthy mismatch pattern occurs mated sequencer (Model 377). The software used for analy with deletions and substitutions. An alteration such as a base sis of the resulting data is “SEQUENCE NAVIGATOR” substitution at a single position will be noted as a single purchased through ABI. The BRCA1" sequence is mismatch between the standard and the patient's gene entered into the SEQUENCE Navigator software as the sample. 10 Standard for comparison. The Sequence Navigator software compares the sample sequence to the BRCA1" standard EXAMPLE 4 (SEQ ID. NO: 1), base by base. The Sequence Navigator Determining the Presence of a Mutation in the highlights all differences between the BRCA1" (SEQID. BRCA1 Gene Using BRCA1" and Seven NO: 1) consensus normal DNA sequence and the patient's Polymorphisms for Reference 15 sample sequence. A first technologist checks the computerized results by A person skilled in the art of genetic susceptibility testing comparing visually the BRCA1" standard against the will find the present invention useful for determining the patient's sample, and again highlights any differences presence of a known or previously unknown mutation in the between the standard and the sample. The first primary BRCA1 gene. A list of mutations of BRCA1 is publicly technologist then interprets the sequence variations at each available in the Breast Cancer Information Core at: http:// position along the sequence. Chromatograms from each www.nchgr.nih.gov/dir/lab transfer/bic. This data site sequence variation are generated by the Sequence Navigator became publicly available on Nov. 1, 1995. Friend, S. et al. and printed on a color printer. The peaks are interpreted by Nature Genetics 11:238, (1995). In this example, a mutation the first primary technologist and a second primary tech in exon 11 is characterized by amplifying the region of the 25 nologist. A secondary technologist then reviews the chro mutation with a primer which matches the region of the matograms. The results are finally interpreted by a geneti mutation. Exon 11 of the BRCA1 gene is subjected to direct cist. In each instance, a variation is compared to known dideoxy sequence analysis by asymmetric amplification normal polymorphisms for position and base change. The using the polymerase chain reaction (PCR) to generate a seven known polymorphisms which occur in the Consensus single stranded product amplified from this DNA sample. 30 Normal DNA sequence are: Shuldiner, et al., Handbook of Techniques in Endocrine Research, p. 457-486, DePablo, F, Scanes, C., eds., Aca AGC and AGT at position 2201, demic Press, Inc., 1993. Fluorescent dye is attached for TTG and CTG at position 2430, automated sequencing using the Taq Dye Terminator Kit CCG and CTG at position 2731, (Perkin-Elmer(s) cat 401628). DNA sequencing is per 35 GAA and GGA at position 3232, formed in both forward and reverse directions on an Applied Biosystems, Inc. (ABI) automated sequencer (Model 377). AAA and AGA at position 3667, The software used for analysis of the resulting data is TCT and TCC at position 4427, and "Sequence Navigator" purchased through ABI. 1. Poly AGT and GGT at position 4956. merase Chain Reaction (PCR) Amplification Genomic DNA Mutations are noted by the length of non-matching varia (100 nanograms) extracted from white blood cells of the tion. Such a lengthy mismatch pattern occurs with deletions subject is amplified in a final volume of 25 microliters and substitutions. containing 1 microliter (100 nanograms) genomic DNA, 2.5 5. Result microliters 10XPCR buffer (100 mM Tris, pH 8.3,500 mM Using the above PCR amplification and standard fluores KCl, 1.2 mMMgCl), 2.5 microliters 10X dNTP mix (2 mM 45 cent sequencing technology, The 3888delGA mutation may each nucleotide), 2.5 microliters forward primer (BRCA1 be found. The 3888delGA mutation The BRCA1 gene lies in 11K-F, 10 micromolar solution), 2.5 microliters reverse segment “K” of exon 11. The DNA sequence results dem primer (BRCA1-11K-R, 10 micromolar solution), and 1 onstrate the presence of a two base pair deletion at nucle microliter Taq polymerase (5 units), and 13 microliters of otides 3888 and 3889 of the published BRCA1" water. The PCR primers used to amplify segment Kof exon 50 sequence. This mutation interrupts the normal reading frame 11 (where the mutation is found) are as follows: of the BRCA1 transcript, resulting in the appearance of an BRCA1-11K-F: 5'-GCA AAA GCG TCC AGA AAG in-frame terminator (TAG) at codon position 1265. This GA-3' SEQID NO: 69 mutation is, therefore, predicted to result in a truncated, and BRCA1-11K-R:5'-AGT CTTCCAATTCACTGCAC-3' most likely, non-functional protein. The formal name of the SEQID NO: 70 55 mutation will be 3888delGA. This mutation is named in The primers are synthesized on an DNA/RNA Synthesizer accordance with the suggested nomenclature for naming Model 3946). Thirty-five cycles are performed, each con mutations, Baudet, A et al., Human Mutation 2:245–248, sisting of denaturing (95°C. 30 seconds), annealing (55° C.; (1993). 1 minute), and extension (72°C.; 90 seconds), except during the first cycle in which the denaturing time is increased to 5 60 minutes, and during the last cycle in which the extension TABLE OF REFERENCES time is increased to 5 minutes. PCR products are purified Sanger, F, et al., J. Mol. Biol. 42:1617, (1980). using QIA-QUICKGE PCR purification kits (Qiagen?s), cath Beaucage, et al., Tetrahedron Letters 22:1859-1862, (1981). i. Maniatis, et al. in Molecular Cloning: A Laboratory Manual, Cold 28104; Chatsworth, Calif.). Yield and purity of the PCR Spring Harbor, NY, p 280-281, (1982). product determined spectrophotometrically at ODso on a 65 Conner, et al., Proc. Natl. Acad. Sci. U.S.A. 80:278, (1983) Beckman DU 650 spectrophotometer. Saiki, et al., BioTechnology 3:1008-1012, (1985) 2. Dideoxy Sequence Analysis 5,654,155 19 20 -continued -continued

