WO2007100911A2 - Detecting fetal chromosomal abnormalities using tandem single nucleotide polymorphisms - Google Patents

Detecting fetal chromosomal abnormalities using tandem single nucleotide polymorphisms Download PDF

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Publication number
WO2007100911A2
WO2007100911A2 PCT/US2007/005399 US2007005399W WO2007100911A2 WO 2007100911 A2 WO2007100911 A2 WO 2007100911A2 US 2007005399 W US2007005399 W US 2007005399W WO 2007100911 A2 WO2007100911 A2 WO 2007100911A2
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seq
compl
sequence
single nucleotide
pair
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PCT/US2007/005399
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French (fr)
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WO2007100911A3 (en
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Aoy Tomita Mitchell
Michael Mitchell
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University Of Louisville Research Foundation
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Priority to EP07752121A priority Critical patent/EP1996728B1/en
Priority to AU2007220991A priority patent/AU2007220991C1/en
Priority to CA2647793A priority patent/CA2647793C/en
Priority to DK07752121.9T priority patent/DK1996728T3/en
Priority to DE602007014335T priority patent/DE602007014335D1/en
Priority to AT07752121T priority patent/ATE508209T1/en
Priority to SI200730667T priority patent/SI1996728T1/en
Publication of WO2007100911A2 publication Critical patent/WO2007100911A2/en
Publication of WO2007100911A3 publication Critical patent/WO2007100911A3/en
Priority to HK09104895.9A priority patent/HK1126254A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • certain embodiments of the present invention provide a method for determining whether a fetus has at least one chromosomal abnormality, comprising using tandem single nucleotide polymorphisms to compare fetal DNA to maternal DNA so as to determine whether the fetus has at least one chromosomal abnormality.
  • Figure 1 depicts an example of a tandem SNP.
  • Figure 2 depicts a DNA melting map of a constant denaturant capillary electrophoresis target sequence covering a tandem SNP.
  • Figure 3 depicts an example of a constant denaturant capillary electrophoresis electropherogram output.
  • Figure 3A depicts results from a sample from a maternal buccal swab.
  • Figure 3B depicts results from a sample from maternal serum.
  • Figure 3C depicts results from a sample from maternal serum.
  • fetal cells in maternal blood might be used to assess the genetic status of a developing embryo.
  • the extremely small amount of fetal cells in maternal blood (about 1 cell per ml) has proven a difficult obstacle to overcome when trying to isolate these cells for widespread clinical testing.
  • cell-free fetal DNA is present in circulating maternal serum at higher percentages than fetal cells and has the potential to be assessed for chromosomal or gene defects.
  • Cell-free fetal DNA can range from 1-47 % of total DNA in maternal blood.
  • a critical limitation that has yet to be successfully overcome is that maternal DNA contamination makes it difficult to differentiate fetal from maternal DNA.
  • tandem single nucleotide polymorphisms to detect chromosomes, e.g., to detect fetal chromosomal abnormalities.
  • the tandem SNPs are combined with a sensitive DNA separation technology, e.g., high-fidelity PCR and constant denaturant capillary electrophoresis (CDCE), to detect fetal chromosomal abnormalities, e.g., through the simple sampling and comparison of maternal DNA to fetal DNA 5 e.g., from maternal serum and maternal buccal swabs.
  • a sensitive DNA separation technology e.g., high-fidelity PCR and constant denaturant capillary electrophoresis (CDCE)
  • CDE constant denaturant capillary electrophoresis
  • certain embodiments of the present invention provide a method for determining whether a fetus has at least one chromosomal abnormality, comprising using tandem single nucleotide polymorphisms to compare fetal DNA to maternal DNA so as to determine whether the fetus has at least one chromosomal abnormality.
  • fetal DNA is obtained from maternal blood. In certain embodiments of the invention, fetal DNA is cell-free fetal DNA. In certain embodiments of the invention, maternal DNA is obtained from a biological sample, e.g., maternal blood, hi certain embodiments of the invention, maternal DNA is obtained from a buccal swab] ' In certain embodiments of the invention, maternal DNA is obtained from a biological sample that does not comprise fetal DNA.
  • fetal DNA is obtained from maternal blood, maternal urine, maternal sweat, maternal cells, or cell free DNA from the mother.
  • the biological sample is biological fluid.
  • the biological sample is a maternal biological sample.
  • samples may be whole blood, bone marrow, blood spots, blood serum, blood plasma, buffy coat preparations, saliva, cerebrospinal fluid, buccal swabs, solid tissues such as skin and hair, body waste products, such as feces and urine.
  • samples may be lysates, homogenates, or partially purified samples of biological materials.
  • biological materials can include crude or partially purified mixtures of nucleic acids.
  • the biological sample is serum, urine, sweat, cells, or cell
  • the comparison step comprises using high-fidelity PCR and constant denaturant capillary electrophoresis to compare the fetal DNA to maternal DNA. In certain embodiments of the invention, the comparison step comprises using at least about 96 tandem single nucleotide polymorphisms.
  • the method further comprises the step of converting the nucleic acid molecules to a homoduplex state, as opposed to being in heteroduplex form. This can be accomplished, e.g., by using an excess of primers and can aid in the tandem SNP analysis.
  • methods such as mutation detection technologies can be used to analyze the tandem SNPs.
  • methods such as denaturing HPLC, denaturing capillary electrophoresis, cycling temperature capillary electrophoresis, allele- specific PCRs, quantitative real time PCR approaches such as TaqMan® PCR system, polony PCR approaches, and microarray approaches can be used to analyze the tandem SNPs.
  • the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 250 basepairs apart.
  • the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 200 basepairs apart. In certain embodiments of the invention, the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 150 basepairs apart. In certain embodiments of the invention, the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 100 basepairs apart. In certain embodiments of the invention, the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 50 basepairs apart.
  • At least one tandem single nucleotide polymorphism is located on the p arm of chromosome 21. In certain embodiments of the invention, at least one tandem single nucleotide polymorphism is located on the q arm of chromosome 21.
  • the chromosomal abnormality is chromosomal aneuploidy. In certain embodiments of the invention, the chromosomal abnormality is trisomy 13, 18 or 21. In certain embodiments of the invention, the chromosomal abnormality is trisomy 21. In certain embodiments of the invention, the chromosomal abnormality is an insertion mutation ⁇ e.g., a large insertion (>3 megabasepair) or small insertion ( ⁇ 3 megabasepair). In certain embodiments of the invention, the chromosomal abnormality is a deletion mutation (e.g., a large deletion (>3 megabasepair) or small deletion ( ⁇ 3 megabasepair)). The deleted region could include a deleted gene.
  • the methods can be used to detect copy number polymorphisms and/or copy number variants in the genome. In certain embodiments of the invention, the methods can be used to detect chromosome 22ql 1 deletion syndrome, which is associated with cardiac defects.
  • Chromosomal abnormalities include deletions associated with genetic syndromes and disorders such as the 22ql 1 deletion syndrome on chromosome 22, which is associated with cardiac defects.
  • Other examples of chromosomal abnormalities include the Hq deletion syndrome on chromosome 11 and 8p deletion syndrome on chromosome 8, both of which are also associated with cardiac defects.
  • the fetus is a male fetus. In certain embodiments of the invention, the fetus is a female fetus. In certain embodiments of the invention, the fetus is a mammal. In certain embodiments of the invention, the fetus is a human. In certain embodiments of the invention, the fetus is a non-human mammal. In certain embodiments of the invention, the fetus has been determined to be at an elevated risk for having a chromosomal abnormality.
  • the method further comprises using tandem single nucleotide polymorphisms to compare paternal DNA to the fetal and/or maternal DNA.
  • the fetal DNA is subjected to an enrichment step. In certain embodiments of the invention, the fetal DNA is not subjected to an enrichment step.
  • Certain embodiments of the present invention provide a method for identifying chromosomes, comprising comparing tandem single nucleotide polymorphisms on the chromosomes so as to identify the chromosomes.
  • the methods of the present invention are not limited to maternal-fetal analysis, but can also be applied to other situations, e.g., forensic analysis of blood samples.
  • the methods further comprises, prior to the comparison step, determining a set of tandem single nucleotide polymorphisms for a specific chromosome.
  • Certain embodiments of the present invention provide a system comprising packaging material and primers that specifically hybridize to each of the single nucleotide polymorphisms of at least one of the tandem single nucleotide polymorphisms identified herein.
  • Certain embodiments of the present invention provide a system comprising packaging material and primers that specifically hybridize flanking sequences of at least one of the tandem single nucleotide polymorphisms of the invention.
  • Certain embodiments of the present invention provide a system comprising packaging material and at least one oligonucleotide that specifically hybridizes to at least one of the tandem single nucleotide polymorphisms of the invention.
  • Certain embodiments of the present invention provide the use of high- fidelity PCR (HiFi-PCR) to amplify SNPs or tandem SNPs for the purpose of, e.g., determining chromosomal abnormalities.
  • HiFi-PCR high- fidelity PCR
  • Certain embodiments of the present invention provide the use of HiFi- PCR to amplify nucleic acids, e.g., DNA, isolated, e.g., from a maternal biological sample to analyze fetal DNA for chromosomal abnormalities.
  • nucleic acids e.g., DNA
  • isolated e.g., from a maternal biological sample to analyze fetal DNA for chromosomal abnormalities.
  • HiFi-PCR is used to detect aneuploidy and large (>3 megabasepairs) or small ( ⁇ 3 megabasepairs) deletions and/or insertions.
  • the maternal biological sample is serum, urine, sweat, cells, or cell free DNA.
  • Certain embodiments of the present invention provide an isolated nucleic acid sequence comprising at least one of SEQ ID NOs 1-357.
  • Certain embodiments of the present invention provide an isolated nucleic acid sequence of the invention (e.g., a nucleic acid sequence comprising a tandem SNP or a primer; e.g. , at least one of SEQ ID NOs 1-357) for use in medical treatment or diagnosis.
  • an isolated nucleic acid sequence of the invention e.g., a nucleic acid sequence comprising a tandem SNP or a primer; e.g. , at least one of SEQ ID NOs 1-357
  • the nucleic acid sequences may be, e.g., isolated nucleic acid sequences and may be, e.g., about 1000 or fewer, e.g., about 900 or fewer, e.g., about 800 or fewer, e.g., about 700 or fewer, e.g., about 600 or fewer, e.g., about 500 or fewer, e.g., about 400 or fewer, e.g., about 300 or fewer, e.g., about 250 or fewer, e.g., about 200 or fewer, e.g., about 150 or fewer, e.g., about 100 or fewer, or e.g., about 50 or fewer nucleic acids in length.
  • tandem SNPs for chromosome 21 are identified, heterozygosity of the tandem SNPs determined, the ability to detect fetal DNA from maternal serum demonstrated, and the ability to detect fetal chromosomal abnormalities in maternal serum demonstrated.
  • tandem SNPs have already been identified. These tandem SNPs are useful in the diagnosis of chromosomal abnormalities, for example, of trisomy 21.
  • certain embodiments of the invention provide the specific tandem SNPs, or combinations thereof, as well as their use in diagnostic and therapeutic applications.
  • tandem SNP assays based on a set of tandem SNPs for chromosome 21
  • These diagnostics are sensitive and specific.
  • the tandem SNP assay is particularly suited for fetal DNA analysis because fetal DNA present in maternal serum is generally present as short fragments (e.g., an average of 300 basepairs or fewer).
  • certain embodiments of the present invention are directed to each of these tandem SNPs individually, and certain embodiments are directed to combinations of any and/or all of the tandem SNPs.
  • Certain embodiments of the invention are directed to methods of using the tandem SNPs for diagnosing chromosomal abnormalities. Certain embodiments of the invention are directed to compilations of the tandem SNPs (e.g., reference tables) that are useful for diagnosing chromosomal abnormalities. Certain embodiments of the invention are also directed to primers for each of these tandem SNPs individually, and certain embodiments are directed to combinations of primers for any and/or all of the tandem SNPs. Certain embodiments of the invention provide isolated nucleic acid sequences that comprise at least one of the tandem SNPs and compositions that comprise the isolated nucleic acid sequences. Prenatal Screening
  • nuchal translucency test An ultrasound screening called the nuchal translucency test is becoming more common. However, this test has an overall sensitivity of 77% for trisomy 21 with a false positive rate of 6% (Malone et al, Obstet Gynecol, 2003. 102(5 Pt 1): p. 1066-79).
  • the most advanced serum marker test is the "quad" screen, which measures the levels of alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), unconjugated estriol (E3), and inhibin-A. The biological reason for these markers to be elevated or reduced in a percentage of mothers carrying children with trisomy 21 is not understood.
  • test is only capable of assigning risk categories (i.e., 1 in 250, 1 in 100, 1 in 10), and not in making specific diagnoses.
  • the quad screen is associated with a false positive rate of 7% and a sensitivity of less than 80%, rates which do not approach those achieved by invasive prenatal diagnostic tests (WaId et ah, Lancet, 2003. 361(9360): p. 835-6).
  • PCR-based approach for detecting aneuploidy relies on a method called quantitative fluorescent polymerase chain reaction (QF-PCR) of short tandem repeats (STRs).
  • QF-PCR quantitative fluorescent polymerase chain reaction
  • STRs short tandem repeats
  • CDCE combined with high-fidelity PCR (HiFi-PCR) was developed to allow researchers to detect and quantify low frequency somatic mutations present in heterogeneous cell populations (Khrapko et al, Nucleic Acids Res, 1994. 22(3): p. 364-9).
  • CDCE permits the highest resolution separation of DNA sequences differing by even a single base pair. The separation is based on differences in the melting temperature and the resulting electrophoretic mobility differences as the DNA molecules migrate through a linear polyacrylamide matrix under partially denaturing conditions (Khrapko et al, 1994).
  • CDCE coupled with HiFi-PCR has been demonstrated to detect mutations in ⁇ 100 bp sequences with a sensitivity of at least 2 x 10 "6 in human cells and tissues (Li-Sucholeiki et al., Nucleic Acids Res, 2000. 28(9): p. E44).
  • this technology can be applied to single nucleotide polymorphisms (SNPs) 5 natural single basepair variations present in the genome, to separate alleles.
  • SNPs single nucleotide polymorphisms
  • CDCE is used in the present invention to screen tandem SNPs to increase the informativeness (or heterozygosity) of each CDCE assay by increasing the number of possible alleles (or haplotypes) available.
  • tandem SNPs Through the use of tandem SNPs, a highly specific and sensitive assay for detecting fetal chromosomal abnormalities by simply comparing maternal serum to maternal buccal swabs has been created.
  • High-Fidelity PCR is an amplification method resulting in an error rate
  • Pfii (in per basepair doubling) equal to or better than standard PCR.
  • Taq polymerase has an error rate of ⁇ 10 "4 per basepair doubling.
  • Pyrococcus furiosus (Pfu) is a high-fidelity polymerase. The published error rate for Pfii is 1.3 x 10 per basepair doubling (Cline et al, Nucleic Acids Res. 1996 September 15; 24(18): 3546-3551).
  • Methods for improving PCR fidelity include, among others: A) using a high-fidelity polymerase enzyme; and B) the addition of chemical reagents (e.g., betaine) that can lower temperatures required during the PCR process.
  • chemical reagents e.g., betaine
  • the prolonged heating of DNA and nucleotides during PCR can lead to damaged products, such as deaminated cytosines (uracils) and thus lead to misincorporation errors and miscopying errors during PCR (Andre, Kim, Khrapko, Thilly. Genome Res. 1997 7: 843-852. Zheng, Khrapko, Coller,
  • amplification e.g., HiFi-PCR
  • primers being in molar excess (e.g., 10 12 copies/ ⁇ l of primer vs 10 6 or less of the template) so that it is more likely that primers will anneal with template DNA than with each other (see, e.g., Li-Sucholeiki XC, Thilly WG. Nucleic Acids Res. 2000 May 1 ;28(9):E44; Thompson JR, Marcelino L, PoIz M. Nucleic Acids Res. 2002 May 1; 30(9): 2083-2088.).
  • This can significantly reduce the creation of heteroduplexes.
  • a “single nucleotide polymorphism (SNP)" is a single basepair variation in a nucleic acid sequence.
  • a “tandem SNP” is a pair of SNPs that are located in a nucleic acid sequence, e.g. on a chromosome, in a manner that allows for the detection of both of the SNPs.
  • the distance between SNPs generally is about 250 basepairs or fewer, e.g., about 200 basepairs or fewer, e.g., about 150 basepairs or fewer, e.g., about 100 basepairs or fewer, e.g., about 50 basepairs or fewer.
  • the tandem SNPs can be detected by a variety of means that are capable of detecting the tandem SNPs.
  • constant denaturant capillary electrophoresis (CDCE) can be combined with high-fidelity PCR (HiFi-PCR) to detect the tandem SNP.
  • hybridization on a microarray is used.