TABLE OF REFERENCES TABLE OF REFERENCES

6. Landgren, et al., Science 241:1007, (1988) 5 14. Baudet, Aet al., Human Mutation 2:245–248, (1993). 7 . Landgren, et. al., Science 242:229-237, (1988). 8. PCR. A Practical Approach, ILR Press, Eds. M. J. McPherson, 15. Friend, S. et al. Nature Genetics 11:238, (1995). P. Quirke, and G. R. Taylor, (1992). 9. Easton et al., American Journal of Human Genetics 52:678-701, (1993). 10. U.S. Pat No. 4458.066. 9 Although the invention has been described with reference . i. S., et al., American Journal of Human Genetics 55:861-865, to the presently preferred embodiments, it should be under 12. Miki, Y. et al., Science 266:66-71, (1994). stood that various modifications can be made without 13. Friedman, L. et al., Nature Genetics 8:399-404, (1994). departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

SEQUENCE LISTING

( 1) GENERAL INFORMATION: { i i i ) NUMBER OF SEQUENCES: 74

( 2) INFORMATION FOR SEQID No:1: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5711 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: cDNA ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Homo sapiens ( B) STRAIN: BRCA1 ( v i i i )POSITION IN GENOME: (A) CHROMOSOME/SEGMENT: 17 (B) MAP POSITION: 17q21 ( x i SEQUENCE DESCRIPTION: SEQID NO:1:

AG C T C GCT GA GA CT C C G G A C C C C GCAC C A G G CT G T G G G GT T. CT CAGA TAACGG GCC 6 0

CCT GC GCT CA G GA. G. G. CCTT C A C C C T C T G C T C T G G G T AAA G TT CAT G GAA CAGAAAGAAA 20

G GATT TAT C E G CT C C GC GTT GAA GAAG TA CAAAA GT CAT AAT G. CT A T G CAGAAAA 1 80

TCTTAGA G T G T C C CAT CT GE C T G GA GT, GA. T CAAG GAA C C T G CT C CACA AA GT G T G ACC 2 4 0

A CATA TTT G CAAATTT GC AT G. C. G.A.A.A. C. TT CT CAA C CA. GAAGAAAG G G CCT CACA GT 3 O O

GT C CTTA T G T AAGAA. GAT ATAAC CAAAA G GAGCC TACA, AGAAA GTA. C. G. AGAT TA GT C 3 60

AACT G T G A A GA G CTAT G AAAAT CA. T G T G CTTTECA G CTTGACA CA. GGTT G GA GT 4 20

A T G CAA ACA G CTATA ATTT G CAAAAAAG G AAAAAA CTC T C CT GAA CAT CAAAAGAT G. 48 0

AA GTTT CAT CAT C CAAA GT ATGG GCT ACA GAAA C C G T G C CAAAA GACE CACA GA GTG 5 4 0

AA C C C GAAAA CCTT C C T G CAG GAAA CCA GT C T C A G T G T C CAA C CTCT AAC CTTG GAA. 60 0

C T G T G A GAA C T C GAG GACA AA G CA. GCG GA TA CAA C CT CA. AAA GAC GT C GT CTA CATTG 6 6 O

AATT G G GAT C GATT C T C T GAA GATA C C G T TAATAAG GC AACTATTG. C. A G T G T G G GAG 7 20

ATCAAGAA T G T A CAAATC A C C C C CAAG GAA C CAGGGA T GAA. A. CAG T G GA. T. CT G. 780

CAAAAAAG G C T G C T G GAA TTT T. CT GAGA CG GA T G T AAC AAAT ACT GAA CAT CAT CAA. C. 8 4 O

C CA GT AATAA T GATTTGAAC ACC ACT GAGA A G C G T G CAG C T G A GA. G. G. CAT C CAGAAAA GT 9 O O

A CA. G. G. G. AG TT CT GTTTCA AACTT G CAT G T G GAGCCA T G T G GCA CAAAT ACT CAGCCA 9 6 O

G CE CATTACA G CAT GA GAA C A G CA GT TAT TACT CACTA A A GACA GAA T G AA T G TAGAAA 1 O 2

5,654,155 2S 26 -continued ( 2) INFORMATION FOR SEQID NO:2: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1863 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant ( i i ) MOLECULETYPE: protein ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Homo sapiens (B) STRAIN: BRCA1 ( v i i i ). POSITION IN GENOME: (A) CHROMOSOMEISEGMENT: 17 (B) MAP POSITION: 17q21 ( xi ) SEQUENCE DESCRIPTION: SEQID NO:2: Me t A s p Leu Se r A la L. eu A rig V a 1 G 1 u G 1 u V a 1 G 1 in As in V a I 1 e A s in 1. 5 0 A a Met G 1 in Lys I le Le u G 1 u Cy s Pro I le Cy s L. eu G 1 u Le u I e Lys 20 25 3 O G 1 u Pro V a 1 Ser Thr Lys Cy s A s p H is I 1 e Phe Cys Lys Ph. e. Cys Me t 35 40 a 5 Le u Lys Le u L. eu As in G 1 n Lys Lys G 1 y P r c Ser G 1 in Cy s Pro Le u Cy s 5 0 55 6 0 Lys As n A s p I le Thr Lys Arg Ser L. eu G 1 in G i u Se r Thr Arg Ph e S e I 65 7 O 75 8 O G 1 in Le u Wa G 1 u G 1 u Le u L. eu Lys I e I le Cy S A a Ph e G 1 in L. eu As p 85 90 95 Thr G y L. eu G 1 u Ty r A 1 a. As n Ser Ty r A s in Phe A1 a Lys Lys G 1 u As in 1 00 1 O 5 1 0 As n Sier Pro G 1 u H is Le u Lys As p G 1 u Wa 1 Ser I I e I le G 1 in Ser Me t 1 15 1 20 25

G 1 y Ty A. 8 As in Arg A a Lys A rig Le u Le u G 1 in Ser G 1 u Pro G 1 u As in 1 30 35 1 4 0 Pro Ser Le u G n G 1 u Thir Ser L. eu Se r V a 1 G n Le u Ser As in Le u G 1 y 45 1 s 0 55 6 Thr Val Arg Th r Le u Arg T hr Lys G 1 in Arg I I e G 1 in Pro G 1 in Lys Thr 65 1 1 75 Ser Wall Tyr I 1 e G 1 u L. eu G 1 y Ser Asp Ser Ser G u A s p Thr Val As in 1 80 85 90 Lys A 1 a Thr Ty r Cys Ser V a 1 G1 y A s p G 1 in G 1 u Le u Le u G 1 in I e Thr 95 20 0 205 Pro G 1 in G y Thr Arg A s p G 1 u I e Sier Le u A s p Ser A a Lys Lys A 1 a 2 1 0 2 15 2, 20 A a Cys G 1 u Ph e Sier G 1 u Th r A s p Wall Thr As in Thr G 1 u H is His G 1 a 225 23 O 23 5 24 O Pro Ser As in As in A s p L. eu As in Th r Thr G 1 u Lys Arg A 1 a. A 1 a G 1 u Arg 2 SO 255 H is Pro G 1 u Lys Tyr G | n G 1 y Ser Ser V a 1 Ser As n Le u H is V a 1 G1 u 2 60 2 65 27 O Pro Cys G 1 y Thr As in Th r H is Al a Ser Ser Le u G 1 in H is G i u As n Sle r 2 75 28 0 285 Ser L. eu Le u L. eu. Thir Lys A s p Arg Mie t As in Wall G 1 u Lys A 1 a G 1 u Ph e 290 295 3 00 Cys As in Lys Ser Lys G 1 in Pro G i y Le u A 1 a. A rig Ser G 1 in H is As in Arg 3 O 5 3 1 0 3 15 320 5,654,155

-continued Trip A 1 a G 1 y Ser Lys G 1 u Thr Cy S. As in A s p A rig Arg Thr Pro Ser Thr 3 25 3 3 O 3 35 Glu Lys Lys V a 1 A s p L. eu As in A 1 a. A s p Pro Le u Cy s G 1 u A rig Lys G 1 u 3 - 0 3 4 5 35 0. T rp As n Lys G 1 in Lys Le u Pro Cy s Ser G 1 u As in Pro Arg A s p Thr G 1 u 3 S 5 3 60 3 55 A s p Val Pro Trip I 1 e Thr L. eu As n Sle r S e r I 1 e G l n Lys V a 1. As in G 1 u 3 7 O 3 75 3 8 O Trip Ph e S e r A rig S e r A s p G i u I e u L. eu G 1 y S e r A s p A s p Ser H is As p 3. 85 3 9 O 3 95 40 G l y Glu Se r G 1 u Ser As in A 1 a Lys Wa 1. A 1 a. A s p Wall L. eu A s p V a 1 Le u 4 O 5 4 1 O 4 15 As in G 1 u V a 1 A s p G 1 u Ty I Ser G 1 y Ser Ser G 1 u Lys I e A s p Le u Le u 2 O 4, 25 43 O. A 1 a Se r A s p Pro His G 1 u A 1 a Le u I 1 e Cy s Lys Ser G 1 u A rig V a 1 H is 35 4 4 4 45 S e r Lys Ser Val G 1 u Se r A is in I e Gi u A s p Lys I le P he G 1 y Lys Thr 45 O 4 55 4. 6 O Ty r A rig Lys Ly is . A la Ser Le u Pro A. s in L. eu Ser H is Wa i Th r G i u As in 4 6 5 a 7 O 4 75 48 0 Le u I e I le G 1 y A 1 a P he V a 1 T hr G 1 u Pro G | n I le I 1 e G 1 in G 1 u Arg 4 85 4, 9 O 495 Pro Le u Tiar As n Lys Le u Lys Arg Lys Arg Arg Pro Thr er G 1 y Le u. 5 0 0 5 O 5 5 O His Pro G 1 u A s p Ph e i 1 e Lys Lys Al a A s p Le u A 1 a Wa i G 1 in Lys Thr 5 15 52 O 5 25

Pro Glu Me t 1 e As in G 1 in G y Thr As in G 1 in Thr G 1 u G 1 n As in G y G 1 in 53 0 5 35 5 4 O Wal Me t As n I 1 e Thr As in Ser G 1 y H is G 1 u As in Lys Thr Lys G 1 y As p 5 4 S 5 5 O 555 5 6 O Se r I le G 1 in As n G 1 u Lys As in Pro As in Pro I 1 e G 1 u Ser Le u G 1 u Lys 5 65 5 7 O 575 G 1 u Ser Al a Ph e Lys Thr Lys A1 a G 1 u Pro I 1 e Sier Ser Ser I 1 e Sier 58 0 5 85 59 0 As in Met G i u Le u G i u L. eu As n I le H is As in Ser Lys A 1 a Pro Lys Lys is 95 60 0 6 05 As in A rig L. eu A rig A rig Lys Ser Ser Thr Arg His I le H is A 1 a L. eu G 1 u 6 1 0 6 15 62. O Le u Val V a l Ser Arg As in Le u Se r Pro Pro As n Cys Thr G 1 u Le u G 1 in 625 6 3 O 6 35 6 4. O I e A s p Ser Cy s Ser Ser Ser G 1 u G 1 u I 1 e Lys Lys Lys Lys Ty r A s in 6 4.5 5 O 555 G 1 in Me t Pro V a 1 Arg H is Ser Arg As in Le u G 1 n Le u Me t G 1 u Gi y Lys 6 60 6 65 6 0 G 1 u Pro A 1 a Thr G 1 y A 1 a Lys Lys Ser As in Lys Pro As in G 1 u G 1 in Thr 6 5 68 0 685 Ser Lys Arg H is Asp Ser A s p Thr Phe Pro G 1 u L. eu Lys Le u Thir As in 6 9 O 695 70 0 A 1 a Pro G 1 y Ser Phe Thr Lys Cys Ser As in Thr Ser G 1 u Le u Lys G 1 u 7 O 5 7 7 15 7 20 Ph e V a 1 As n Pro Ser Le u Pro Arg G 1 u G 1 u Lys G 1 u G 1 u Lys Le u G 1 u 7 25 73 0 7 35

Th T V a 1 Lys Val Ser As in As in A a G 1 u A s p Pro Lys A s p Le u Me t Le u 74 0 7 45 7 5 O 5,654,155 29 30 -continued