  • high-fidelity PCR is used and another method capable of detecting SNPs present at low frequencies is used (e.g., denaturing HPLC, denaturing capillary electrophoresis, cycling temperature capillary electrophoresis, allele-specific PCRs, quantitative real time PCR approaches such as TaqMan® PCR system, polony sequencing approaches, microarray approaches, and mass spectrometry).
  • high- throughput sequencing approaches e.g. , at a single molecule level, are used.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, made of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also encompasses conservatively modified variants thereof ⁇ e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
  • nucleotide sequence refers to a polymer of DNA or RNA which can be single- stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • nucleic acid refers to a polymer of DNA or RNA which can be single- stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • nucleic acid nucleic acid molecule
  • polynucleotide are used interchangeably.
  • an "isolated” or “purified” DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment and is therefore not a product of nature.
  • An isolated DNA molecule or RNA molecule may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
  • an "isolated” or “purified” nucleic acid molecule is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence,” (b) “comparison window,” (c) “sequence identity,” (d) “percentage of sequence identity,” and (e) “substantial identity.”
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions ⁇ i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters.
  • CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters.
  • CLUSTAL program is well described by Higgins et al (Higgins et al, CABIOS, 5, 151 (1989)); Corpet et al (Corpet et al, Nucl. Acids Res., 16, 10881 (1988)); Huang et al (Huang et al, CABIOS, 8, 155 (1992)); and Pearson et al (Pearson et al, Meth. MoI. Biol., 24, 307 (1994)).
  • the ALIGN program is based on the algorithm of Myers and Miller, supra.
  • the BLAST programs of Altschul et al (Altschul et al, JMB, 215, 403 (1990)) are based on the algorithm of Karlin and Altschul supra.
  • HSPs high scoring sequence pairs
  • a scoring matrix is used to calculate the cumulative score. Extension of the word, hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative- scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, less than about 0.01, or even less than about 0.001.
  • Gapped BLAST in BLAST 2.0
  • PSI-BLAST in BLAST 2.0
  • the default parameters of the respective programs e.g., BLASTN for nucleotide sequences, BLASTX for proteins
  • the BLASTN program for nucleotide sequences
  • W wordlength
  • E expectation
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. Alignment may also be performed manually by inspection.
  • comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein may be made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program.
  • equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the program.
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non- conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. ⁇
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • stringent conditions encompass temperatures in the range of about 1°C to about 20 0 C, depending upon the desired degree of stringency as otherwise qualified herein.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)).
  • An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • certain embodiments of the invention provide nucleic acid molecules that are substantially identical to the nucleic acid molecules described herein.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture ⁇ e.g., total cellular) DNA or RNA.
  • Bod(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as
  • Tm The thermal melting point
  • Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
  • T n can be approximated from the equation of Meinkoth and Wahl (1984); T n , 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity.
  • the T m can be decreased 10 0 C.
  • stringent conditions are selected to be about 5°C lower than the T m for the specific sequence and its complement at a defined ionic strength and pH.
  • severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the T m ;
  • moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10 0 C lower than the T m ;
  • low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20 0 C lower than the T ra .
  • hybridization and wash compositions those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a temperature of less than 45°C (aqueous solution) or 32°C (formamide solution), the SSC concentration is increased so that a higher temperature can be used. Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the T m for the specific sequence at a defined ionic strength and pH.
  • An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2 x SSC wash at 65 0 C for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1 x SSC at 45°C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.5 M, less than about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C and at least about 60 0 C for long probes ⁇ e.g., >50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a signal to noise ratio of 2 x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent conditions for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1 x SSC at 60 to 65°C.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5 x to 1 x SSC at 55 to 60°C.
  • hybridization data-sets e.g. microarray data
  • treatment or “treating,” to the extent it relates to a disease or condition includes preventing the disease or condition from occurring, inhibiting the disease or condition, eliminating the disease or condition, and/or relieving one or more symptoms of the disease or condition.
  • 96 allelic markers on chromosome 21 are selected by examining tandem SNPs. These tandem SNPs will cover both q and p arms of the chromosome. Using heterozygosity data available through dbSNP, DCC Genotype Database and through the HapMap Project, SNPs that appear to be promising for high heterozygosity (>25%) are selected. Because all four possibilities may not exist in nature due to haplotype blocks in regions of low recombination, those that suggest less than three haplotypes are screened out.
  • Target sequences covering tandem SNPs are designed using Vector NTI and WinMelt software.
  • the melting map of a CDCE target covering two tandem SNPs (dbSNP rs2839416 and rs2839417) on chromosome 21 was calculated using WinMelt according to the algorithm of Lerman and Silverstein (Lerman et at, Methods Enzymol, 1987. 155: p.482-501) and is depicted in Figure 2.
  • Figure 2 depicts a DNA melting map of a CDCE target sequence covering tandem SNPs. All four haplotypes can be theoretically separated according to DNA melting temperature.
  • the black line indicates haplotype 1 (G,A).
  • the yellow line indicates haplotype 2 (T 5 A).
  • the red line indicates haplotype 3 (G 5 G).
  • the green line indicates haplotype 4 (T 5 G).
  • HiFi PCR optimization for each target sequence is performed using Pfu polymerase.
  • One of primers flanking the target sequence is ⁇ 20 bases in length and labeled 5' with a fluorescein molecule.
  • the other primer is about 74 bases including a ⁇ 20-base target specific sequence and the 54-base clamp sequence.
  • a standard HiFi PCR condition is applied to all target sequences, varying only annealing temperatures. These PCR amplicons are subjected to CDCE electrophoretic separation. The resulting electropherogram are analyzed for yield and purity of the PCR products. The purity is evaluated by comparing the peak area of the desired products to that of the byproducts and nonspecific amplification.
  • Target sequences that can be amplified with a high PCR efficiency (> 45% per cycle) and low levels of byproducts and nonspecific amplification ( ⁇ 0.1% of the desired products) are immediately be subjected to CDCE optimization.
  • increasing amounts of Mg +2 concentrations (up to about 7 mM) in combination with different annealing temperatures are tested.
  • primer positions are changed and the entire optimization process is repeated.
  • the relevant haplotypes are created for the targets.
  • the optimal separation condition for each haplotype should provide the greatest resolution among the observed peaks.
  • Initial optimization is done around the theoretical melting temperature (T m ) in a 2 0 C temperature range in increments of 0.2 0 C which covers (T n , — 1°C ⁇ a predetermined offset) to (T n , + 1 0 C ⁇ a predetermined offset).
  • Electropherogram and peak measurements are transferred to a spreadsheet for analysis. To ensure the quality of the data, minimum and maximum peak heights are used. Individual markers are failed if electrophoretic spikes occur. Peak areas are used to calculate allele ratios. A check for allelic preferential amplification is performed on all 96 tandem SNPs. . Results
  • the neighboring sequences for each of the tandem SNPs are imported into a software program, e.g., Sequencher (Gene Codes, Ann Arbor, MI) and/or Vector NTI (Invitrogen, Carlsbad, CA) for sequence alignment and primer design, and into Winmelt (Medprobe, Norway) or Tru software (available on the world wide web at biophys.uniduesseldorf.de ⁇ ocal/POLAND/poland.html) where the algorithm for computing DNA melting temperatures given the Gotoh-Tagashira values for the enthalpy of melting DNA sequences are used to calculate melting temperatures of target sequences.
  • CDCE candidates generally have a high melting region adjacent to a low melting region, lie in a low melting region, melting temperatures of the low melting region fall below 80 0 C, and no "valleys" occur between the high melting region and the low melting region.
  • Primer sequences for these 118 tandem SNP/CDCE targets have been designed. These will be optimized as described herein using HiFi PCR and CDCE. These optimizations are described herein and include the creation of relevant haplotypes for all targets, a check for allelic preferential amplification during HiFi PCR, and obtaining the greatest resolution among peaks during CDCE. Haplotypes may be separated as homoduplex peaks. However, if certain targets cannot be separated out as homoduplexes, maternal DNA can be separated from fetal DNA as heteroduplexes.
  • Example 2 Determining Heterozygosity of the Tandem SNPs As a complement to Example 1, genomic DNA samples from 300 anonymous subjects have been obtained from healthy young adults who are less than 35 years old. The samples are anonymous as the only data obtained were the geographic location of the Red Cross blood donor center, donor gender, and whether or not the donor was 35 and under. These samples were spot-checked to look for the haplotypes seen in the HapMap project.
  • Example 3 Detecting Fetal DNA from Maternal Serum A cohort of patients who have been confirmed to have trisomy 21 by traditional karyotype analysis are examined. Tandem SNPs are used to demonstrate detection of trisomy in patients. DNA from 20 patients who have been characterized by traditional karyotype analysis to have trisomy 21 are analyzed with the tandem SNP panel. Biological samples, including a buccal (cheek) swab and a blood sample are collected from a cohort of pregnant women. Maternal buccal swab samples are compared to maternal serum to demonstrate that a third (paternal) peak is observed in several of the tandem SNP assays. Approximately 20 maternal buccal swab to maternal serum comparisons are made.
  • genomic DNA samples from maternal buccal swabs are utilized for each target sequence.
  • the buccal samples are subjected to the process in parallel with the maternal blood sample. Any artifacts generated by the CDCE/HiFi-PCR procedure (including nonspecific PCR amplification and polymerase-induced mutations) are revealed as background peaks in the buccal swab samples.
  • Example 4 Detecting Fetal Chromosomal Abnormalities A blinded study is performed where the goal is to detect 20 known trisomy 21 fetuses by assaying maternal serum from 40 patients (previously determined by amniocentesis or CVS) (see Figure 3).
  • Figure 3 depicts an example of a CDCE electropherogram output with the peaks at full scale.
  • Figure 3 A depicts a sample from maternal buccal swab. Markers exhibiting two alleles are pursued. A baby with trisomy is expected to show either three alleles, evident by three peaks in a 1:1 :1 ratio or two alleles in a 2: 1 ratio.
  • Figure 3B depicts a sample from maternal serum. Markers exhibiting three alleles are informative.
  • Maternal serum from a woman carrying a baby with trisomy is expected to exhibit three alleles, evident by two equal peaks with a third smaller peak if the trisomy occurred during meiosis I (75% of T21 cases) or three alleles with different areas if the trisomy occurred during meiosis II (20% of T21 cases) where areas are: peak, x, and peak + 2x.
  • Figure 3C depicts analysis of a sample from maternal serum. Markers exhibiting three alleles are informative. Maternal serum from a woman with a normal baby with three alleles has three different areas where areas are: peak, x, and peak + x.
  • the allele frequencies at each SNP loci are expected to be 85% and 15% for the majority and minority alleles, respectively, assuming Hardy- Weinberg equilibrium.
  • the desired third haplotype is expected to be present at an average of 6.4 markers (15%) of per maternal-fetal sample tested. Because most loci have a heterozygosity value greater than 25%, for every maternal-fetal sample tested using the panel of 96 tandem SNP assays, greater than about 6.4 markers are most informative. Thus, while a panel of 96 tandem SNPs may be used, 6 or 7 of those tandem SNPs may be informative for any one specific maternal-fetal sample tested, and a 'positive' result from any one of those tandem SNPs is informative.
  • tandem SNPs should be identified on both the p and the q arm of chromosome 21. Because of the comparative nature of the basic approach, the tandem SNP assay is predicted to have a detection rate of 95% (those that occur during maternal meiosis) for trisomy 21. If paternal samples are available, non-disjunctions that occur during paternal meiosis can also be detected. Thus, detection rates would be higher (about ⁇ 99%) with a 0% false positive rate.
  • Tandem SNPs and Primers Table 2 provides exemplary tandem SNPs of the invention and primers that can be used in the methods of the invention to detect the tandem SNPs.
  • Certain embodiments of the present invention provide primers that can be used to amplify at least one of the SNPs. Certain embodiments of the present invention provide nucleic acid sequences that comprise at least one of the SNPs, e.g., at least one of the tandem SNPs. Table 2
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Abstract

The invention provides tandem single nucleotide polymorphisms and methods for their use, for example, in diagnosing Down Syndrome.

Description

DETECTING FETAL CHROMOSOMAL ABNORMALITIES USING TANDEM SINGLE NUCLEOTIDE POLYMORPHISMS ■ Related Applications)
This patent document claims the benefit of priority of U.S. application serial No. 60/777,865, filed February 28, 2006, which application is herein incorporated by reference.
Background About 6.4 million women become pregnant in the U.S. each year, and about 70% of those women have maternal serum screening and/or an ultrasound test in an attempt to determine risks for common birth defects, such as those resulting from trisomy 13, 18, and 21 (Down Syndrome). Both the sensitivity and specificity of these common non-invasive screening tools are extremely poor. The best current non-invasive tests lead to a false positive rate between 7 and 20%. This high false positive rate has two catastrophic consequences for American families and society. First, it creates a large market for the two invasive diagnostic tests, chorionic villus sampling (CVS) and amniocentesis, which each carry a fetal loss rate of 0.5% -1 %. These invasive tests directly result in the loss of thousands of normal fetuses annually. Second, the high false positive rate heightens maternal anxiety and stress in the large and fixed proportion of pregnant American women who receive false positive results. However, prenatal diagnosis are critical in managing a pregnancy with chromosomal abnormalities and localized genetic abnormalities, as the diagnosis can allow for interventional care during delivery and can prevent devastating consequences for the neonate. Thus there is a tremendous need for the development of a sensitive and specific non-invasive prenatal diagnostic test for chromosomal abnormalities.
Summary of Certain Embodiments of the Invention Accordingly, certain embodiments of the present invention provide a method for determining whether a fetus has at least one chromosomal abnormality, comprising using tandem single nucleotide polymorphisms to compare fetal DNA to maternal DNA so as to determine whether the fetus has at least one chromosomal abnormality.
Brief Description of the Figures Figure 1 depicts an example of a tandem SNP. Figure 2 depicts a DNA melting map of a constant denaturant capillary electrophoresis target sequence covering a tandem SNP.
Figure 3 depicts an example of a constant denaturant capillary electrophoresis electropherogram output. Figure 3A depicts results from a sample from a maternal buccal swab. Figure 3B depicts results from a sample from maternal serum. Figure 3C depicts results from a sample from maternal serum.
Detailed Description
For years, it has been hoped that the use of fetal cells in maternal blood might be used to assess the genetic status of a developing embryo. Unfortunately, the extremely small amount of fetal cells in maternal blood (about 1 cell per ml) has proven a difficult obstacle to overcome when trying to isolate these cells for widespread clinical testing. However, cell-free fetal DNA is present in circulating maternal serum at higher percentages than fetal cells and has the potential to be assessed for chromosomal or gene defects. Cell-free fetal DNA can range from 1-47 % of total DNA in maternal blood. However, a critical limitation that has yet to be successfully overcome is that maternal DNA contamination makes it difficult to differentiate fetal from maternal DNA.
As described herein, this limitation has been overcome by identifying tandem single nucleotide polymorphisms (SNPs) to detect chromosomes, e.g., to detect fetal chromosomal abnormalities. The tandem SNPs are combined with a sensitive DNA separation technology, e.g., high-fidelity PCR and constant denaturant capillary electrophoresis (CDCE), to detect fetal chromosomal abnormalities, e.g., through the simple sampling and comparison of maternal DNA to fetal DNA5 e.g., from maternal serum and maternal buccal swabs. This approach substantially eliminates false positives and significantly reduces false negatives. Accordingly, certain embodiments of the present invention provide a method for determining whether a fetus has at least one chromosomal abnormality, comprising using tandem single nucleotide polymorphisms to compare fetal DNA to maternal DNA so as to determine whether the fetus has at least one chromosomal abnormality.
In certain embodiments of the invention, fetal DNA is obtained from maternal blood. In certain embodiments of the invention, fetal DNA is cell-free fetal DNA. In certain embodiments of the invention, maternal DNA is obtained from a biological sample, e.g., maternal blood, hi certain embodiments of the invention, maternal DNA is obtained from a buccal swab]' In certain embodiments of the invention, maternal DNA is obtained from a biological sample that does not comprise fetal DNA.
In certain embodiments of the invention, fetal DNA is obtained from maternal blood, maternal urine, maternal sweat, maternal cells, or cell free DNA from the mother.
In certain embodiments, the biological sample is biological fluid. In certain embodiments, the biological sample is a maternal biological sample. In certain embodiments, samples may be whole blood, bone marrow, blood spots, blood serum, blood plasma, buffy coat preparations, saliva, cerebrospinal fluid, buccal swabs, solid tissues such as skin and hair, body waste products, such as feces and urine. In other embodiments, samples may be lysates, homogenates, or partially purified samples of biological materials. In other instances, biological materials can include crude or partially purified mixtures of nucleic acids. In certain embodiments, the biological sample is serum, urine, sweat, cells, or cell
In certain embodiments of the invention, the comparison step comprises using high-fidelity PCR and constant denaturant capillary electrophoresis to compare the fetal DNA to maternal DNA. In certain embodiments of the invention, the comparison step comprises using at least about 96 tandem single nucleotide polymorphisms.