Ser G y G 1 u A rig V a 1 Le u G 1 in Thr G 1 u Arg Ser V a 1 G 1 u Ser Ser Ser 7 55 7 60 7 6 5 I 1 et Ser Le u Wa Pro G 1 y Th r A s p Tyr G 1 y Thr G 1 in G 1 u Se r I 1 e Ser 7 70 7 7 5 7 80 Le u Le u G 1 u Wa Ser Thr Le u G 1 y Lys A 1 a Lys Thr G 1 u Pro As in Lys 785 79 0 7 95 800 Cys V a 1 Ser G 1 in Cys A 1 a. A 1 a Phe G 1 u As n Pro Lys G 1 y Le u I le H is 8 O 5 8 0 8 5 G y Cys Ser Lys A s p As in Arg As in A s p Thr G 1 u G y P he Lys Ty r P ro 8 2 O 8 25 8 3 O Le u G 1 y H is G 1 u V a 1. As n H is Ser Arg G 1 u T h r S e I I I e G 1 u Me t G 1 u 83 5 8 4 O 8. A 5 G 1 u Ser G i u Le u A s p A a G 1 in Tyr Le u G 1 in As in Th r P he Lys Wal Ser 8 5 O 855 8 60 Lys Arg G 1 in Ser Ph e A a L. eu Ph e Ser As in Pro G 1 y As in A a G 1 u G 1 u 8 65 8 0. 8 75 8 80 G 1 u Cys A 1 a Thr Ph e Ser A 1 a His Ser G 1 y Ser L. eu Lys Lys G 1 in Ser 8 85 89 0 895 Pro Lys V a 1 T hr P he G 1 u Cy s G i u G n Lys G 1 u G 1 u As in G 1 in G 1 y Lys 9 OO 9 05 9 1 0. As in G 1 u Ser As in I 1 e Lys Pro Wa G 1 in Th T V a 1. As n I I e Thr A a G 1 y 9 5 92 0 9 2 5 Phe Pro V a 1 W. a 1 G1 y G 1 in Lys As p Lys Pro Wa 1 A s p As in A1 a Lys Cys 9 30 9 35 9 4 0 Ser I 1 e Lys G 1 y G 1 y Ser Arg Phe Cys Leu Se r S e r G 1 in Ph e Arg G y 9 45 950 955 9 60 As in G 1 u Thr G 1 y Le u e Thr Pro As in Lys His G y Le u Le u G 1 in As in 9 65 9 70 9 75 Pro Ty r A rig E 1 e Pro Pro Le u Ph e Pro I e Lys Ser P he V a 1 Lys Thr 8 9 85 99 O Lys Cy s Lys Lys As n Le u Le u G 1 u G 1 u As in P he G 1 u G 1 u H is Ser Me t 995 O O O 1 O O 5 Ser Pro G 1 u Arg G 1 u Me t G 1 y As in G 1 u As n I i e Pro Ser Thr V a 1 Ser 1 0 1 0 0 15 1 O 20 Thr I le Ser Arg As in As n I 1 e Arg G 1 u As n V a 1 P he Lys G 1 y A la Ser 1 O 25 1 0 3 0 1 0 35 0 40 Ser Ser As n I le As in G i u Wa 1 G1 y Ser Ser Th I. As n G 1 u Val G 1 y Ser O 45 1 O 50 05 5 Se r I 1 e As in G 1 u I le G y Ser Ser A s p G 1 u As n I e G 1 in A1 a G 1 u L. eu 1 O 6 O 1 O 65 O 70 G y Arg As in Arg G 1 y Pro Lys L. eu. As in A i a Me t L. eu. A rig L. eu G 1 y Val 1 0 75 1 08 0. 1 0 85 Le u G 1 in Pro G 1 u Val Tyr Lys G 1 in Ser Le u Pro G 1 y Se As in Cys Lys O 9 0 1 0 95 1 1 0 O H is Pro G 1 u I 1 e Lys Lys G 1 in G 1 u Tyr G 1 u G 1 u v a 1 V a 1 G 1 in Thr V a 1 1 1 O 5 1 1 1 0. 1 1 S 20 As in Th r A s p Ph e Sier Pro Ty r Le u I 1 e Ser A s p As in L. eu G 1 u G 1 in Pro 1 2 5 1 13 O 1 35 Me t G y Ser Ser H is A 1 a Ser G 1 in V a 1 Cys Ser G i u Thr Pro A s p As p 1 4 0 45 1 150 Le u Le u A s p A s p G 1 y G i u I e Lys G 1 u A s p Th r S er Phe A a G 1 u As in 1 55 1 60 1, 1 65 A s p I le Lys G 1 u Se r S e r A 1 a V a 1 P h e Ser Lys Ser Val G 1 in Arg G y 5,654,155

-continued

1 1 7 O 175 1 1 80 G 1 u L. eu Ser Arg Ser Pro Ser Pro Phe T hr H is Thr His Le u A 1 a G 1 in 1 1 85 90 95 1 20 O G 1 y Ty r A rig Arg G 1 y Al a Lys Lys Le u G 1 u Se r S e r G 1 u G 1 u As n Le u 12 O 5 1 2 1 0 12 15 Ser Ser G 1 u A s p G 1 u G 1 u Le u Pro Cy s P he G 1 in H is Le u Le u Ph e G 1 y 22 O 1 225 23 O Lys V a 1 As n As n I 1 e Pro Ser G in Ser Thr Arg His Ser Thr V a 1. A a 1 2 3 5 1 2 4 0 2 4 5 Thr G i u Cy s L. eu Ser Lys As in Thr G 1 u G 1 u As a Le u Le u Ser Le u Lys 1 2 50 255 1 2 6 O