In certain embodiments of the invention, the method further comprises the step of converting the nucleic acid molecules to a homoduplex state, as opposed to being in heteroduplex form. This can be accomplished, e.g., by using an excess of primers and can aid in the tandem SNP analysis.
In certain embodiments of the invention, methods such as mutation detection technologies can be used to analyze the tandem SNPs. In certain embodiments of the invention, methods such as denaturing HPLC, denaturing capillary electrophoresis, cycling temperature capillary electrophoresis, allele- specific PCRs, quantitative real time PCR approaches such as TaqMan® PCR system, polony PCR approaches, and microarray approaches can be used to analyze the tandem SNPs. In certain embodiments of the invention, the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 250 basepairs apart. In certain embodiments of the invention, the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 200 basepairs apart. In certain embodiments of the invention, the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 150 basepairs apart. In certain embodiments of the invention, the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 100 basepairs apart. In certain embodiments of the invention, the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 50 basepairs apart.
In certain embodiments of the invention, at least one tandem single nucleotide polymorphism is located on the p arm of chromosome 21. In certain embodiments of the invention, at least one tandem single nucleotide polymorphism is located on the q arm of chromosome 21.
In certain embodiments of the invention, the chromosomal abnormality is chromosomal aneuploidy. In certain embodiments of the invention, the chromosomal abnormality is trisomy 13, 18 or 21. In certain embodiments of the invention, the chromosomal abnormality is trisomy 21. In certain embodiments of the invention, the chromosomal abnormality is an insertion mutation {e.g., a large insertion (>3 megabasepair) or small insertion (< 3 megabasepair). In certain embodiments of the invention, the chromosomal abnormality is a deletion mutation (e.g., a large deletion (>3 megabasepair) or small deletion (< 3 megabasepair)). The deleted region could include a deleted gene.
In certain embodiments of the invention, the methods can be used to detect copy number polymorphisms and/or copy number variants in the genome. In certain embodiments of the invention, the methods can be used to detect chromosome 22ql 1 deletion syndrome, which is associated with cardiac defects.
Chromosomal abnormalities include deletions associated with genetic syndromes and disorders such as the 22ql 1 deletion syndrome on chromosome 22, which is associated with cardiac defects. Other examples of chromosomal abnormalities include the Hq deletion syndrome on chromosome 11 and 8p deletion syndrome on chromosome 8, both of which are also associated with cardiac defects.
In certain embodiments of the invention, the fetus is a male fetus. In certain embodiments of the invention, the fetus is a female fetus. In certain embodiments of the invention, the fetus is a mammal. In certain embodiments of the invention, the fetus is a human. In certain embodiments of the invention, the fetus is a non-human mammal. In certain embodiments of the invention, the fetus has been determined to be at an elevated risk for having a chromosomal abnormality.
In certain embodiments of the invention, the method further comprises using tandem single nucleotide polymorphisms to compare paternal DNA to the fetal and/or maternal DNA.
In certain embodiments of the invention, the fetal DNA is subjected to an enrichment step. In certain embodiments of the invention, the fetal DNA is not subjected to an enrichment step.
Certain embodiments of the present invention provide a method for identifying chromosomes, comprising comparing tandem single nucleotide polymorphisms on the chromosomes so as to identify the chromosomes. Thus, the methods of the present invention are not limited to maternal-fetal analysis, but can also be applied to other situations, e.g., forensic analysis of blood samples. In certain embodiments of the invention, the methods further comprises, prior to the comparison step, determining a set of tandem single nucleotide polymorphisms for a specific chromosome.
Certain embodiments of the present invention provide a system comprising packaging material and primers that specifically hybridize to each of the single nucleotide polymorphisms of at least one of the tandem single nucleotide polymorphisms identified herein.
Certain embodiments of the present invention provide a system comprising packaging material and primers that specifically hybridize flanking sequences of at least one of the tandem single nucleotide polymorphisms of the invention.
Certain embodiments of the present invention provide a system comprising packaging material and at least one oligonucleotide that specifically hybridizes to at least one of the tandem single nucleotide polymorphisms of the invention.
Certain embodiments of the present invention provide the use of high- fidelity PCR (HiFi-PCR) to amplify SNPs or tandem SNPs for the purpose of, e.g., determining chromosomal abnormalities.
Certain embodiments of the present invention provide the use of HiFi- PCR to amplify nucleic acids, e.g., DNA, isolated, e.g., from a maternal biological sample to analyze fetal DNA for chromosomal abnormalities.
In certain embodiments, HiFi-PCR is used to detect aneuploidy and large (>3 megabasepairs) or small (<3 megabasepairs) deletions and/or insertions.
In certain embodiments, the maternal biological sample is serum, urine, sweat, cells, or cell free DNA.
Certain embodiments of the present invention provide an isolated nucleic acid sequence comprising at least one of SEQ ID NOs 1-357.
Certain embodiments of the present invention provide an isolated nucleic acid sequence of the invention (e.g., a nucleic acid sequence comprising a tandem SNP or a primer; e.g. , at least one of SEQ ID NOs 1-357) for use in medical treatment or diagnosis. In certain embodiments, the nucleic acid sequences may be, e.g., isolated nucleic acid sequences and may be, e.g., about 1000 or fewer, e.g., about 900 or fewer, e.g., about 800 or fewer, e.g., about 700 or fewer, e.g., about 600 or fewer, e.g., about 500 or fewer, e.g., about 400 or fewer, e.g., about 300 or fewer, e.g., about 250 or fewer, e.g., about 200 or fewer, e.g., about 150 or fewer, e.g., about 100 or fewer, or e.g., about 50 or fewer nucleic acids in length.
Thus, short haplotypes are used to detect fetal chromosomal abnormalities in maternal serum, e.g., for the most common of these defects, trisomy 21. To demonstrate this method, tandem SNPs for chromosome 21 are identified, heterozygosity of the tandem SNPs determined, the ability to detect fetal DNA from maternal serum demonstrated, and the ability to detect fetal chromosomal abnormalities in maternal serum demonstrated. 118 tandem SNPs have already been identified. These tandem SNPs are useful in the diagnosis of chromosomal abnormalities, for example, of trisomy 21. Thus, certain embodiments of the invention provide the specific tandem SNPs, or combinations thereof, as well as their use in diagnostic and therapeutic applications.
The output of these experiments, e.g., assays based on a set of tandem SNPs for chromosome 21, can be used in the clinic as an alternative to invasive diagnostic tests like amniocentesis and CVS, using, e.g., CDCE or other techniques capable of detecting the tandem SNPs. These diagnostics are sensitive and specific. The tandem SNP assay is particularly suited for fetal DNA analysis because fetal DNA present in maternal serum is generally present as short fragments (e.g., an average of 300 basepairs or fewer). Thus, certain embodiments of the present invention are directed to each of these tandem SNPs individually, and certain embodiments are directed to combinations of any and/or all of the tandem SNPs. Certain embodiments of the invention are directed to methods of using the tandem SNPs for diagnosing chromosomal abnormalities. Certain embodiments of the invention are directed to compilations of the tandem SNPs (e.g., reference tables) that are useful for diagnosing chromosomal abnormalities. Certain embodiments of the invention are also directed to primers for each of these tandem SNPs individually, and certain embodiments are directed to combinations of primers for any and/or all of the tandem SNPs. Certain embodiments of the invention provide isolated nucleic acid sequences that comprise at least one of the tandem SNPs and compositions that comprise the isolated nucleic acid sequences. Prenatal Screening
An increasing number of fetal medical conditions can be successfully managed during the neonatal period if an early diagnosis is made. A variety of prenatal screening tools are available for chromosomal and birth defects. The two most commonly utilized non-invasive tools are ultrasound and measurements of maternal serum markers. Both of these "tests" have inadequate sensitivity and specificity for screening the most common of the defects, Down Syndrome (trisomy 21).
An ultrasound screening called the nuchal translucency test is becoming more common. However, this test has an overall sensitivity of 77% for trisomy 21 with a false positive rate of 6% (Malone et al, Obstet Gynecol, 2003. 102(5 Pt 1): p. 1066-79). The most advanced serum marker test is the "quad" screen, which measures the levels of alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), unconjugated estriol (E3), and inhibin-A. The biological reason for these markers to be elevated or reduced in a percentage of mothers carrying children with trisomy 21 is not understood. Further, the test is only capable of assigning risk categories (i.e., 1 in 250, 1 in 100, 1 in 10), and not in making specific diagnoses. The quad screen is associated with a false positive rate of 7% and a sensitivity of less than 80%, rates which do not approach those achieved by invasive prenatal diagnostic tests (WaId et ah, Lancet, 2003. 361(9360): p. 835-6).
Because of the inadequate sensitivity and specificity of currently available non-invasive tools, amniocentesis and chorionic villus sampling (CVS), both invasive procedures, remain the standard for the definitive detection of fetal chromosomal abnormalities. Both of these procedures carry a 0.5%-l% fetal loss rate, which translate into the death of thousands of normal fetuses annually. To solve this problem and meet the overwhelming need for an accurate non-invasive test, several strategies have been previously proposed by other investigators. However, those studies have been limited by their ability to detect and differentiate fetal DNA from maternal DNA.
A PCR-based approach for detecting aneuploidy relies on a method called quantitative fluorescent polymerase chain reaction (QF-PCR) of short tandem repeats (STRs). However, polymerase errors are frequently made in the repeat sequences, generating a high background "noise" for each STR assay. These PCR errors (stutters) make peak area measurements difficult and thus the detection and quantification of low frequency fetal DNA in maternal serum not possible (Dhallan et al, JAMA, 2004. 291(9): p. 1114-9). In 1994, a technology called constant denaturant capillary electrophoresis
(CDCE) combined with high-fidelity PCR (HiFi-PCR) was developed to allow researchers to detect and quantify low frequency somatic mutations present in heterogeneous cell populations (Khrapko et al, Nucleic Acids Res, 1994. 22(3): p. 364-9). Compared to other DNA separation methods, CDCE permits the highest resolution separation of DNA sequences differing by even a single base pair. The separation is based on differences in the melting temperature and the resulting electrophoretic mobility differences as the DNA molecules migrate through a linear polyacrylamide matrix under partially denaturing conditions (Khrapko et al, 1994). CDCE coupled with HiFi-PCR has been demonstrated to detect mutations in ~100 bp sequences with a sensitivity of at least 2 x 10"6 in human cells and tissues (Li-Sucholeiki et al., Nucleic Acids Res, 2000. 28(9): p. E44). As described herein, this technology can be applied to single nucleotide polymorphisms (SNPs)5 natural single basepair variations present in the genome, to separate alleles. CDCE is used in the present invention to screen tandem SNPs to increase the informativeness (or heterozygosity) of each CDCE assay by increasing the number of possible alleles (or haplotypes) available. Through the use of tandem SNPs, a highly specific and sensitive assay for detecting fetal chromosomal abnormalities by simply comparing maternal serum to maternal buccal swabs has been created. High-Fidelity PCR is an amplification method resulting in an error rate
(in per basepair doubling) equal to or better than standard PCR. For example, Taq polymerase has an error rate of ~ 10"4 per basepair doubling. As an example, Pyrococcus furiosus (Pfu) is a high-fidelity polymerase. The published error rate for Pfii is 1.3 x 10 per basepair doubling (Cline et al, Nucleic Acids Res. 1996 September 15; 24(18): 3546-3551).
Methods for improving PCR fidelity include, among others: A) using a high-fidelity polymerase enzyme; and B) the addition of chemical reagents (e.g., betaine) that can lower temperatures required during the PCR process. The prolonged heating of DNA and nucleotides during PCR can lead to damaged products, such as deaminated cytosines (uracils) and thus lead to misincorporation errors and miscopying errors during PCR (Andre, Kim, Khrapko, Thilly. Genome Res. 1997 7: 843-852. Zheng, Khrapko, Coller,
ThUIy, Copeland. Mutat Res. 2006 JuI 25;599(1-2):11-20). Examples of high- fidelity enzymes include Pfu and its derivations, or other enzymes with similar proofreading 3'->5' exonucleases.
In certain embodiments of the invention, amplification, e.g., HiFi-PCR, is performed with primers being in molar excess (e.g., 1012 copies/μl of primer vs 106 or less of the template) so that it is more likely that primers will anneal with template DNA than with each other (see, e.g., Li-Sucholeiki XC, Thilly WG. Nucleic Acids Res. 2000 May 1 ;28(9):E44; Thompson JR, Marcelino L, PoIz M. Nucleic Acids Res. 2002 May 1; 30(9): 2083-2088.). This can significantly reduce the creation of heteroduplexes.
A "single nucleotide polymorphism (SNP)" is a single basepair variation in a nucleic acid sequence. A "tandem SNP" is a pair of SNPs that are located in a nucleic acid sequence, e.g. on a chromosome, in a manner that allows for the detection of both of the SNPs. The distance between SNPs generally is about 250 basepairs or fewer, e.g., about 200 basepairs or fewer, e.g., about 150 basepairs or fewer, e.g., about 100 basepairs or fewer, e.g., about 50 basepairs or fewer. The tandem SNPs can be detected by a variety of means that are capable of detecting the tandem SNPs. In one embodiment of the invention, constant denaturant capillary electrophoresis (CDCE) can be combined with high-fidelity PCR (HiFi-PCR) to detect the tandem SNP. In another embodiment, hybridization on a microarray is used. In another embodiment, high-fidelity PCR is used and another method capable of detecting SNPs present at low frequencies is used (e.g., denaturing HPLC, denaturing capillary electrophoresis, cycling temperature capillary electrophoresis, allele-specific PCRs, quantitative real time PCR approaches such as TaqMan® PCR system, polony sequencing approaches, microarray approaches, and mass spectrometry). In another embodiment, high- throughput sequencing approaches, e.g. , at a single molecule level, are used.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, made of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof {e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
The term "nucleotide sequence" refers to a polymer of DNA or RNA which can be single- stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" are used interchangeably.
Certain embodiments of the invention encompass isolated or substantially purified nucleic acid compositions. In the context of the present invention, an "isolated" or "purified" DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or RNA molecule may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence," (b) "comparison window," (c) "sequence identity," (d) "percentage of sequence identity," and (e) "substantial identity."
(a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions {i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well-known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (Myers and Miller, CABIOS, 4, 11 (1988)); the local homology algorithm of Smith et al. (Smith et al., Adv. Appl. Math., 2, 482 (1981)); the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)); the search-for-similarity-method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85, 2444 (1988)); the algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87, 2264 (1990)), modified as in Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90, 5873 (1993)).
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters. The
CLUSTAL program is well described by Higgins et al (Higgins et al, CABIOS, 5, 151 (1989)); Corpet et al (Corpet et al, Nucl. Acids Res., 16, 10881 (1988)); Huang et al (Huang et al, CABIOS, 8, 155 (1992)); and Pearson et al (Pearson et al, Meth. MoI. Biol., 24, 307 (1994)). The ALIGN program is based on the algorithm of Myers and Miller, supra. The BLAST programs of Altschul et al (Altschul et al, JMB, 215, 403 (1990)) are based on the algorithm of Karlin and Altschul supra.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word, hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative- scoring residue alignments, or the end of either sequence is reached.
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, less than about 0.01, or even less than about 0.001.
To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. Alignment may also be performed manually by inspection.
For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein may be made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the program.
(c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non- conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.). (d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. ■
(e)(i) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, 80%, 90%, or even at least 95%.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1°C to about 200C, depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. (e)(ϋ) The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. In certain embodiments, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Thus, certain embodiments of the invention provide nucleic acid molecules that are substantially identical to the nucleic acid molecules described herein.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
As noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture {e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
"Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as
Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. The thermal melting point (Tm) is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tn, can be approximated from the equation of Meinkoth and Wahl (1984); Tn, 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is reduced by about 1°C for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 100C. Generally, stringent conditions are selected to be about 5°C lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the Tm; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 100C lower than the Tm; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 200C lower than the Tra. Using the equation, hybridization and wash compositions, and desired temperature, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a temperature of less than 45°C (aqueous solution) or 32°C (formamide solution), the SSC concentration is increased so that a higher temperature can be used. Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the Tm for the specific sequence at a defined ionic strength and pH.
An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2 x SSC wash at 650C for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1 x SSC at 45°C for 15 minutes. For short nucleotide sequences (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, less than about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C and at least about 600C for long probes {e.g., >50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2 x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent conditions for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1 x SSC at 60 to 65°C. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1 x to 2 x SSC (20 x SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5 x to 1 x SSC at 55 to 60°C.
In addition to the chemical optimization of stringency conditions, analytical models and algorithms can be applied to hybridization data-sets (e.g. microarray data) to improve stringency.
The term "treatment" or "treating," to the extent it relates to a disease or condition includes preventing the disease or condition from occurring, inhibiting the disease or condition, eliminating the disease or condition, and/or relieving one or more symptoms of the disease or condition.
The invention will now be illustrated by the following non-limiting Examples. Example 1 Tandem SNPs for Chromosome 21
96 allelic markers on chromosome 21 are selected by examining tandem SNPs. These tandem SNPs will cover both q and p arms of the chromosome. Using heterozygosity data available through dbSNP, DCC Genotype Database and through the HapMap Project, SNPs that appear to be promising for high heterozygosity (>25%) are selected. Because all four possibilities may not exist in nature due to haplotype blocks in regions of low recombination, those that suggest less than three haplotypes are screened out. Figure 1 depicts an example of tandem SNPs (SNP 1 = rs2839416, average estimated heterozygosity 0.444 and SNP2 = rs2839417, average estimated heterozygosity 0.414).
Target sequences covering tandem SNPs are designed using Vector NTI and WinMelt software. As an example, the melting map of a CDCE target covering two tandem SNPs (dbSNP rs2839416 and rs2839417) on chromosome 21 was calculated using WinMelt according to the algorithm of Lerman and Silverstein (Lerman et at, Methods Enzymol, 1987. 155: p.482-501) and is depicted in Figure 2.
Figure 2 depicts a DNA melting map of a CDCE target sequence covering tandem SNPs. All four haplotypes can be theoretically separated according to DNA melting temperature. The black line indicates haplotype 1 (G,A). The yellow line indicates haplotype 2 (T5A). The red line indicates haplotype 3 (G5G). The green line indicates haplotype 4 (T5G).
HiFi PCR optimization for each target sequence is performed using Pfu polymerase. One of primers flanking the target sequence is ~20 bases in length and labeled 5' with a fluorescein molecule. The other primer is about 74 bases including a ~20-base target specific sequence and the 54-base clamp sequence. A standard HiFi PCR condition is applied to all target sequences, varying only annealing temperatures. These PCR amplicons are subjected to CDCE electrophoretic separation. The resulting electropherogram are analyzed for yield and purity of the PCR products. The purity is evaluated by comparing the peak area of the desired products to that of the byproducts and nonspecific amplification. Target sequences that can be amplified with a high PCR efficiency (> 45% per cycle) and low levels of byproducts and nonspecific amplification (< 0.1% of the desired products) are immediately be subjected to CDCE optimization. For those target sequences that do not have acceptable PCR products in the first stage, increasing amounts of Mg+2 concentrations (up to about 7 mM) in combination with different annealing temperatures are tested. For the remaining target sequences that still do not work, primer positions are changed and the entire optimization process is repeated.
For CDCE optimization, the relevant haplotypes are created for the targets. The optimal separation condition for each haplotype should provide the greatest resolution among the observed peaks. Initial optimization is done around the theoretical melting temperature (Tm) in a 20C temperature range in increments of 0.20C which covers (Tn, — 1°C ± a predetermined offset) to (Tn, + 10C ± a predetermined offset). Electropherogram and peak measurements are transferred to a spreadsheet for analysis. To ensure the quality of the data, minimum and maximum peak heights are used. Individual markers are failed if electrophoretic spikes occur. Peak areas are used to calculate allele ratios. A check for allelic preferential amplification is performed on all 96 tandem SNPs. . Results
In the fall of 2005, the International HapMap Project publicly released genotypes and frequencies from 270 people of four ethnic populations. Chromosome 21 haplotype data from approximately 40,000 SNPs genotyped across four populations, including U.S. residents with northern and western European ancestry, residents of Ibadan, Nigeria, of Tokyo, Japan, and of Beijing, China, were downloaded (2005-10-24: HapMap Public Release #19) and converted to the + orientation. Tandem SNP candidates fell within 100 basepairs from each other and at least three haplotypes existed in all four ethnic populations. CDCE target sequences and primers are designed for the tandem SNPs identified through the HapMap Project. The neighboring sequences for each of the tandem SNPs are imported into a software program, e.g., Sequencher (Gene Codes, Ann Arbor, MI) and/or Vector NTI (Invitrogen, Carlsbad, CA) for sequence alignment and primer design, and into Winmelt (Medprobe, Oslo, Norway) or Poland software (available on the world wide web at biophys.uniduesseldorf.deΛocal/POLAND/poland.html) where the algorithm for computing DNA melting temperatures given the Gotoh-Tagashira values for the enthalpy of melting DNA sequences are used to calculate melting temperatures of target sequences. CDCE candidates generally have a high melting region adjacent to a low melting region, lie in a low melting region, melting temperatures of the low melting region fall below 800C, and no "valleys" occur between the high melting region and the low melting region.
All of the 40,000 genotypes on chromosome 21 have been analyzed for tandem SNP/CDCE marker suitability. 118 tandem SNPs/CDCE targets meeting our requirements have been identified (see Table 1 for the first 42 identified and Table 2 for all 118).
Primer sequences for these 118 tandem SNP/CDCE targets have been designed. These will be optimized as described herein using HiFi PCR and CDCE. These optimizations are described herein and include the creation of relevant haplotypes for all targets, a check for allelic preferential amplification during HiFi PCR, and obtaining the greatest resolution among peaks during CDCE. Haplotypes may be separated as homoduplex peaks. However, if certain targets cannot be separated out as homoduplexes, maternal DNA can be separated from fetal DNA as heteroduplexes.
Table 1
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Example 2 Determining Heterozygosity of the Tandem SNPs As a complement to Example 1, genomic DNA samples from 300 anonymous subjects have been obtained from healthy young adults who are less than 35 years old. The samples are anonymous as the only data obtained were the geographic location of the Red Cross blood donor center, donor gender, and whether or not the donor was 35 and under. These samples were spot-checked to look for the haplotypes seen in the HapMap project.
Example 3 Detecting Fetal DNA from Maternal Serum A cohort of patients who have been confirmed to have trisomy 21 by traditional karyotype analysis are examined. Tandem SNPs are used to demonstrate detection of trisomy in patients. DNA from 20 patients who have been characterized by traditional karyotype analysis to have trisomy 21 are analyzed with the tandem SNP panel. Biological samples, including a buccal (cheek) swab and a blood sample are collected from a cohort of pregnant women. Maternal buccal swab samples are compared to maternal serum to demonstrate that a third (paternal) peak is observed in several of the tandem SNP assays. Approximately 20 maternal buccal swab to maternal serum comparisons are made. To control for experimental artifacts, genomic DNA samples from maternal buccal swabs are utilized for each target sequence. The buccal samples are subjected to the process in parallel with the maternal blood sample. Any artifacts generated by the CDCE/HiFi-PCR procedure (including nonspecific PCR amplification and polymerase-induced mutations) are revealed as background peaks in the buccal swab samples.
Example 4 Detecting Fetal Chromosomal Abnormalities A blinded study is performed where the goal is to detect 20 known trisomy 21 fetuses by assaying maternal serum from 40 patients (previously determined by amniocentesis or CVS) (see Figure 3).
Figure 3 depicts an example of a CDCE electropherogram output with the peaks at full scale. Figure 3 A depicts a sample from maternal buccal swab. Markers exhibiting two alleles are pursued. A baby with trisomy is expected to show either three alleles, evident by three peaks in a 1:1 :1 ratio or two alleles in a 2: 1 ratio. Figure 3B depicts a sample from maternal serum. Markers exhibiting three alleles are informative. Maternal serum from a woman carrying a baby with trisomy is expected to exhibit three alleles, evident by two equal peaks with a third smaller peak if the trisomy occurred during meiosis I (75% of T21 cases) or three alleles with different areas if the trisomy occurred during meiosis II (20% of T21 cases) where areas are: peak, x, and peak + 2x. Figure 3C depicts analysis of a sample from maternal serum. Markers exhibiting three alleles are informative. Maternal serum from a woman with a normal baby with three alleles has three different areas where areas are: peak, x, and peak + x. Interpretation of Results
For the case of the minimum heterozygosity, where both SNPl and SNP2 are hetero∑ygous at their respective loci at a rate of 25%, if 96 tandem SNPs are assayed, an average of 43 markers (44.5%) are expected to be heterozygous (two haplotypes) in the mother. The mother's expected heterozygosity is calculated using the following formula:
H = I- Z p1-2 for i =1 to k alleles where pj = estimated allele frequency.
The allele frequencies at each SNP loci are expected to be 85% and 15% for the majority and minority alleles, respectively, assuming Hardy- Weinberg equilibrium. The desired third haplotype is expected to be present at an average of 6.4 markers (15%) of per maternal-fetal sample tested. Because most loci have a heterozygosity value greater than 25%, for every maternal-fetal sample tested using the panel of 96 tandem SNP assays, greater than about 6.4 markers are most informative. Thus, while a panel of 96 tandem SNPs may be used, 6 or 7 of those tandem SNPs may be informative for any one specific maternal-fetal sample tested, and a 'positive' result from any one of those tandem SNPs is informative.
Finally, in order to diagnose a trisomy, a "positive" tandem SNPs should be identified on both the p and the q arm of chromosome 21. Because of the comparative nature of the basic approach, the tandem SNP assay is predicted to have a detection rate of 95% (those that occur during maternal meiosis) for trisomy 21. If paternal samples are available, non-disjunctions that occur during paternal meiosis can also be detected. Thus, detection rates would be higher (about ~ 99%) with a 0% false positive rate.
Example 5 Tandem SNPs and Primers Table 2 provides exemplary tandem SNPs of the invention and primers that can be used in the methods of the invention to detect the tandem SNPs.
Certain embodiments of the present invention provide primers that can be used to amplify at least one of the SNPs. Certain embodiments of the present invention provide nucleic acid sequences that comprise at least one of the SNPs, e.g., at least one of the tandem SNPs. Table 2
1) Whole sequence ::: rs432114 - rs365433 CC/CT/GC/GT
AACAAATCTTCATCTTGGAATAGCCTGTGAGAATGCCTAATCATCTACGAATgTTACTTT GGCACCATCTACTGGACAgATTAAATAACAACCAACTCACTGTGGATTAGACCTACTTCT ATTTCAG (SEQ ID NO:1)
OLIGO start len tm gc% any 31 seq (SEQ ID NOs: 2, 3)