As in Ser Le u. As in A s p Cy s Ser As in G 1 in V a I le Le u A la Lys A1 a Ser 2 65 2 7 O 1 2 is 28 O G 1 in G 1 u H is H is L. eu Ser G 1 u G 1 u Tar Lys Cy s Ser A la Ser Le u P he 1 285 1 90 2 9 5 Ser Ser G 1 in Cys Ser G 1 u Le u G 1 u A s p Le u Thir A 1 a. As in Thr As n Thr 3 00 13 O 5 3 1. O G 1 in A s p Pro P he Le u I le G 1 y Ser Ser Lys G in Me t Arg His G 1 in Ser 1. 3 15 1. 3 2 O 3 2 5 G 1 u Ser G 1 in G y V a 1 G1 y L. eu Se r A s p Lys G 1 u Le u V a 1 Ser A s p As p 1 33 0 13 3 5 1. 3 4 O Glu G i u A rig G 1 y Thr G 1 y Le u G 1 u G 1 u As in As in G 1 in G 1 u G 1 u G 1 in Ser 3 4 5 1. 3 5 O 355 13 6 O Me t A s p Ser As n Le u G 1 y G. I u. A a A la Ser G i y Cys G 1 u Ser G 1 u Thr 13 6 is 13 7 O 1. 3 75 Ser V a Ser G 1 u A s p Cy s Ser G y L. eu Ser Ser G 1 in Ser A s p I 1 e Le u 1. 3 8 O 3 85 13 9 Th I Thr G in G 1 in Arg A s p Thr Me t G 1 in H is As in Le u I e Lys Le u G 1 in 395 4 0 0 1 4 0 5 G l n G 1 u Me t A 1 a G Iu Le u G 1 u A 1 a V a l L. eu G 1 u G 1 in H is G 1 y Ser G 1 n 1 4 1 0 1 4 15 1 4 20 Pro Ser As n Ser Ty r Pro Se r I 1 e I 1 e Ser Asp Ser Ser A1 a Le u G 1 u 1 4, 25 1 4 3 0 1 4 35 1 4 4 O A s p L. eu A rig As in Pro G 1 u G 1 n Ser Thr Ser G 1 u Lys A 1 a V a 1 Le u Thr 1 4 45 1 4 50 1 4 55 Ser G 1 in Lys Ser Ser G 1 u Tyr Pro I 1 e Ser G 1 in As n Pro G 1 u G 1 y Le u 4 6 O 1 4 6 5 1 4 S e r A 1 a. A s p Lys P he G i u Val Ser A 1 a. A s p Ser Ser T h r S e r Lys As in 1 4 5 1. 4- 8 0 1 4 35 Ly s G 1 u Pro G 1 y V a 1 G 1 u Arg Ser Ser Pro Ser Lys Cy s Pro Ser Le u 4 90 1 495 50 0 A s p A s p Arg Tr p Tyr Me t H is Ser Cys Ser G 1 y Ser Le u G 1 in As n Arg 15 O 5 1 5 1 O 1 5 5 5 20 As n Ty I Pro Ser G 1 in G i u G 1 u Le u I 1 e Lys Val V a 1 A s p V a 1 G 1 u G 1 u 15 2S 53 0. 1 5 35 G 1 in G 1 in Le u G 1 u G 1 u Ser G i y Pro His A s p Le u Thr G 1 u Thir Ser Tyr 5 4 O 1 5 45 550 Le u Pro Arg G in A s p Le u G 1 u G l y Thr Pro Ty r Le u G 1 u Ser G 1 y I 1 e 555 15 60 15 65 Ser Le u Ph e Ser A s p A s p Pro G 1 u Se r A s p Pro Ser G 1 u A s p Arg A 1 a 1 5 70 1 5 75 158 0 Pro G 1 u Se r A la Arg Wa 1 G i y As n I 1 e Pro Se r S e T Thir Ser A1 a Le u 585 5 9 O 595 1 60 0 5,654,155 33 34 -continued Ly is V a 1 Pro G | n Le u Lys V a 1 A 1 a G 1 u Se r A a G 1 in G y Pro A 1 a A 1 a 6 O 5 1 6 1 0 1 6 1 5

A a His Thr Thr Asp Thr A a G 1 y Tyr As in A 1 a Me t G i u G 1 u Sle r Wa 1 620 625 1 630 Ser Arg G 1 u Lys Pro G i u L. eu Thir A 1 a Ser Thr G 1 u Arg Wa I. As n Lys 635 1 6 4 O 6 45

A rig Me t Ser Me t Val V a 1 Ser G 1 y L. eu Thr Pro G 1 u G 1 u Ph e Me t L. eu 65 0 65 s 6 6 O

Wa 1 Ty r Lys Phe A a A rig Lys His His E 1 e i hr L. eu Th As in Le u I e 1 6 65 6 7 O 675 1 68 O

Thr G 1 u G 1 u Thir Thr H is V a 1 V a 1 Me t Lys Th r A s p A a G 1 u P he Wa 1 685 1 5 9 O 1 695 Cy s G 1 u Arg Th r Le u Lys Ty r P he Le u G 1 y I 1 e A 1 a G 1 y G 1 y Lys T r p 1 0 0 7 O 5 1 7 10

Wa 1 Wa 1 Ser Tyr Phe Trip V a l Thr G 1 in Ser I 1 e Lys G 1 u Arg Lys Me t 1 7 5 1 20 1 725 Le u As in G 1 u H is A s p P he G 1 u Va. 1 Arg G 1 y A s p V a V a As in G y Arg 73 0. 7 35 4 O

As in H is G 1 in G 1 y Pro Lys Arg A a Arg G 1 u Ser G 1 in A s p A rig Lys I 1 e 1745 1 750 755 1 7 6 O

Ph e Arg G 1 y Le u G 1 u I 1 e Cy s Cy s Tyr G 1 y Pro Phe Thr As a Me t P ro 7 6 5 1 7 0. 1775

Thr As p G n Le u G 1 u Trip Me t w a 1 G1 in Le u Cy s G 1 y A a Ser V a 1 Wa 1 8 O 785 1790

Ly is G 1 u Le u Ser Ser Phe Thr Le u G 1 y Thr G 1 y Val H is Pro I 1 e Wa 1795 1 800 8 O 5

Wa 1 Wa 1 G 1 in Pro A s p A a Trip Thr G 1 u A s p As in G 1 y Ph e H is A 1 a e 8 1 0 18 15 1. 82 0.