LEFT PRIMER 20 20 55.08 45.00 3.00 2.00 ATAGCCTGTGAGAATGCCTA
RIGHT PRIMER 107 20 55.30 45.00 5.00 0.00 ATCCACAGTGAGTTGGTTGT SEQUENCE SIZE: 127
INCLUDED REGION SIZE: 127
PRODUCT SIZE: 88, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00 1 AACAAATCTTCATCTTGGAATAGCCTGTGAGAATGCCTAATCATCTACGAATgTTACTTT
61 GGCACCATCTACTGGACAgATTAAATAACAACCAACTCACTGTGGATTAGACCTACTTCT
««««««««««
121 ATTTCAG 2) Whole sequence : : : rs7277033-rs2110153 CC/CT/TC/TT PCR did not work
TTCCTGGAAAACAAAAGTATTTCTTTCATAGCCCAGCTAGCAtGATAAATCAGCgAGTCA GAATTCTAGCTTTGTTGTAAGGTT (SEQ ID NO: 4)
OLIGO start len tm qc% any 3 ' seq (SEQ ID NOs: 5, 6) LEFT PRIMER 2 20 51.63 30.00 5.00 3.00 TCCTGGAAAACAAAAGTATT
RIGHT PRIMER 84 21 51.36 33.33 4.00 0.00 AACCTTACAACAAAGCTAGAA SEQUENCE SIZE: 84 INCLUDED REGION SIZE: 84 PRODUCT SIZE: 83, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 2.00
1 TTCCTGGAAAACAAAAGTATTTCTTTCATAGCCCAGCTAGCAtGATAAATCAGCgAGTCA
61 GAATTCTAGCTTTGTTGTAAGGTT
3) Whole sequence : : : rs2822654-rsl882882 AA/AG/CA/CG CACTAAGCCTTGGGGATCCAGCTGCTTaAGGACTAAGACCgTATCTAGCTCCTTTTAGTA TTTCCACAGCA (SEQ ID NO: 7)
OLIGO start len tm gel any 3 ' seq (SEQ ID NOs: 8, 9) LEFT PRIMER 2 20 60.46 55.00 6.00 2.00 ACTAAGCCTTGGGGATCCAG
RIGHT PRIMER 71 21 54.78 38.10 3.00 0.00 TGCTGTGGAAATACTAAAAGG SEQUENCE SIZE: 71 INCLUDED REGION SIZE: 71 PRODUCT SIZE: 70, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00
1 CACTAAGCCTTGGGGATCCAGCTGCTTaAGGACTAAGACCgTATCTAGCTCCTTTTAGTA
61 TTTCCACAGCA
4) Whole sequence ::: rs368657-rs376635 AA/AG/GA/GG TCCTCCAGAGGTAATCCTGTGATCAGCACTAACaCCACATACCAGCCCTTTCATCAGCTT GTTGGAGAAGCATCTTTACTTCCCgCCAAGCAGTGACCTagataccatCtcacaccagtt agaatcaggatcattaaaaagtcaagaaaaaacag (SEQ ID NO: 10)
OLIGO start len tm 2c% _ any 3' seq (SEQ ID NOs : 11, 12) L LEEFFTT PPRRIIMMEERR 3 3 2 200 5 555. .2200 5 500. .0000 5 5..0000 3 3..000 CTCCAGAGGTAATCCTGTGA
RIGHT PRIMER 117 21 55 .10 47 .62 5.00 2 2..000 tggtςtgagatggtatctAGG
SEQUENCE SIZE: 155
INCLUDED REGION SIZE: 155 PRODUCT SIZE: 115, PAIR ANY COMPL: 3.00, PAIR 31 COMPL: 2.00
1 TCCTCCAGAGGTAATCCTGTGATCAGCACTAACaCCACATACCAGCCCTTTCATCAGCTT »»»»»»»»»»
61 GTTGGAGAAGCATCTTTACTTCCCgCCAAGCAGTGACCTagataccatctcacaccagtt
121 agaatcaggatcattaaaaagtcaagaaaaaacag
5) Whole sequence ::: rs2822731-rs2822732 AA/AG/GA/GG
• TCCAAGTATAATCCATGAATCTTGTTTAAATATAGATCAAaTAAACCACTATACCAAAAA CATCAAAAGACAACTGGGTAAATTTTTTAAATGACTAGCTATTTGATGTTAAgGAAGTAA TGTTACTCTCTTATATACAATTTGAA (SEQ ID NO: 13)
OLIGO start len tm gc% any 3J_ seg (SEQ ID NOs: 14, 15) LEFT PRIMER 6 22 50.35 27.27 6.00 3.00 GTATAATCCATGAATCTTGTTT
RIGHT PRIMER 146 22 45.69 22.73 6.00 1.00 TTCAAATTGTATATAAGAGAGT SEQUENCE SIZE: 146 INCLUDED REGION SIZE: 146 PRODUCT SIZE: 141, PAIR ANY .COMPL: 4.00, PAIR 3' COMPL: 2.00
1 TCCAAGTATAATCCATGAATCTTGTTTAAATATAGATCAAaTAAACCACTATACCAAAAA
' 61 CATCAAAAGACAACTGGGTAAATTTTTTAAATGACTAGCTATTTGATGTTAAgGAAGTAA
121- TGTTACTCTCTTATATACAATTTGAA «««««««««««
6) Whole sequence ::: rs6516899-rs4S5221 CC/CT/TC/TT
ATGGAACCGAAACTTCAAGTAGTTTCATAcGTATCACATTGACAGTTTTCTCTAAGTTTT CtGGTCTTATGACTCGTTGTTTCATTATTAAAACTGTGCCAGTGTATGCATAGGGCTTAG AAATTTTTTAAT (SEQ ID NO: 16)
OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 17, 18)
LEFT PRIMER 1 18 53.87 38.89 4.00 3.00 ATGGAACCGAAACTTCAA RIGHT PRIMER 91 22 52.84 27.27 5.00 1.00 TTAATAATGAAACAACGAGTCA SEQUENCE SIZE: 132 INCLUDED REGION SIZE: 132
PRODUCT SIZE: 91, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00
1 ATGGAACCGAAACTTCAAGTAGTTTCATACGTATCACATTGACAGTTTTCTCTAAGTTTT »»»»»»»»»
61 CtGGTCTTATGACTCGTTGTTTCATTATTAAAACTGTGCCAGTGTATGCATAGGGCTTAG 121 AAATTTTTTAAT
5
7) Whole sequence ::: rs7275381-rsl2627144 GA/GG/TA/TG acaggatccttcctgaagacaccaccttggggagggtgaagGataaagaatttgatcaga aatcaagggtggtgagatacatgttaaggatgaataaactggccttttaggattcttgct -10 aaaAttagacaatgcagaggcaaccacagagtccaag (SBQ ID NO: 19)
OLIGO start len tm gc% any 3' sβq (SEQ ID HOs: 20, 21)
LEFT PRIMER 10 19 55.-53 47.37 4.00 0.00 ttcctgaagacaccacctt RIGHT PRIMER 157 18 54.94 55.56 3.00 2.00 cttggactctgtggttgc 15 SEQOENCK SIZE: 157
INCLUDED REGION SIZE: 157
PRODUCT SIZE: 148, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00 20 1 acaggatccttcctgaagacaccaccttggggagggtgaagGataaagaatttgatcaga
61 aatcaagggtggtgagatacatgttaaggatgaataaactggccttttaggattcttgct
25
121 aaaAttagacaatgcagaggcaaccacagagtccaag
8) Whole sequence : : : rsl999288-rs208897 CC/CT/TC
30
AATTTCCATTAAATCTTGTTCGTTGCTTTACTGAGGCACTGAAGTTACCAATGTTcCACT
GGTTGACCTGC'GGGGCTATCTCTAGGTTATGTTACTCCAGAAAATGAATTGTGTATAAAA GAGGCCTTGGAGGAAGGCGTTTTATTCACATCAGTTGTTTTGCACATTGCTTA (SEQ ID NO: 22)
35
OLIGO start len tm gc% any 3J_ seq (SEQ ID NOs: 23, 24)
LEFT PRIMEE 30 20 54.40 50.00 4.00 2.00 ACTGAGGCACTGAAGTTACC
RIGHT PRIMER 173 20 54.96 35.00 4.00 0.00 TAAGCAATGTGCAAAACAAC SEQUENCE SIZE: 173 40 INCLUDED REGION SIZE: 173
PRODUCT SIZE: 144, PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 0.00
1 AATTTCCATTAAATCTTGTTCGTTGCTTTACTGAGGCACTGAAGTTACCAATGTTCCACT . ■ •
45 >»»»>»»»»»»
61 GGTTGACCTGCGGGGCTATCTCTAGGTTATGTTACTCCAGAAAATGAATTGTGTATAAAA
50 121 GAGGCCTTGGAGGAAGGCGTTTTATTCaCATCAGTTGTTTTGCACATTGCTTA
9) Whole sequence ::: rsl475881-rs7275487 CA/CG/GA/GG PCR did not work CTTTGTGATCCTAGGTTAAAAAACATATTCAAGATAGCTTCAGAATGTTTGGTATACAAgTAGGTCTGGCTAAATATAAGT GTTAGCTTT CTCAAGCATC TAAATGCTGG (SEQ ID NO: 25)
OLIGO start len tm qc3 any 3' seq (SEQ ID NOs: 26, 27)
LEFT PRIMER IQ 20 48.49 25.00 5.00 3.00 GCAGGAAAGTTATTTTTAAT RIGHT PRIMER 179 21 54.70 38.10 4.00 1.00 TGCTTGAGAAAGCTAACACTT SEQUENCE SIZE: 191 INCLUDED REGION SIZE: 191
PRODUCT SIZE: 170, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00
1 TCGGTTTCAGCAGGAAAGTTATTTTTAATAACTTCCCTGTATTTcTTGGTTTCAGTTATT »»»»»»»»»»
61 AATTAACTCATTAATGCTAAACTTTGTGATCCTAGGTTAAAAAACATATTCAAGATAGCT
121 TCAGAATGTTTGGTATACAAgTAGGTCTGGCTAAATATAAGTGTTAGCTTTCTCAAGCAT
181 CTAAATGCTGG
ALTERNATIVE: : ( LESS THAN 5 bp APART )
AAGTTATTTTTAATAACTTCCCTGTATTTcTTGGTTTCAGTTATTAATTAACTCATTAAT GCTAAACTTTGTGATCCTAGGTTAAAAAACATATTCAAGATAGCTTCAGAATGTTTGGTA TACAAgTAGGTCTGGCTAAATATAAGTGTTAGCTTTCTCAAGCATC {SEQ ID NO: 28)
OLIGO start len tm get any 3' seq (SEQ ID NOs: 29, 30)
LEFT PRIMER 6 20 47 .68 25 .00 6 .00 0.00 ATTTTTAATAACTTCCCTGT
RIGHT PRIMER 148 20 49 .30 40 .00 4 .00 0.00 CACTTATATTTAGCCAGACC
SEQUENCE SIZE: 166
INCLUDED REGION SIZE: 166
PRODUCT SIZE: 143, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00
1 AAGTTATTTTTAATAACTTCCCTGTATTTcTTGGTTTCAGTTATTAATTAACTCATTAAT
61 GCTAAACTTTGTGATCCTAGGTTAAAAAACATATTCAAGATAGCTTCAGAATGTTTGGTA'
121 TACAAgTAGGTCTGGCTAAATATAAGTGTTAGCTTTCTCAAGCATC
10) Whole sequence ::: rsl735976-rs2827016 AA/AC/GA/GC
ATTCATTGTGTAGAAAGTGCCTGACTCAGTGTTTGGAAATTGTCTGACTTTTCCTCATAT aTAGTGTGGTTTCATGTTATTGTATATAAGAaCTGACATGAACTCTGTTTACAATAATCT CCCAGTGCCATAAAGACCATAATAAATAATAT (SEQ ID NO: 31) OLIGO start len tm qc% any 3' 3eg (SEQ ID NOs: 32, 33)
LEFT PRIMER 27 20 54.11 - 40.00 4.00 1.00 CAGTGTTTGGAAATTGTCTG
RIGHT PRIMER 129 20 55.17 45.00 3.00 2.00 GGCACTGGGAGATTATTGTA SEQUENCE SIZE: 152
INCLUDED REGION SIZE: 152
PRODUCT SIZE: 103, PAIR ANY COMPL: 5.00, PAIR 3" COMPL: 1.00 1 ATTCATTGTGTAGAAAGTGCCTGACTCAGTGTTTGGAAATTGTCTGACTTTTCCTCATAT
61 aTAGTGTGGTTTCATGTTATTGTATATAAGAaCTGACATGAACTCTGTTTACAATAATCT
121 CCCAGTGCCATAAAGACCATAATAAATAATAT
2nd group ot primers
11) Whole sequence ::: rs447349-rs2824097 CT/TC/TT ( 156 long )
CACTGGGTCCTGTTGTTAAGTACACATAATACCACaCAGGAGAAAATCAGGCTAATTGTA AATGGGCAACCTACTTAATTGTTTCATTAAAAAGCATACAGATTACATTTACACTAtAGC TAGTCTTGTTTGTTTTTTTATTTTGCAAAAGTAATTACGGCCC (SEQ ID NO: 34)
OLIGO start len tm Seg (SEQ ID NOs: 35', 36)
LEFT PRIMER 8 20 47.79 TCCTGTTGTTAAGTACACAT
RRIIGGHHTT PPRRIIMMEERR 163 18 53.29
Figure imgf000032_0001
GGGCCGTAATTACTTTTG ■ SEQUENCE SIZE: 163 INCLUDED REGION SIZE: 163
"PRODUCT SIZE: 156, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00
1 CACTGGGTCCTGTTGTTAAGTACACATAATACCACaCAGGAGAAAATCAGGCTAATTGTA
61 AATGGGCAACCTACTTAATTGTTTCATTAAAAAGCATACAGATTACATTTACACTAtAGC
121 TAGTCTTGTTTGTTTTTTTATTTTGCAAAAGTAATTACGGCCC
12) Whole sequence ::: rs418989- rsl3047336 AC/AT/CC
CTACTCAGTAGGCACTTTGTGTCTAGAAACTTCTGTGTCAACgGTTTTCCCTCTCTCTGG AATTCaTCAGGACAGAAGTGATTGGTGTGGTGGAAGAGGGTTGTGSTA (SEQ ID NO: 37)
OLIGO 3' seg (SEQ ID NOs: 38, 39) LEFT PRIMER 3.00 ACTCAGTAGGCACTTTGTGTC RIGHT PRIMER
Figure imgf000032_0002
0.00 TCTTCCACCACACCAATC SEQUENCE SIZE: 108 INCLUDED REGION SIZE: 108
PRODUCT SIZE: 95, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00 1 CTACTCAGTAGGCACTTTGTGTCTAGTiAACTTCTGTGTCAACgGTTTTCCCTCTCTCTGG
61 AATTCaTCAGGACAGAAGTGATTGGTGTGGTGGAAGAGGGTTGTGSTA
«««««««««
13) Whole sequence ::: rs987980- rs987981 AG/GG/GT
TGGCTTTTCAAAGGTAAAATTTACTaAGTGTATTAATATTTTACCAATTTCCAGCCAGGA GAGTATGAATGTTGCATTATTACATTGCTTTGAAACAAAGCATTAgTCTTAATTCAGAAG TTTAAATTCAGATGTTAACGTTGC (SEQ ID NO: 40)
OLIGO start len tm gc% any 3J_ seq (SEQ ID NOs: 41, 42)
LEFT PRIMER 1 19 53.67 31.58 6.00 2.00 TGGCTTTTCAAAGGTAAAA
RIGHT PRIMER 144 21 54.59 33.33 6.00 3.00 GCAACGTTAACATCTGAATTT SEQUENCE SIZE: 144
INCLUDED REGION SIZE: 144
PRODUCT SIZE: 144, PAIRANY COMPL: 6.00, PAIR 3' COMPL: 3.00 1 TGGCTTTTCAAAGGTAAAATTTACTaAGTGTATTAATATTTTACCAATTTCCAGCCAGGA
61 GAGTATGAATGTTGCATTATTACATTGCTTTGAAACAAAGCATTAgTCTTAATTCAGAAG
121 TTTAAATTCAGATGTTAACGTTGC
14) Whole sequence ::: rs4143392- rs4143391 CA/CG/GA/GG
TAAGTATTGAAGAAAGGAGAATTTAAATTACTTCATATACctgataaaggaaaacatata CAAGGCAAATAAACATCTTAGATCATGACATATAAAATAATAGATTATTA (SEQ ID NO: 43)
OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 44, 45) LEFT PRIMER 7 20 49.56 25.00 4.00 4.00 TTGAAGAAAGGAGAATTTAA
RIGHT PRIMER 98 22 45.86 22.73 6.00 3.00 ATTTTATATGTCATGATCTAAG SEQUENCE SIZE: 110 INCLUDED REGION SIZE: 110 PRODUCT SIZE: 92, PAIRANY COMPL: 4.00, PAIR 3' COMPL: 1.00
1 TAAGTATTGAAGAAAGGAGAATTTAAATTACTTCATATACctgataaaggaaaacatata
61 CAAGGCAAATAAACATCTTAGATCATGACATATAAAATAATAGATTATTA
15) Whole sequence ::: rsl691324- rsl3050434 CG/TA/TG ( 4 bp apart for right primer) TGCAGAGATTACAGGTGTGAGCCACCGTGCCCAGCCTCATAACcGTTTCAACTACTTTTT CACTTGACAAGCAGATGTGAAGTTAACAAAGTCACCCATATTTGAAATAAAGATAGTATA TTCCTGGGGtAGGCAGAGGCAGTTGAGGATCATGAAATAACTATG (SEQ ID HO: 46)
OLIGO start len tm qc% any 3' βeq (SEQ ID HOs: 47, 48)
LEET PRIMER 4 19 49.78 47.37 4.00 4.00 AGAGATTACAGGTGTGAGC
RIGHT PRIMER 153 19 54.61 47.37 4.00 0.00 ATGATCCTCAACTGCCTCT SEQUENCE SIZE: 165 INCLUDED REGION SIZE: 165
PRODUCT SIZE: 150, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00
1 TGCAGAGATTACAGGTGTGAGCCACCGTGCCCAGCCTCATAACCGTTTCAACTACTTTTT »»»»»»>»?>»
61 CACTTGACAAGCAGATGTGAAGTTAACAAAGTCACCCATATTTGAAATAAAGATAGTATA
121 TTCCTGGGGtAGGCAGAGGCAGTTGAGGATCATGAAATAACTATG
16) Whole sequence : : : rsll909758-rs9980111 (159 bp long ) AG/AT /GT TGCAATGAAACTCAAAAGAGAAAAGTTAACAGGTGCAAaAGGTAGTTTTATTATAAAAGG AGGGTAGGCAACAAGAATATGTTTAATTTTTCTTCCTTTTCATGAGTAAGGACAAGAGTg TCATATATGTGaatatttttatttaattttaaGTAGAAATCTGTTTTTAAAATATGGG (SEQ ID NO: 49)
OLIGO start len tm ctc% any 3' seq (SEQ ID NOs: 50, 51) LEFT PRIMER 6 20 49.91 30.00 3.00 0.00 TGAAACTCAAAAGAGAAAAG
RIGHT PRIMER 164 20 42.77 20.00 6.00 4.00 ACAGATTTCTACttaaaatt SEQUENCE SIZE: 178 INCLUDED REGION SIZE: 178 PRODUCT SIZE: 159, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 3.00
1 TGCAATGAAACTCAAAAGAGAAAAGTTAACAGGTGCAAaAGGTAGTTTTATTATAAAAGG
61 AGGGTAGGCAACAAGAATATGTTTAATTTTTCTTCCTTTTCATGAGTAAGGACAAGAGTg
121 TCATATATGTGaatatttttatttaattttaaGTAGAAATCTGTTTTTAAAATATGGG
««««««««««
17) Whole sequence : : : rs854613-rs854614 AA/AG/TG
CCACCATTCATCAAAACTTTGATACTGGACTCAATTGTGAATTTGaCTTGAAATTTGATA ATGCTTTTGTTTTACTgTTCTGCTCAGCAAAATAGTACATGT (SEQ ID NO: 52)
OLIGO start len tm gc% any 3^_ seq (SEQ ID NOs: 53, 54)
LEFT PRIMER 12 20 49.40 35.00 6.00 1.00 CAAAACTTTGATACTGGACT
RIGHT PRIMER 102 19 46.05 31.58 6.00 1.00 ACATGTACTATTTTGCTGA .