G y G 1 in Met Cy s G 1 u A a Pro V a Wa Thr Arg G 1 u Trip V a 1 Le u As p 825 1 83 0 1 83 5 1 8 40

Ser Wa A 1 a Le u Tyr G in Cys G 1 in G 1 u L. eu A s p Thr Ty r Le u I le P ro 1845 85 0 855 G 1 n e Pro H is Ser H is Ty r 8 6. 0

( 2) INFORMATION FOR SEQID NO:3: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE; mucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B ) STRAIN: 2F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:3:

GAA GTT GT CA TTTTATAAA C CTTT 2 4

( 2) INFORMATION FOR SEQID NO:4: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) 5,654,155 35 36 -continued ( v i ) ORIGINAL SOURCE: ( B STRAIN: 2R primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:4:

T GT CTTTT CT T C ccT AG TAT GT 22

( 2) INFORMATION FOR SEQID NO:5; ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B) STRAIN: 3F primer ( xi ) SEQUENCE DESCRIPTION: SEQID No.5:

CCT GACACA G CAGA CATT. A 2 1

( 2) INFORMATION FOR SEQID NO:6: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 21 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 3R primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:6:

TT G GATTTTC GTTCT CAC TT A 2

( 2) INFORMATION FOR SEQID NO:7: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 5F primer ( x i ). SEQUENCE DESCRIPTION: SEQID No.:7:

CT CTAAG G G CA GT G T GAG 20

( 2) INFORMATION FOR SEQID NO:8: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B ) STRAIN:5R-M13* primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:8:

TT CCTACT G T G G T G CTTCC 2 O 5,654,155 37 38 -continued ( 2 INFORMATION FOR SEQID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 67F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:9: CTTATTTTAG T G T C CTTAAA AG G 23

( 2) INFORMATION FOR SEQID No:10: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 6R ( xi ) SEQUENCE DESCRIPTION: SEQID NO:10:

TT CAT G GAC A G CA CTTGA G T G 22

( 2) INFORMATION FOR SEQID No:11: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 7F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:11:

CA CAA CAAAG AG CATA CATA GGG 2 3

( 2) INFORMATION FOR SEQID No:12: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B) STRAIN: 67R primer ( xi ) SEQUENCE DESCRIPTION: SEQID No:12:

TCGGGTT CAC T C T G TAGAAG 20

( 2) INFORMATION FOR SEQID No:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear 5,654,155 39 40 -continued ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 8F1 primer ( x i SEQUENCE DESCRIPTION: SEQID No:13:

TT C T CTT CAG GAG (GAAAAGC A 2 1

( 2) INFORMATION FOR SEQID No:14: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B ) STRAIN: 8R1 primer ( xi ) SEQUENCE DESCRIPTION: SEQID No:14:

GCT GCC TAC C A CAAATA CAA A 2 1

( 2) INFORMATION FOR SEQID No:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B ) STRAIN: 9F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:15:

C CA CAG TAGA T G C T CAG TAA ATA 2 3

( 2) INFORMATION FOR SEQID No:16: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 9R primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:16:

TAGGAAAATA C C A G CTT CAT AGA 2 3

( 2) INFORMATION FOR SEQID NO:17: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B, ) STRAIN: 10F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:17:

T G GT CAG CTT T. CT GTAATCG 2 O 5,654,155 41 42 -continued

(2) INFORMATION FOR SEQID No:18: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 10R primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:18:

GAT CTA C C C A CTCTCTCT CAG 2 4

( 2) INFORMATION FOR SEQID No:19: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11AF primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:19:

C CAC CTCCAA G G T G TAT CA 19

(2) INFORMATION FOR SEQID NO:20: ( i) SEQUENCE CHARACTERISTICS: (A) LENGH: 20 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11AR primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:20:

GT TAT G T T G GCTC CTTG CT 20

( 2) INFORMATION FOR SEQID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B, ) STRAIN: 11BF1 primer ( x i ). SEQUENCE DESCRIPTION: SEQID NO:21:

CA CTA AAG AC A GAATGAAC TA 22

( 2) INFORMATION FOR SEQID NO:22: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant 5,654,155 43 -continued (D) TOPOLOGY: Finear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B ) STRAIN: 11BR1 primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:22:

GAA GAA C CAG AAA TT CATC A 22

( 2) INFORMATION FOR SEQID NO:23: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant ( D.) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11CF1 primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:23:

T GAT GGG GAG T C T GAAT CAA 20

( 2) INFORMATION FOR SEQID NO:24: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Einear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B, STRAIN: 11CR1 primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:24:

T C T G CTT T. CT T GATAAAATC CT 22

( 2) INFORMATION FOR SEQID NO:25: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: iiDF1 primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:25:

AGC GT C C C C T CACAAATAAA 2 O

( 2) INFORMATION FOR SEQID NO:26: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: Not Relevant ( D.) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11DR1 primer ( x i SEQUENCE DESCRIPTION: SEQID NO:26: 5,654,155 45 -continued

TCA A G C G CAT GAA. TAT G. C. CT 20

(2) INFORMATION FOR SEQID NO:27: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant ( D TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11EF primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:27: GTATA A G CAA TAT G GAA CTC GA 22

( 2) INFORMATION FOR SEQID NO:28: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11ER primer ( x i ). SEQUENCE DESCRIPTION: SEQID NO:28: TTAAGTT CACT G GTATTT GAA CA 2 3

( 2) INFORMATION FOR SEQID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11FF primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:29: GA CAG CGATA CT T C C CAGA 20

( 2) INFORMATION FOR SEQID NO:30: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11FR primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:30:

TG GAA CAA, CC AT GAA TA GT C 21

( 2) INFORMATION FOR SEQID NO:31: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs 5,654,155 47 48 -continued (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11GF primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:31:

G GAA GTE AGC ACC TAG G GA 20

( 2) INFORMATION FOR SEQID NO:32: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant ( D TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11GR primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:32:

GCA GT GATAT TAA CT G T C T G T A 22

( 2) INFORMATION FOR SEQID NO:33: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11HF primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:33:

TGG GT CCTTA AAGAAACAAA GT 22

( 2) INFORMATION FOR SEQID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11HR primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:34:

T CAG G T GACA TT GAA. T. CTTC C 2

( 2) INFORMATION FOR SEQID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11IF primer 5,654,155 49 50 -continued

( xi ) SEQUENCE DESCRIPTION: SEQID NO:35:

C CA CTTTTT C C CAT CAA GT C A 2, 1

( 2 INFORMATION FOR SEQID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11IR primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:36:

T CAG GAT G. CT TACAATTACT C 22

( 2) INFORMATION FOR SEQID NO:37: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11JF primer ( xi ) SEQUENCEDESCRIPTION: SEQID NO:37;