SEQUENCE SIZE: 102 INCLUDED REGION SIZE: 102
PRODUCT SIZE: 91, PAIR ANY COMPL: 5.00, PAIR 31 COMPL: 1.00 1 CCACCATTCATCAAAACTTTGATACTGGACTCAATTGTGAATTTGaCTTGAAATTTGATA
61 ATGCTTTTGTTTTACTgTTCTGCTCAGCAAAATAGTACATGT
3rd group order primers from 18 - 25
18) Whole sequence ::: rs2826225-rs2826226 AA/GA/GC GCCTGCATAAAGTGAGGATGGTGTAGTAATTGGGTATCTCCAGTTATAAACACAAaAAGC ATGATAGAGCTGGGAcTGTGATTGCAGGAAAGCAATAGTCACTCCAAAAGGAGATCCTCA TGATATGAATACGGAAGAAACAATATTTCCTGCTAATGTAGTAGCC (SEQ ID NO: 55)
OLIGO start len tin qc% any 3J_ seg (SEQ ID NOs: 56, 57) LEFT PRIMER 2 20 58.17 50.00 4.00 0.00 CCTGCATAAAGTGAGGATGG
RIGHT PRIMER 120 21 59.27 47.62 6.00 0.00 TGAGGATCTCCTTTTGGAGTG SEQUENCE SIZE: 166 INCLUDED REGION SIZE: 166 PRODUCT SIZE: 119, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 3.00
1 GCCTGCATAAAGTGAGGATGGTGTAGTAATTGGGTATCTCCAGTTATAAACACAAaAAGC
61 ATGATAGAGCTGGGAcTGTGATTGCAGGAAAGCAATAGTCACTCCAAAAGGAGATCCTCA
121 TGATATGAATACGGAAGAAACAATATTTCCTGCTAATGTAGTAGCC
19) Whole sequence ::: rs2826842-rs232414 CA/CG/TA/TG
GCAAAGGGGTACTCTATGTAATGAAcATgacctggcagtactgacatctcctgagggact gttagaagtgcagactcttgtatcttttctcaagtctatgaaatctagacttcattttaa caagatgacccgatatttacatacacattaaagt (SEQ ID NO: 58)
OLIGO start len tra gc% any 3' seq (SEQ ID NOs: 59, 60)
LEFT PRIMER 1 20 52.04 45.00 4.00 2.00 GCAAAGGGGTACTCTATGTA
RIGHT PRIMER 135 20 53.29 35.00 4.00 3.00 tatcgggtcatcttgttaaa SEQUENCE SIZE: 154
INCLUDED REGION SIZE: 154
PRODUCT SIZE: 135, PAIR ANY COMPL: 3.00, PAIR 31 COMPL: 2.00 1 GCAAAGGGGTACTCTATGTAATGAAcATgacctggcagtactgacatctcctgagggact »»»»»»»»»»
61 gttagaagtgcagactcttgtatcttttctcaagtctatgaaatctagacttcattttaa
<«« 121 caagatgacccgatatttacatacacattaaagt
20) Whole sequence ::: rsl980969-rsl980970 AA/AG/TA/TG
GTATCTAACAAAGCTCTGTCCAAAATTTTGAATTTCTCGTTAAAaGCATCATGATTATAG AACAGAGGTTACAATCAATTATTCAGTCACACAATCACTCTCATCAGTCATTAAGGTGCg TACCTGGTGTTCCAGTTATTCAGTGTGGTATAACAAACTACCTGGAACTTAATG (SEQ ID NO: 61>
OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 62, 63)
LEFT PRIMER 4 22 56.88 36.36 8.00 2.00 TCTAACAAAGCTCTGTCCAAAA
RIGHT PRIMER 148 21 56.12 42.86 3.00 1.00 CCACACTGAATAACTGGAACA SEQUENCE SIZE: 174 INCLUDED REGION SIZE: 174
PRODUCT SIZE: 145, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00
1 GTATCTAACAAAGCTCTGTCCAAAATTTTGAATTTCTCGTTAAAaGCATCATGATTATAG
61 AACAGAGGTTACAATCAATTATTCAGTCACACAATCACTCTCATCAGTCATTAAGGTGCg
121 TACCTGGTGTTCCAGTTATTCAGTGTGGTATAACAAACTACCTGGAACTTAATG
4th group 21) Whole sequence ::: rsl89900-rs2221492
AGAGTGGTTAAGTGACTTGATCAATTCCTCA GGTGGGGATTCAAGCTCTTAAAGCTGTAG ACTATGTCGTCCAAACAAACACTGACATGAATATGACTTCCAATAGGCAAGAAAAGAGGC CTAGGTCgAGATACTGCAAGACATGCAAGCAATCTAGTAATGGCATAAAACCTGCTATCC GAATTGGCTAAAATTATGTATT (SEQ ID NO: 64)
OLIGO start len tm gel any 3' seq (SEQ ID NOs: 65, 66)
LEFT PRIMER 32 20 59.13 50.00 4.00 2.00 GGTGGGGATTCAAGCTCTTA RIGHT PRIMER 180 22 59.38 40.91 5.00 3.00 GGATAGCAGGTTTTATGCCATT SEQUENCE SIZE: 202
INCLUDED REGION SIZE: 202
PRODUCT SIZE: 149, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00 1 AGAGTGGTTAAGTGACTTGATCAATTCCTCAGGTGGGGATTCAAGCTCTTAAAGCTGTAG
61 ACTATGTCGTCCAAACAAAcACTGACATGAATATGACTTCCAATAGGCAAGAAAAGAGGC
121 CTAGGTCgAGATACTGCAAGACATGCAAGCAATCTAGTAATGGCATAAAACCTGCTATCC
181 GAATTGGCTAAAATTATGTATT 22) Whole sequence ::: rs2827920-rs2827921 TTCTTTCTCACACAATGGGTTCCATTCCCACTACTACTCCATTCAAATTGAAGTGCCTTC AATGATTATTAAAAAACTCTCTTTAAAATAGCTCACGTAACCTTACATCCTTTGACTGAG GCTCAACTCATGTCAATGCTTCAGTATCAACTTTTC (SEQ ID KO: 67)
OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 68, 69) LEFT PRIMER 14 21 59.93 47.62 7.00 0.00 AATGGGTTCCATTCCCACTAC RIGHT PRIMER 125 20 58.96 50.00 7.00 1.00 TGAGCCTCAGTCAAAGGATG SEQUENCE SIZE: 156 INCLUDED REGION SIZE: 156 PRODUCT SIZE: 112, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00
1 TTCTTTCTCACACAATGGGTTCCATTCCCACTACTACTCCATTCAAATTGAAGTGCCTTC
61 aATGATTATTAAAAAACTCTCTTTAAAATAGCTCAcGTAACCTTACATCCTTTGACTGAG
121 GCTCAACTCATGTCAATGCTTCAGTATCAACTTTTC <««
23) Whole sequence : : : rsl98047-rs2827935
ATTTGTAATAACATTTAGTAAGTATTTATTTGAGGAGTTTGAATTTTGTTCTTGTTTATC TTGTTCTCTTTCTTcGTAGATTAGTTGGTGTTAACATCAATAGGATAACCCTTTCTTTCA GCATATGTGAATGAAATaAACCAATTATTGCCACTTTCCAGGTTAACCAGAATATACATA GATACGAGGACAGTGGACTGTT (SEQ ID NO: 70)
OLIGO . start "len tm qc% any 3' seq (SEQ ID NOs: 71, 72)
LEFT PRIMER 30 22 56.07 31.82 4.00 1.00 TTGAGGAGTTTGAATTTTGTTC RIGHT PRIMER 164 20 57.22 40.00 3.00 1.00 AACCTGGAAAGTGGCAATAA SEQUENCE SIZE: 202 INCLUDED REGION SIZE: 202
PRODUCT SIZE: 135, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 2.00
1 ATTTGTAATAACATTTAGTAAGTATTTATTTGAGGAGTTTGAATTTTGTTCTTGTTTATC
61 TTGTTCTCTTTCTTcGTAGATTAGTTGGTGTTAACATCAATAGGATAACCCTTTCTTTCA
121 GCATATGTGAATGAAATaAACCAATTATTGCCACTTTCCAGGTTAACCAGAATATACATA
181 GATACGAGGACAGTGGACTGTT
24) Whole sequence ::: rs9978999-rs9979175 tagggcagagagagcaagcaagctctctaccttctcatataagggcactaatcccaccat gaaggcgccactgtcatgacctgattatgtcacaaagaccccggggcaaatattaccact Gtgaggagtacagttttagcatgtgaattttggaagaacacaaacatttag (SEQ ID NO: 73) OLIGO start len tm gc4 any 3' seq (SEQ ID HOs: 74, 75)
LEFT PRIMER 14 21 58.50 52.38 4.00 0.00 gcaagcaagctctctaccttc RIGHT PRIMER 160 22 59.98 36.36 4.00 2.00 tgttcttccaaaattcacatgc SEQUENCE SIZE: 171 INCLUDED REGION SIZE: 171
PRODUCT SIZE: 147, PAIR ANY COMPL: 3.00, PAIR 31 COMPL: 0.00
1 tagggcagagagagcaagcaagctctctaccttctcatataagggcactaatcccaccat
61 gaaggcgccactgtcatgacCtgattatgtcacaaagaccccggggcaaatattaccact
121 Gtgaggagtacagttttagcatgtgaattttggaagaacacaaacatttag . <««««««««««<
25) Whole sequence ::: rsl034346-rsl2481852
ATTCTAATTTTAAATATCATTGATGTAGAACATTCTATTTCACTATTCCTTCATTTTATT aTTATGGGAAATTATATACAGTTCTCCAGATTTTTAAAGCCTTGCTAACATGTTTTAAGT CACACAAATATTCTcCTGTGGGAAAATGACAGTAATTTAGTGTGCAACAATTATATAGAA CTATTTTTCAAACTT (SEQ ID NO: 76)
OLIGO start len tm get any 3' seq (SEQ ID NOs: 77, 78) LEFT PRIMER 37 21 50.04 23.81 2.00 0.00 ATTTCACTATTCCTTCATTTT
RIGHT PRIMER 173 22 50.19 27.27 6.00 3.00 TAATTGTTGCACACTAAATTAC SEQUENCE SIZE: 195 INCLODED REGION SIZE: 195 PRODUCT SIZE: 137, PAIR ANY COMPL: 4.00, PAIR 3" COMPL: 2.00
1 ATTCTAATTTTAAATATCATTGATGTAGAACATTCTATTTCACTATTCCTTCATTTTATT
61 aTTATGGGAAATTATATACAGTTCTCCAGATTTTTAAAGCCTTGCTAACATGTTTTAAGT
121 CACACAAATATTCTcCTGTGGGAAAATGACAGTAATTTAGTGTGCAACAATTATATAGAA
181 CTATTTTTCAAACTT
5th group
26) Whole sequence : : : rs7509629-rs2828358
ACTGTCATGGACTTAAACAATTGTCTTTGAATTGTCTTTTTTCATACTTTTATTTGCATC TTTcCACTAAAAAGATGgCACAAAGTAATCCTAGTTTACATTTTTTACCATGTAATTCCA TATTACTTTTTCCTGAJiA (SEQ ID NO: 79)
OLIGO start len tm gc% any 3' seq (SEQ ID NOs: 80, 81)
LEFT PRIMER 1 20 50.46 35.00 4.00 0.00 ACTGTCATGGACTTAAACAA RIGHT PRIMER 137 22 53.49 27.27 4.00 0.00 TTCAGGAAAAAGTAATATGGAA SEQUENCE SIZE: 138 INCLUDED REGION SIZE: 138
PRODUCT SIZE: 137, PAIRANY COMPL: 4.00, PAIR 31 COMPL: 0.00
1 ACTGTCATGGACTTAAACAATTGTCTTTGAATTGTCTTTTTTCATACTTTTATTTGCATC
61 TTTCCACTAAAAAGATGgCACAAAGTAATCCTAGTTTACATTTTTTACCATGTAATTCCA ««<
121 TATTACTTTTTCCTGAAA
6th group
27) Whole sequence ::: rs4817013-rs7277036 aaagaaaaaaaagccacagaaatcagtcctagagaaaacCgatctatgagctgcctgaAa ataattataaaataactatcataaaaatgcccagtgagatataagaaaacacagacaac (SEQ ID NO: 82)
OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 83, 84)
LEFT PRIMER 8 21 56.10 38.10 4.00 2.00 aaaaagccacagaaatcagtc
RIGHT PRIMER 107 22 55.60 36.36 4.00 2.00 ttcttatatctcactgggcatt SEQUENCE SIZE: 119
INCLUDED REGION SIZE: 119
PRODUCT SIZE: 100, PAIR ANY COMPL: 5.00, PAIR 31 COMPL: 1.00 1 aaagaaaaaaaagccacagaaatcagtcctagagaaaacCgatctatgagctgcctgaAa
61 ataattataaaataactatcataaaaatgcccagtgagatataagaaaacacagacaac
28) Whole sequence ::: rs9981121-rs2829696
CAAGGTCAGAGAAGTTATCTTGGATGGTAGAAGAGAAGAAAGGAGAAGAAaGGATAAGCA GAAAATCAAAAAGGGCATAAAAAAATTACTGGgGAAAATAATTCTTAGTCACTCACCATT TCTTATGTTTGTGAAAACAGAAA (SEQ ID NO: 85)
OLIGO start len tin gcS any 3_^ seq (SEQ ID NOs: 86, 87)
LEFT PRIMER 22 22 56.24 45.45 2.00 0.00 GGATGGTAGAAGAGAAGAAAGG
RIGHT PRIMER 134 22 55.74 31.82 4.00 1.00 TCACAAACATAAGAAATGGTGA SEQUENCE SIZE: 143 . . . . INCLUDED REGION SIZE: 143
PRODUCT SI2E: 113, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00 1 CAAGGTCAGAGAAGTTATCTTGGATGGTAGAAGAGAAGAAAGGAGAAGAAaGGATAAGCA
»»»»»»»»»»»
61 GAAAATCAAAAAGGGCATAAAAAAATTACTGGgGAAAATAATTCTTAGTCACTCACCATT
5 ««««
121 TCTTATGTTTGTGAAAACAGAAA «««««««
10 29) Whole sequence ::: rs455921-rs2898102 gaccacaattcacaaatgcaaagatgcagaaccaacctaagtggccaCtgactaatgaga ggataaagaagatgtggcatatataTatcagggactactactcagccattacaaggaaca aaataatgtcttttgc (SEQ ID NO: 88)
15
OLIGO start len tm gc% any 3' seq (SEQ ID NOs: 89, 90)
LEFT PRIMER 17 20 59.85 45.00 4.00 0.00 tgcaaagatgcagaaccaac RIGHT PRIMER 123 22 59.63 36.36 2.00 1.00 ttttgttccttgtaatggctga SEQUENCE SIZE: 136 20 INCLUDED REGION SIZE: 136
PRODUCT SIZE: 107, PAIR ANY COMPL: S.00, PAIR 31 COMPL: 1.00
1 gaccacaattcacaaatgcaaagatgcagaaccaacctaagtggccactgactaatgaga
61 ggataaagaagatgtggcatatataTatcagggactactactcagccattacaaggaaca
30 121 aaataatgtcttttgc
30) Whole sequence ::: rs2898102- rs458848
35 gaccacaattcacaaatgcaaagatgcagaaccaacctaagtggccactgactaatgaga ggataaagaagatgtggcatatataCatcagggactactTctcagccattacaaggaaca aaataatgtcttttgcaacaacttggatagagctggaggc (SEQ ID NO: 91)
OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 92, 93)
40 LEFT PRIMER 17 20 59.85 45.00 4.00 0.00 tgcaaagatgcagaaccaac RIGHT PRIMER 160 21 59.86 52.38 4.00 3.00 gcctccagctctatccaagtt SEQUENCE SIZE: 160 INCLUDED REGION SIZE: 160
45 PRODUCT SIZE: 144, PAIRANY COMPL: 4.00, PAIR 3' COMPL: 3. 00
1 gaccacaattcacaaatgcaaagatgcagaaccaacctaagtggccactgactaatgaga
50 61 ggataaagaagatgtggcatatataCatcagggactactTctcagccattacaaggaaca
121 aaataatgtcttttgcaacaacttggatagagctggaggc 31) Whole sequence : : : rs961301-rs2830208
AATCCTAGACCTTGGATTGCAAGAGACTCCTTAATATCTTCCCATGTCCACATTTCCTTC 5 ACATAGTTTGAATGTGGCTTCTATTATATACAGATACAAGATTCAAATCCAACCTCTAtG ATGACTGGTCTTGTGAATAAGCAGAAGAGGCACTAACAAT (SEQ ID NO: 94)
OLIGO start len tm get any 3' seq (SEQ ID NOs: 95, 96)
LEFT PRIMER 29 22 57.95 40.91 4.00 2.00 CCTTAATATCTTCCCATGTCCA 10 RIGHT PRIMER 160 22 57.35 40.91 3.00 0.00 ATTGTTAGTGCCTCTTCTGCTT SEQUENCE SIZE: 160 INCLUDED REGION SIZE: 160
PRODUCT SIZE: 132, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00
15
1 AATCCTAGACCTTGGATTGCAAGAGACTCCTTAATATCTTCCCATGTCCACATTTcCTTC
»»»»»»»»»»»
61 ACATAGTTTGAATGTGGCTTCTATTATATACAGATACAAGATTCAAATCCAACCTCTAtG
20
121 ATGACTGGTCTTGTGAATAAGCAGAAGAGGCACTAACAAT
25 32) Whole sequence ::: rs2174536-rs458076
AAGAGAAGTGAGGTCAGCAGCTGCAAGCCACCTCCGTCATTTAGAAAAGCTTCaTGATGT AGTGTGTCGTTTCGATGTGACACTGTCTCACAGAGTTAAAATGATGTtAAGGAACTGTTC AATGGAAATTTAGAAATTTCTCTTTTTCTCAATTTTAGTGTA (SEQ ID NO: 97)
30
OLIGO start len tin gc3 any 3^_ seq (SEQ ID NOs: 98, 99)
LEFT PRIMER 3 20 57.31 55.00 5.00 5.00 GAGAAGTGAGGTCAGCAGCT
RIGHT PRIMER 136 22 53.92 27.27 6.00 2.00 TTTCTAAATTTCCATTGAACAG SEQUENCE SIZE: 162 35 INCLUDED REGION SIZE: 162
PRODUCT SIZE: 134, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00
1 AAGAGAAGTGAGGTCAGCAGCTGCAAGCCACCTCCGTCATTTAGAAAAGCTTCaTGATGT ΛCt
61 AGTGTGTCGTTTCGATGTGACACTGTCTCACAGAGTTAAAATGATGTtAAGGAACTGTTC
45 121 AATGGAAATTTAGAAATTTCTCTTTTTCTCAATTTTAGTGTA
33) Whole sequence : : : rs432557-rsl012766
50 ATGGCTGAATAGTATTCCCTTGTGTATATATCTaTTTATCCTTTTATTCATTGATGGACA CTTAGGCTGATTTTCTCTCTTCTCATGGCTGGCTTCTCATCACCCTTTGGTCCTCCTGTA TCCTCgTGTAATAAAGCTCTTCCCCAATATCTCGATAGAT (SEQ ID NO: 100)
OLIGO start len tm gc% any 3' seq (SEQ ID MOs: 101, 102) LEFT PRIMER 3 22 57.77 45.45 9.00 0.00 GGCTGAATAGTATTCCCTTGTG
RIGHT PRIMER 155 20 59.22 50.00 4.00 2.00 TCGAGATATTGGGGAAGAGC SEQUENCE SIZE: 160 INCLUDED REGION SIZE: 160
PRODUCT SIZE: 153, PAIR ANY COMPL: 5.00, PAIR 31 COMPL: 1.00
1 ATGGCTGAATAGTATTCCCTTGTGTATATATCTaTTTATCCTTTTATTCATTGATGGACA »»»»»»»»»»»
61 CTTAGGCTGATTTTCTCTCTTCTCATGGCTGGCTTCTCATCACCCTTTGGTCCTCCTGTA
121 TCCTCgTGTAATAAAGCTCTTCCCCAATATCTCGATAGAT ««««««««««
34) Whole sequence ::: rsl0222076-rsl0222075 catfcttaacttgatta cctccacaaagactafctccagaataaggttatgttctgaggtat taggggttacAacttcaacatatgaattttgagtggacacaattcaacccatagcaCCTC CGTGTAAGAGCTGGGAAGGGAAAGTGGCTAAGTTGTGCAAATGTGCACATTGGTTGGAGA TGATTAACTTCTGGCATGT (SEQ ID NO: 103)
OLIGO start len tm gel any 3' seq (SEQ ID NOs: 104, 105) LEFT PRIMER 17 22 58.32 45.45 4.00 2.00 cctccacaaagactattccaga RIGHT PRIMER 146 20 60.76 55.00 4.00 2.00 CACTTTCCCTTCCCAGCTCT SEQUENCE SIZE: 199 INCLUDED REGION SIZE: 199 PRODUCT SIZE: 130, PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 3.00
1 cattttaacttgattacctccacaaagactattccagaataaggttatgttctgaggtat
61 taggggttacAacttcaacatatgaattttgagtggacacaattcaacccatagcaCCTC
121 CGTGTAAGAGCTGGGAAGGGAAAGTGGCTAAGTTGTGCAAATGTGCACATTGGTTGGAGA ««««««««««
181 TGATTAACTTCTGGCATGT
35) Whole sequence ::: rsll088023-rsll088024 agggggaaattggcaatctgattctaaaattcataCggaaaaaaacaatggagttagaat aactaaaacaagtccgaaaaagaaaaagaaatggaggactaatgctacctgatttcaagt cttatcTtataaatctacatcaataaaggacaagttg (SEQ ID NO: 106) O OLLIIGGOO ssttaarrtt lleenn ttmm ggcc%% _ aannyy 3 3J'. seq (SEQ ID NOs: 107, 108)
LEFT PRIMER 6 20 54 .34 35.00 7. 00 3 3..000 gaaattggcaatctgattct
RIGHT PRIMER 157 21 51 .94 33.33 5. 00 0 0..000 caacttgtcctttattgatgt
SEQUENCE SIZE: 157
INCLUDED REGION SIZE: 157 PRODUCT SIZE: 152,' PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 0.00
1 agggggaaattggcaatctgattctaaaattcataCggaaaaaaacaatggagttagaat >>»»»»»»»»»
61 aactaaaacaagtccgaaaaagaaaaagaaatggaggactaatgctacctgatttcaagt
121 cttatcTtataaatctacatcaataaaggacaagttg
36) Whole sequence ::: rsl011734-rsl011733 TCTGTGTTTGTCTATGTTGATAAAACATTGAAATGCCAaATAGCTCAAAGGTCATTCACT TAAGAAATCTAAGTACTGATAACATCTTAGCCCCGATTCTTCATAGGCATTGTTAAGCCT ATTATAATTTTGGTtCAGAGAGAAGGTAAACTATATTCCAGACAGGCATATAA (SEQ ID NO: 109)
OLIGO start len tm qc% any 3J_ se^ (SEQ ID NOs: 110, 111)
LEFT PRIMER 12 22 50.06 22.73 6.00 2.00 CTATGTTGATAAAACATTGAAA
RIGHT PRIMER . 167 20 51.09 40.00 4.00 2.00 GCCTGTCTGGAATATAGTTT SEQUENCE SIZE: 173 INCLUDED REGION SIZE: 173
PRODUCT SIZE: 156, PAIR ANY COMPL: 5.00, PAIR 31 COMPL: 3.00
1 TCTGTGTTTGTCTATGTTGATAAAACATTGAAATGCCAaATAGCTCAAAGGTCATTCACT
61 TAAGAAATCTAAGTACTGATAACATCTTAGCCCCGATTCTTCATAGGCATTGTTAAGCCT
121 ATTATAATTTTGGTtCAGAGAGAAGGTAAACTATATTCCAGACAGGCATATAA ««««««««<«<
37) Whole sequence ::: rs2831244-rs9789838
TGCAGGGCATATAATCTAAGCTGTAAACGTCCTGTcAGAAGACAACATATTCATCTTGCT AAGGTtTAAGCTATATGACTGGCACTGTGCTCAACTCAGAGTCATTGAATGAACAGTATT TATTTA (SEQ ID NO: 112)
OLIGO start len tm get, any 3^ sβ3 <SE0- ID NOs: 113' 114>
LEFT PRIMER 3 22 55.40 40.91 5.00 3.00 CAGGGCATATAATCTAAGCTGT RIGHT PRIMER 107 21 55.99 47.62 7.00 2.00 CAATGACTCTGAGTTGAGCAC SEQUENCE SIZE: 126 INCLUDED REGION SIZE: 126
PRODUCT SIZE: 105, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00
1 TGCAGGGCATATAATCTAAGCTGTAAACGTCCTGTcAGAAGACAACATATTCATCTTGCT
61 AAGGTtTAAGCTATATGACTGGCACTGTGCTCAACTCAGAGTCATTGAATGAACAGTATT 121 TATTTA .
38) Whole sequence ::: rs8132769-rs2831440
TTCACATTATTCCCTTAAAATAAACTCTCTCCCTCCCCTCTCCCGTCTCAaCCTTGTCCC TTTCTTTATATAATGGGTAATtCGTTAATGTCAGCAGAATAGTTTTGGGGCCATAATGGC AAGTATCACGTG (SEQ ID NO: 115)
OLIGO start len tm qc% any 3J. seq (SEQ ID NOs: 116, 117)
LEFT PRIMER 23 19 56.84 57.89 1.00 0.00 AACTCTCTCCCTCCCCTCT
RIGHT PRIMER 115 20 56.24 40.00 4.00 2.00 TATGGCCCCAAAACTATTCT SEQUENCE SIZE: 132
INCLUDED REGION SIZE: 132
PRODUCT SIZE: 93, PAIR ANY COMPL: 2.00, PAIR 31 COMPL: 0.00 1 TTCACATTATTCCCTTAAAATAAACTCTCTCCCTCCCCTCTCCCGTCTCAaCCTTGTCCC
61 TTTCTTTATATAATGGGTAATtCGTTAATGTCAGCAGAATAGTTTTGGGGCCATAATGGC
121 AAGTATCACGTG
39) Whole sequence ::: rs8134080-rs2831524 .
TCAGGAAGCAACAAGTACTGGGCAGATTGATACTGTAGCTaGGCTCTAGCTCTATACCTC TAGAATaaatgttacaaactagcaacttgaaagctaaacctggcccacag (SEQ ID NO: 118)
OLIGO start len tm 2 £± _ an 2 3' seq (SEQ ID NOs: 119, 120) L LEEFFTT PPRRIIMMEERR 1 111 2 200 5 555. .7755 4 455. .0000 6 6..0 000 2 2..000 ACAAGTACTGGGCAGATTGA
RIGHT PRIMER 104 20 56 .27 45 .00 4. 00 2 2..000 gccaggtttagctttcaagt
SEQUENCE SIZE: 110
INCLUDED REGION SIZE: 110 PRODUCT SIZE: 94, PAIR ANY COMPL: 4.00, PAIR 3" COMPL: 3. 00
1 TCAGGAAGCAACAAGTACTGGGCAGATTGATACTGTAGCTaGGCTCTAGCTCTATACCTC
61 TAGAATaaatgttacaaactagcaacttgaaagctaaacctggcccacag
««««««««««
40) Whole sequence ::: rs4817219-rs4817220 tggttcttgagaattttatatcaggagaaacactgtcagtCtgtattgaaaggaacagag aaaatTcgaaattaaagaagactattaaacctccaaaattctggca (SEQ ID NO: 121)
OLIGO • start . len tm gel- any 3J_ seg (SEQ ID NOS: 122, 123)
LEFT PRIMER 14 22 51.54 31.82 4.00 3.00 ttttatatcaggagaaacactg RIGHT PRIMER 104 21 55.03 33.33 8.00 2.00 ccagaattttggaggtttaat SEQUENCE SIZE: 106 INCLUDED REGION SIZE: 106 PRODUCT SIZE: 91, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00
1 tggttcttgagaattttatatcaggagaaacactgtcagtctgtattgaaaggaacagag
61 aaaatTcgaaattaaagaagactattaaacctccaaaattctggca
41) Whole sequence ::: rs2250911-rs2250997 GCATCAAACTACACACTGTCATTCCTCCTTTATCTCCAAAAGCTTGAAAATTCCTCACTT GTaTCTCATTCTTTCTCTCTTAGAAAACTGATCACCTCTGATGAATTAgAACGGAATGAC CAAGCTTTGGGAGAGGCAAAAGAATCTCGGTGTTAAAGACTCAGAGTTTAA(SEQIDNO: 124)
OLIGO start len tm gc% any 3J_ seq (SEQ ID NOs: 125, 126) LEHT PRIMER 17 22 58.65 40.91 3.00 0.00 TGTCATTCCTCCTTTATCTCCA
RIGHT PRIMER 144 20 59.42 45.00 4.00 2.00 TTCTTTTGCCTCTCCCAAAG SEQUENCE SIZE: 171 INCLUDED REGION SIZE: 171 PRODUCT SIZE: 128, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00
1 GCATCAAACTACACACTGTCATTCCTCCTTTATCTCCAAAAGCTTGAAAATTCCTCACTT
61 GTaTCTCATTCTTTCTCTCTTAGAAAACTGATCACCTCTGATGAATTAgAACGGAATGAC
121 CAAGCTTTGGGAGAGGCAAAAGAATCTCGGTGTTAAAGACTCAGAGTTTAA ««««««««««
42) Whole sequence ::: rs2831899-rs2831900
TTGAAAATTAAGAAACCCTGGCACAGTGTTGACTGGAGCCaCTTACCTTAATAGAAAATA
AAGCTCACATATATCCATAATGAAAAGCAGAGACCAGCACAACCATAGTCACCTGACAGT TTtAAAATCCAAGGCCAGGATCTTCTCAACTCAGGCCCACTCA (SEQ ID NO: 127)
OLIGO start len tin gcS any 3' sβq (SEQ ID NOs: 128, 129)
LEFT PRIMER 15 20 60.63 55.00 6.00 2.00 ACCCTGGCACAGTGTTGACT RIGHT PRIMER 159 20 59.80 50.00 4.00 2.00 TGGGCCTGAGTTGAGAAGAT SEQOENCE SIZE: 163
INCLUDED REGION SIZE: 163
PRODUCT SIZE: 145, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00 1 TTGAAAATTAAGAAACCCTGGCACAGTGTTGACTGGAGCCaCTTACCTTAATAGAAAATA
»»»»»»»»»»
61 AAGCTCACATATATCCATAATGAAAAGCAGAGACCAGCACAACCATAGTCACCTGACAGT 121 TTtAAAATCCAAGGCCAGGATCTTCTCAACTCAGGCCCACTCA
43) Whole sequence ::: rs2831902-rs2831903
CACATAACTAATAAATTTGTAAGTATGTGCAACGGCTCACaCTTGCTTCCAGAATGGCAC CTAAAAAACAGATTTACCTCTCCCCAAATTCAGATATGGAATTAAATGTAATGTCAGGAA AAcTGTCTAAGAGTTGGAAATGGGAAAAAAATGTTCTTTTGGT (SEQ ID NO: 212)
OLIGO start len tm gcS any 3' seq (SEQ ID NOs: 213, 130)
LEFT PRIMER 14 21 53.16 33.33 4.00 2.00 AATTTGTAAGTATGTGCAACG RIGHT PRIMER 149 20 56.27 35.00 2.00 0.00 TTTTTCCCATTTCCAACTCT SEQaENCE SIZE: 163 INCLUDED REGION SI2E: 163
PRODDCT SIZE: 136, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00
1 CACATAACTAATAAATTTGTAAGTATGTGCAACGGCTCACaCTTGCTTCCAGAATGGCAC >»»»»»»>»»»>
61 CTAAAAAACAGATTTACCTCTCCCCAAATTCAGATATGGAATTAAATGTAATGTCAGGAA
121 AAcTGTCTAAGAGTTGGAAATGGGAAAAAAATGTTCTTTTGGT
44) Whole sequence ::: rsll088086-rs2251447 AAAAAAAAAGATGAGACAGGCAGGTGCGAAAGAAATAAAAGTCAaAACTGATCCAGTTGG GAAACTCAGAATTGACAGTTAcGTGTCCTTTCATTTATTGATATTTTGAGATTCACAGGG GT (SEQ ID NO: 131)
OLIGO start len tm gc% any 3' seq (SEQ ID NOs: 132, 133) LEFT PRIMER 6 20 56.41 45.00 2.00 2.00 AAAAGATGAGACAGGCAGGT
RIGHT PRIMER 122 20 55.99 40.00 5.00 2.00 ACCCCTGTGAATCTCAAAAT SEQUENCE SI2E: 122 INCLUDED REGION SIZE: 122 PRODUCT SIZE: 117, PAIRANY COMPL: 5.00, PAIR 3' COMPL: 2.00
1 AAAAAAAAAGATGAGACAGGCAGGTGCGAAAGAAATAAAAGTCAaAACTGATCCAGTTGG
61 GAAACTCAGAATTGACAGTTACGTGTCCTTTCATTTATTGATATTTTGAGATTCACAGGG
121 GT «
45) Whole sequence : : : rs2832040-rsll088088
GAGTTAAATAAAGCACTTGCTTCTATTGTTTGTACCTAAACTTAACAGAAcACAGTAAGT AACAAGTCATTGGGATGCAGAAAAGAAAAAAGAGAGTGAAGGAAGGAGAaAAGGTGAAGG GAGAATGGAAGAGAGGAAGGGAGGGAGGAA (SEQ ID NO: 134)
OLIGO start Ien tm gc6 any 3' seq (SEQ ID. NOs: 135, 136)
LEFT PRIMER 13 21 54.81 38.10 4.00 0.00 GCACTTGCTTCTATTGTTTGT RIGHT PRIMER 141 20 57.37 50.00 2.00 0.00 CCCTTCCTCTCTTCCATTCT SEQDENCE SIZE: 150 INCLUDED REGION SIZE: 150
PRODUCT SIZE: 129, PAIR ANY COMPL: 2.00, PAIR 3' COMPL: 0.00
1 GAGTTAAATAAAGCACTTGCTTCTATTGTTTGTACCTAAACTTAACAGAAcACAGTAAGT ' ■
61 AACAAGTCATTGGGATGCAGAAAAGAAAAAAGAGAGTGAAGGAAGGAGAaAAGGTGAAGG
121 GAGAATGGAAGAGAGGAAGGGAGGGAGGAA
46) Whole sequence ::: rs2832141-rs2246777 aaacgagccaccagtgggAGCACTGCAGGTATCTGTGTGAGACCcGTACTTCACAACTCC
TGCTTTCCCTCCATAAAGtAGCTTGCATTTTCCACATTGACTTTGCAGTTCTTTGGTATC TGTATTGGT (SEQ ID NO: 137)
OLIGO start Ien tm gc% any 31 seq (SEQ ID NOs: 138, 139)