CAAAATT GAA T G CTAT G. CTT AGA 2 3

( 2) INFORMATION FOR SEQID NO:38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B) STRAIN: 11JR primer ( x i ) SEQUENCE DESCRIPTION: SEQID NO:38:

TCG GAA C C C T GAGCCAAAT 2 O

( 2) INFORMATION FOR SEQID NO:39: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: mucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11KF primer ( xi ) SEQUENCE DESCRIPTION: SEQED NO:39:

GCAA AA G CGT C CAGAAAG GA 2 0

( 2) INFORMATION FOR SEQID No:40: 5,654,155 S1 52 -continued ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 11KR-1 primer ( xi ) SEQUENCE DESCRIPTION: SEQID No:40:

TATTT G CA GT CAA GT CTT C C AA 22

( 2) INFORMATION FOR SEQID NO:41: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B) STRAIN: 11LF-1 primer ( x i ) SEQUENCE DESCRIPTION: SEQID NO:41:

GT AATA T G G CAAA. G. G. CAT C T 2

( 2) INFORMATION FOR SEQID NO:42: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B) STRAIN: 11LR primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:42:

TAAAA T G T G C T C C C CAAAA G CA 22

( 2) INFORMATION FOR SEQID NO:43: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant ( D.) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B, ) STRAIN: 12F primer ( x i ). SEQUENCE DESCRIPTION: SEQID NO:43:

GT C C T G CCAA T G A GAAGAAA 2 O

( 2) INFORMATION FOR SEQED NO:44: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 21 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) 5,654,155 53 54 -continued ( v i ) ORIGINAL SOURCE: ( B ) STRAIN: 12R primer ( xi ) SEQUENCE DESCRIPTION: SEQED NO:44:

T GT CAG CAAA C CTA A GAA T G 2

( 2) INFORMATION FOR SEQID NO:45: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (c) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B, ) STRAIN: 13F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:45:

AA T G GAA AGC CT CAAA GT A 2 1

( 2) INFORMATION FOR SEQID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 13R primar ( xi ) SEQUENCE DESCRIPTION: SEQID NO:46:

A T G TT G GA. G. C. A. G. G. C. CTTA. C. 2

( 2 INFORMATION FOR SEQID NO:47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B) STRAIN: 14F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:47:

CTA ACC GAA TAT CACTA CA 22

( 2) INFORMATION FOR SEQID NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 14R primer ( x i ) SEQUENCE DESCRIPTION: SEQID NO:48:

G GTATAAA T G CCT G T A GC A 2 5,654,155 SS 56 -continued ( 2) INFORMATION FOR SEQID NO:49: ( i) SEQUENCE CHARACTERISTICs: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 15F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:49:

T G G CT G C C C A G GAA GT ATG 19

( 2) INFORMATION FOR SEQID NO:50: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B) STRAIN: 15R primer ( xi ) SEQUENCE DESCRIPTION: SEQID No:50:

AA C C A GAATA TCTTTA T G T A G GA 2 3

( 2) INFORMATION FOR SEQID NO:51: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 16F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:51:

AATT CTTAAC AGAGA C CAGA AC 22

( 2) INFORMATION FOR SEQID NO:52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 16R primer ( xi ) SEQUENCE DESCRIPTION: SEQID No:52:

AAAA CT CTTT C C A GAA T G TT GT 22

( 2) INFORMATION FOR SEQID No.53: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear 5,654,155 57 58 -continued ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 17F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:53:

G T G T A GAA CG G CAG GATT G 20

( 2) INFORMATION FOR SEQID NO:54: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 17R primer ( x i ) SEQUENCE DESCRIPTION: SEQID NO:54:

C GC CT CAT G T G GTTTA 8

( 2) INFORMATION FOR SEQID No.55: ( i.) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B ) STRAIN: 18F primer ( x i ) SEQUENCE DESCRIPTION: SEQID No.55:

G. G. CT CTA G CTTCTTAGGA C 21

(2) INFORMATION FOR SEQID NO:56: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (BSTRAIN: 18R primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:56:

GAGA C CATT T C C C A G CATC 2 O

( 2) INFORMATION FOR SEQED NO:57: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 19F primer ( xi ) SEQUENCE DESCRIPTION: SEQID No:57:

CT GT CATT CT T C CT G T G CTC 2 0. 5,654,155 59 60 -continued

( 2) INFORMATION FOR SEQID NO:58: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B) STRAIN: 19R primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:58:

CAT G T TAAG GAAA G T G G T G C 2 1

( 2) INFORMATION FOR SEQID NO:59: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 20F primer ( x i SEQUENCE DESCRIPTION: SEQID NO:59:

AA GAC GT G T C T G C T C CAC 20

( 2) INFORMATION FOR SEQID NO:60: (i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 20R primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:60:

GG GAA C CAA ATTACA CA. GC 20

( 2) INFORMATION FOR SEQID NO:61: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 21F primer ( xi ) SEQUENCE DESCRIPTION: SEQID No.61:

AACS C T C T C C TTTTT GAAA G TC 22

( 2) INFORMATION FOR SEQID NO:62: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH; 22 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant 5,654,155 61 62 -continued (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 21R primer ( xi ) SEQUENCE DESCRIPTION: SEQID No.62:

GT AGAGAAA. A GAAT AG CCT CT 22

( 2) INFORMATION FOR SEQID NO:63: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 22F primer ( xi ) SEQUENCE DESCRIPTION: SEQID No.63:

T C C CAT GAG AG GT C T G CT 20

( 2) INFORMATION FOR SEQID NO:64: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Einear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 22R primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:64: GA GAAG ACT CT GA. GGCTAC 20

( 2) INFORMATION FOR SEQID NO:65: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B, ) STRAIN: 23F-1 primer ( x i SEQUENCE DESCRIPTION: SEQID NO:65:

T GAA GT GACA GTTC CAGAG. T 21

( 2) INFORMATION FOR SEQID No.66: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 23R-1 primer ( xi ) SEQUENCE DESCRIPTION: SEQID No.66: 5,654,155 63 -continued

CAT TT TA GCC AT CAT CAA CAA 2 3

( 2) INFORMATION FOR SEQID NO:67: ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant ( D.) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: ( B) STRAIN; 24F primer ( xi ) SEQUENCE DESCRIPTION: SEQID NO:67:

A T GAATTGAC ACTA A C C T G C 22

(2) INFORMATION FOR SEQID NO:68: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: DNA (genomic) ( v i ) ORIGINAL SOURCE: (B) STRAIN: 24R primer ( x i ) SEQUENCE DESCRIPTION: SEQID NO:68:

GT AGCCA G GA. CAG TAGAA. G. G. A 2 1

( 2) INFORMATION FOR SEQID No.69: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant ( i i MOLECULETYPE: DNA (genomic) ( x i ) SEQUENCE DESCRIPTION: SEQID NO:69:

G CA.A.A.A. GC GT C CAGAAAG GA 20

( 2) INFORMATION FOR SEQID No.:70: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant ( i i ) MOLECULE TYPE: DNA (genomic) ( xi ) SEQUENCE DESCRIPTION: SEQID NO:70:

A GT CTT C CAA TT CAC T G CAC 20

( 2) INFORMATION FOR SEQID NO:71: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant ( i i ) MOLECULETYPE; DNA (genomic) 5,654,155 65 -continued ( xi ) SEQUENCE DESCRIPTION: SEQID No.:71:

GAA CA CA. G. G.A. G.A.A.T 1 4

( 2) INFORMATION FOR SEQID NO:72: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant ( D.) TOPOLOGY: Not Relevant ( i i ) MOLECULETYPE: DNA (genomic) ( xi ) SEQUENCE DESCRIPTION: SEQED NO:72:

TAAGAA CACA G GAG 4

(2) INFORMATION FOR SEQID NO:73: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 16 base pairs (B) TYPE: nucleic acid (c) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant ( i i ) MOLECULETYPE: DNA (genomic) ( xi ) SEQUENCE DESCRIPTION: SEQID NO:73:

GAA CACA GAG GAGAAT 1 6

( 2) INFORMATION FOR SEQID NO:74: (i) SEQUENCE CHARACTERISIICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant ( i i ) MOLECULE TYPE: DNA (genomic) ( xi ) SEQUENCE DESCRIPTION: SEQID NO:74:

TAAGAA CACA GAG GAG 1 6

We claim: TCT and TCC at position 4427, 1. An isolated consensus DNA sequence of the BRCA1 and ACT and GGT at position 4956; coding sequence as set forth in SEQID NO: 1. 45 f) determining any sequence differences between said 2. A method of identifying individuals having a BRCA1 individual's BRCA1 coding sequences and SEQ. ID. gene with a BRCA1 coding sequence not associated with NO: 1 wherein the presence of any of the said poly breast or ovarian cancer comprising: morphic variations and the absence of a polymorphism a) amplifying a DNA fragment of an individual's BRCA1 outside of positions 2201, 2430, 2731, 3232, 3667, coding sequence using an oligonucleotide primer 50 4427, and 4956, is correlated with an absence of which specifically hybridizes to sequences within the increased genetic susceptibility to breast or ovarian cancer resulting from a BRCA1 mutation in the gene; BRCA1 coding sequence. b) sequencing said amplified fragment by dideoxy 3. A method according to claim 2 wherein said oligo sequencing; 55 nucleotide primer is labeled with a radiolabel, a fluorescent c) repeating steps (a) and (b) until said individual's label, a bioluminescentlabel, a chemiluminescent label oran BRCA1 coding sequence is completely sequenced; enzyme label. d) comparing the sequence of said amplified DNA to the 4. A method of detecting an increased genetic suscepti sequence of SEQ. ID. NO: 1; bility to breast and ovarian cancer in an individual resulting e) determining the presence or absence of each of the 60 from the presence of a mutation in the BRCA1 coding following polymorphic variations in said individual's sequence, comprising: BRCA1 coding sequence: a) amplifying a DNA fragment of an individual's BRCA1 AGC and ACT at position 2201, coding sequence using an oligonucleotide primer TTG and CTG at position 2430, which specifically hybridizes to sequences within the CCG and CTG at position 2731, 65 gene; GAA and GGA at position 3232, b) sequencing said amplified fragment by dideoxy AAA and AGA at position 3667, sequencing; 5,654,155 67 68 c) repeating steps (a) and (b) until said individual's TTG or CTG at position 2430, BRCA1 coding sequence is completely sequenced; CCG or CTG at position 2731, d) comparing the sequence of said amplified DNA to the GAA or GGA at position 3232, sequence of SEQ. ID. NO: 1; AAA or AGA at position 3667, e) determining any sequence differences between said TCT or TCC at position 4427, individual's BRCA1 coding sequences and SEQ. D. and AGT or GGT at position 4956; NO: 1 to determine the presence or absence of poly- is correlated with the potential of increased genetic sus morphisms in said individual's BRCA coding ceptibility to breast or ovarian cancer resulting from a sequences wherein a polymorphism which is not any of 10 BRCA1 mutation in the BRCA1 coding sequence. the following: AGC or AGT at position 2201, :* : * : ::

UNITED STATES PATENT ANDTRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,654,155 DATED : August 5, 1997 INVENTOR(S) : Brenda S. Critz, et al. it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: Column 15, line 34, replace "bellow" with --below--; Column 18, line 57, replace "Baudet" with --Beaudet--; Column 20, line 5, replace "Baudet" with --Beaudet--;

Signed and Sealed this Twenty-eighth Day of April, 1998 (a teen

BRUCELEMAN Attesting Officer Commissioner of Patents and Trademarks UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,654,155 Page 1 of 1 APPLICATIONNO. : 08/598591 DATED : August 5, 1997 INVENTOR(S) : Patricia D. Murphy et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 66, line 44 (Claim 2) - please replace “ACT with --AGT--

Signed and Sealed this Thirtieth Day of January, 2007 WDJ

JON. W. DUDAS Director of the United States Patent and Trademark Office

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,654,155 Page 1 of 1 APPLICATIONNO. : 08/598591 DATED : August 5, 1997 INVENTOR(S) : Patricia D. Murphy et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 65, line 63 (Claim 2) - please replace “ACT with --AGT--

Signed and Sealed this Twenty-eighth Day of October, 2008 WDJ

JON. W. DUDAS Director of the United States Patent and Trademark Office