LEFT PRIMER 14 18 58.28 61.11 6.00 2.00 gtgggAGCACTGCAGGTA RIGHT PRIMER 123 21 55.05 38.10 4.00 2.00 ACAGATACCAAAGAACTGCAA SEQUENCE SIZE: 129 INCLUDED REGION SXZE: 129
PRODUCT SIZE: 110, PAIR ANY COMPL: 5.00, PAIR 31 COMPL: 3.00
1 aaacgagccaccagtgggAGCACTGCAGGTATCTGTGTGAGACCcGTACTTCACAACTCC
61 TGCTTTCCCTCCATAAAGtAGCTTGCATTTTCCACATTGACTTTGCAGTTCTTTGGTATC
121 TGTATTGGT <«
47) Whole sequence ::: rs2832959 -rs9980934 TGGACACCTTTCAACTTAGAAATCATAAACAGATTCATTTcCTTAAAGTTAATGaaaaga attaacagaccctcctcaaaaaagacatatatgcagcctacaatcatatgaaaaaaagtt caacattactgttcagcaaatcaaa (SEQ ID NO: 140)
OLIGO start Ien tm qc% any 3J_ se% (SEQ ID WOs: 141, 142) LEFT PRIMER 1 20 53.30 40.00 3.00 3.00 TGGACACCTTTCAACTTAGA
RIGHT PRIMER 134 22 50.67 27.27 8.00 3.00 gaacagtaatgttgaacttttt SEQUENCE SIZE: 145 INCLUDED REGION SIZE: 145 PKODUCT SIZE: 134, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 3.00
1 TGGACACCTTTCAACTTAGAAATCATAAACAGATTCATTTcCTTAAAGTTAATGaaaaga »»»»»»»»»»
61 attaacagaccctcctcaaaaaagacatatatgcagcctacaatcatatgaaaaaaagtt
121 caacattactgttcagcaaatcaaa «««««««
7th group
48) Whole sequence : : : rs2833734-rs2833735
TGGATACATTCCTAGAAATAGATGGAAACTGCTCTTGCAAAAAGCTTAGCACATGTTAAA aATTTTAGAAACAATTTGCCAAAGTTTATTTAGTCTAGTGATTTtGACAGGTTAAATGGA CCCTTTGAGATCTTTTTTCCTCAAGTACAAAGGCT (SEQ ID NO: 143) OLIGO start len tm gc% any 3J_ seq (SEQ ID NOs: 144, 145)
LEFT PRIMER 33 21 58.90 38.10 6.00 2.00 TCTTGCftAAAAGCTTAGCACA
RIGHT PRIMER 137 21 57.77 38.10 6.00 1.00 AAAAAGATCTCAAAGGGTCCA SEQUENCE SI2E: 155 INCLUDED REGION SIZE: 155
PRODUCT SIZE: 105, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00
1 TGGATACATTCCTAGAAATAGATGGAAACTGCTCTTGCAAAAAGCTTAGCACATGTTAAA
61 aATTTTAGAAACAATTTGCCAAAGTTTATTTAGTCTAGTGATTTtGACAGGTTAAATGGA
<«<
121 CCCTTTGAGATCTTTTTTCCTCAAGTACAAAGGCT «<«««<««<«
49) Whole sequence ::: rs933121-rs933122
GCTTTTGCTGAACATCAAGTGGTGAGCCAGGACTCAAaGCCAGATCTTCTTGTTTCCCTG TTAGGTGTtTGTAGCACAACTGGTATCTGCAGACTATGCTGCTGGAAGGGCTAGCCGTC (SEQ ID NO: 146)
OLIGO start len tm gc% any 3_i sea (SEQ ID NOs: 147, 148)
LEFT PRIMER 1 20 55.61 40.00 6.00 3.00 GCTTTTGCTGAACATCAAGT
RIGHT PRIMER 109 19 55.56 52.63 3.00 3.00 CCTTCCAGCAGCATAGTCT SEQUENCE SIZE: 119
INCLUDED REGION SIZE: 119
PRODUCT SIZE: 109, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00 1 GCTTTTGCTGAACATCAAGTGGTGAGCCAGGACTCAAaGCCAGATCTTCTTGTTTCCCTG »»»»»»»»»»
61 TTAGGTGTtTGTAGCACAACTGGTATCTGCAGACTATGCTGCTGGAAGGGCTAGCCGTC 50) Whole sequence ::: rs2834140-rsl26269S3
ACTGTCCTAGAAAATCCAGGATGTGCAGTGATCAtGTATGAATGCATGGACCTGCACACA CAGGAGTGAACAAAAGACCCACCCCTGCCAGGTCACCACTCATATCTCACCCCAGCCCAC GCTAGCTCACaCTCCTCCCCACACACCACTGACCTCATCAT <SEQ ID NO: 149)
OLIGO 3tart len tm qc% any 3J. seg {SEQ ID NOs: 150, 151)
LEFT PRIMER 12 18 53.64 44.44 7.00 1.00 AAATCCAGGATGTGCAGT RIGHT PRIMER 161 19 53.29 47.37 4.00 0.00 ATGATGAGGTCAGTGGTGT SEQUENCE SIZE: 161 INCLUDED REGION SIZE: 161
PRODUCT SIZE: 150, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00
1 ACTGTCCTAGAAAATCCAGGATGTGCAGTGATCAtGTATGAATGCATGGACCTGCACACA
61 CAGGAGTGAACAAAAGACCCACCCCTGCCAGGTCACCACTCATATCTCACCCCAGCCCAC
121 GCTAGCTCACaCTCCTCCCCACACACCACTGACCTCATCAT
51) Whole sequence ::: rs2834485-rs3453
CACATCACAGATCATAGTAAATGGCTTTAATTTTTTAaCGAAATCTCACTACTGCAAATG CATTGTTGTCCTAGCTAATGAATGCAtAGAGTATTGCCTGCAAAATAATAATTGAGATTC TATT (SEQ ID NO: 152)
OLIGO start len fcm gc% any 3J_ seq (SEQ ID NOs: 153, 154)
LEFT PRIMER 3 22 52.35 36.36 4.00 0.00 CATCACAGATCATAGTAAATGG
RIGHT PRIMER 113 21 53.50 23.81 6.00 4.00 AATTATTATTTTGCAGGCAAT SEQUENCE SIZE: 124 INCLUDED REGION SIZE: 124
PRODUCT SIZE: 111, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2-00
1 CACATCACAGATCATAGTAAATGGCTTTAATTTTTTAaCGAAATCTCACTACTGCAAATG »»»»>»»»»>>»>
61 CATTGTTGTCCTAGCTAATGAATGCAtAGAGTATTGCCTGCAAAATAATAATTGAGATTC
121 TATT
8th group 52) Whole sequence ::: rs9974986-rs2834703
TTATCCTCCACATCCTCATGAGGCAAACACCTTTCCTACCTTACCGCTCCCCAGTGGCCT CCCTGTTGCCTTCTTATTCAAGACTAAGACtCTCTAGAATGTTCTTTATCCTGAGTCCAG CTGATTGTCTATACTAATATCAGTACGGGGT (SEQ ID NO: 155) OLIGO 3' seg (SBQ ID NOs: 156, 157)
LEFT PRIMER 2.00 CATGAGGCAAACACCTTTCC RIGHT PRIMER
Figure imgf000050_0001
0.00 GCTGGACTCAGGATAAAGAftCA SEQUENCE SI2E: 151
INCLUDED REGION SIZE: 151
PRODUCT SIZE: 105, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00
1 TTATCCTCCACATCCTCATGAGGCAAACACCTTTCCTACCTTACCGCTCCcCAGTGGCCT
61 CCCTGTTGCCTTCTTATTCAAGACTAAGACtCTCTAGAATGTrCTTTATCCTGAGrCCAG
121 CTGATTGTCTATACTAATATCAGTACGGGGT <
53) Whole sequence ::: rsl2482353-rs2205032
AGAAGTTCTCGAATCTCCTCCTTAAcAGAGCTGCAGAAGGGAAACACAGACAGGAAGCACCTGTTTGACrCAgACAGCAGC CCTAATGCAGTGCCACTCAGGAGCATTCCCTCATTTGAAGACCCCCCAATTACATGAAATTATCAACCCC (SEQ ID NO: 346)
OLIGO start len tm gc% any 3' seg (SEQ XD NOs: 347, 348)
LEFT PRIMER 56 20 59.74 45.00 4.00 2.00 ACACCCAAAAGCTCTGCAAT RIGHT PRIMER 199 20 60.59 50.00 4.00 2.00 CAAATGAGGGAATGCTCCTG SEQUENCE SIZE: 232 INCLUDED REGION SIZE: 232
PRODUCT SIZE: 144, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00
1 ATCACCTGGTTTGGTGCATCCTCGCAGAAAGAGAGCCATACAGTGAAGTGGAAACACACC »»>
61 CAAAAGCTCTGCAATATTCCTAGAAGTTCTCGAATCTCCTCCTTAAcAGAGCTGCAGAAG
121 GGAAACACAGACAGGAAGCACCTGTTTGACTCAgACAGCAGCCCTAATGCAGTGCCACTC
181 AGGAGCATTCCCTCATTTGAAGACCCCCCAATTACATGAAATTATCAACCCC
54) Whole sequence ::: rs2776266-rs2835001 agggtgcagcactttattatggaagcctgagctgactaatacaGGTGTCTcTATATCTCA CTGAGGGAAAGTGACAGGAAAGTAAGAACCATTTaTGTCCAAGAGTCCAGAGGAGTCAAC CAGATTCTGGGGGAAAAGAAGGTAC (SEQ ID NO: 158)
OLIGO start len tm gc% any 3J_ seg (SEQ ID NOs: 159, 160)
LEFT PRIMER 20 20 58.75 50.00 4.00 1.00 tggaagoctgagctgactaa RIGHT PRIMER 142 20 59.87 50.00 4.00 3.00 CCTTCTTTTCCCCCAGAATC SEQUENCE SIZE: 145 INCLUDED REGION SIZE: 145
PRODUCT SIZE: 123, PAIR ANY COMPL: 4.00, PAIR 3" COMPl: 0.00
5
1 agggtgcagcactttattatggaagcctgagctgactaatacaGGTGTCTcTATATCTCA »»»»»»»»»»
61 CTGAGGGAAAGTGACAGGAAAGTAAGAACCATTTaTGTCCAAGAGTCCAGAGGAGTCAAC
10
121 CAGATTCTGGGGGAAAAGAAGGTAC
15 55) Whole sequence ::: rsl984014-rsl984015
TGAGAAT TTAGGAGAACAGAAGATCAGAGGGCTGCACaGGCTAAACTAGACAATGAGCCC ATGCAAGTAAGTTAAGAGGAGAAGCGGGTAAGTATGCACCTGCTTTGTCTAGGtGACCAG CAAGCATTTAGCAATAGTCTTT TCAAAACAACAG (SEQ ID NO: 161)
20
OLIGO start len tm gc% any 3 ' seq (SEQ ID NOs: 162, 163)
LEFT PRIMER θ 22 53.09 40.91 4.00 1.00 TTAGGAGAACAGAAGATCAGAG
RIGHT PRIMER 142 22 53.52 31.82 4.00 2.00 AAAGACTATTGCTAAATGCTTG SEQUENCE SIZE: 154 25 INCLUDED REGION SIZE: 154
PRODUCT SIZE: 135, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00
1 TGAGAATTTAGGAGAACAGAAGATCAGAGGGCTGCACaGGCTAAACTAGACAATGAGCCC
ΛΛ
61 ATGCAAGTAAGTTAAGAGGAGAAGCGGGTAAGTATGCACCTGCTTTGTCTAGGtGACCAG
35 . 121 CAAGCATTTAGCAATAGTCTTTTCAAAACAACAG
56) Whole sequence ::: rsl014593-rs9305569
40
ATCTTACATGAAGGTATACAGTTTGAAGAAGCCAGTTTGACTCCAATATCTGTGCAATGGAATACTGCTCATTAAAAAGgA (SEQIDNO: 349)
45 OLIGO start len tm gc% any 3J. seg (SEQ ID NOs: 350, 351)
LEFT PRIMER 51 19. 59.86 52.63 2.00 0.00 ACTTCTGCCCCTGTTTGCT
RIGHT PRIMER 198 21 58.84 42.86 4.00 3.00 TGATCTTCACCCATGTTGTGT SEQUENCE SIZE: 239 INCLUDED REGION SIZE: 239
50
PRODUCT SIZE: 148, PAIR ANY COMPL: 2.00, PAIR 3' COMPL: 0.00
1 GAACTGCAGGAGATCCCTGCTGCCTTCCAGTTCATGGGATGATGGCCTCCACTTCTGCCC 61 CTGTTTGCTTCTCCTTTCAaATCTTACATGAAGGTATACAGTTTGAAGAAGCCAGTTTGA
121 CTCCAATATCTGTGCAATGGAATACTGCTCATTAAAAAGgAATTAAACTATTGATACACA
181 CAACATGGGTGAAGATCAAACTGTCTCCTTCCCTTTGATTCAAGGGAATCTGAGAAATG «««««««««
57) Whole sequence ::: rs7281674-rs2835316
AAACAGGCAAAATAAGCGTAGGGCTGTGTGTGCAACAGTTaATCATAAAGCCATCACCAG GAGACgTCACTGGGCGCCTTCTGGAGTCTATCCGTCCTAACTTTGC (SEQ ID NO: 164)
OLIGO start len tm qc% any 3' seq {SEQ ID NOs: 165, 166)
LEFT PRIMER 13 20 59.93 55.00 4.00 0.00 TAAGCGTAGGGCTGTGTGTG RIGHT PRIMER . 97 21 60.08 57.14 3.00 1.00 GGACGGATAGACTCCAGAAGG SEQOEHCE SIZE: 106 INCLUDED REGION SIZE: 106
PRODUCT SIZE: 85, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00
1 AAACAGGCAAAATAAGCGTAGGGCTGTGTGTGCAACAGTTaATCATAAAGCCATCACCAG
61 GAGACgTCACTGGGCGCCTTCTGGAGTCTATCCGTCCTAACTTTGC
58) Whole sequence ::: rsl3047304-rsl3047322 gaatgaccttggcacttttatcaaacatcaactggccacaCacaggtgagtctacttctg gacacttaTcctgttccattcatctgtatatctctatccttacac (SEQ ID NO: 167) OLIGO start len tm gc% any 3' seq (SEQ ID NOs: 168, 169)
LEFT PRIMER 1 23 60.36 39.13 3.00 2.00 gaatgaccttggcacttttatca
RIGHT PRIMER 101 27 57.86. 33.33 4.00 0.00 aaggatagagatatacagatgaatgga
SEQUENCE SIZE: 105 INCLUDED REGION SIZE: 105
PRODUCT SIZE: 101, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00
1 gaatgaccttggcacttttatcaaacatcaactggccacaCacaggtgagtctacttctg »»»»»»»»»>»»
61 gacacttaTcctgttccattcatctgtatatctctatccttacac
59) Whole sequence ::: rs283S545-rs4816551
CTGCTGGAATAGGCTGCTTGGCCATGTTCTTGGAAGCTACCACCATATCAaGGTAATTTC CCACACAACATTCCAGCCCCTGCTTTCCtCTCTGGCCTTATCTAGGGCCATTCCCCAACT CAGGTGAAT (SEQ IDNO: 170) OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 171, 172)
LEFT PRIMEB 20 20 60.21 50.00 4.00 2.00 GGCCATGTTCTTGGAAGCTA RIGHT PRIMER 128 20 60.89 50.00 5.00 0.00 TTCACCTGAGTTGGGGAATG SEQUENCE SIZE: 129
INCLUDED REGION SIZE: 129
PRODUCT SIZE: 109, PAIR ANY COMPL: 3.00, PAIR 31 COMPL: 0.00 1 CTGCTGGAATAGGCTGCTTGGCCATGTTCTTGGAAGCTACCACCATATCAaGGTAATTTC
»»»»»»»»»»
61 CCACACAACATTCCAGCCCCTGCTTTCCtCTCTGGCCTTATCTAGGGCCATTCCCCAACT
121 CAGGTGAAT
60) Whole sequence ::: rs2835735-rs2835736
ACCTTTGTTCCATGCACCGCGCAAATACCTGGGAACCCTTaTTGCCCAACTCAAGAGCCA GAGTCCTCTGTCATCATTTTGCCTCTCTCCTAAGTGAgAGGACTGAGTGCAGACTTGGTG TTTGTGGGTGAGGCATGT (SEQ IDNO: 173) OLIGO start len tm qc% any 3J_ seg (SEQ ID NOs: 174, 175)
LEFT PRIMER 11 18 62.22 5S.56 5.00 0.00 CATGCACCGCGCAAATAC
RIGHT PRIMER 136 19 59.38 52.63 2.00 0.00 ATGCCTCACCCACAAACAC SEQUENCE SIZE: 138 INCLUDED REGION SIZE: 138
PRODUCT SIZE: 126, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00
1 ACCTTTGTTCCATGCACCGCGCAAATACCTGGGAACCCTTaTTGCCCAACTCAAGAGCCA
61 GAGTCCTCTGTCATCATTTTGCCTCTCTCCTAAGTGAgAGGACTGAGTGCAGACTTGGTG
<«
121 TTTGTGGGTGAGGCATGT ««««««««
61) Whole sequence ::: rsl3047608-rs2835826
CTCCTGAGTCCAAGCCCTTCTCACTCACCTCTTTCTTGAACTAATTTCTTcCTGTTTTTT TCCAGTCCTCCCTTCTGTTCATGTCTCTCCTCTGCACACTTCCATTTTgTGGTTCAGAAA ATGTCACCGTCCCAG TCACACTTGCCTTATGGCTGTTGT (SEQ ID NO: 176)
OLIGO start len tm qc% any 3J_ seg (SEQ ID NOs: 177, 178)
LEFT PRIMER 9 20 60.39 55.00 4.00 0.00 TCCAAGCCCTTCTCACTCAC RIGHT PRIMER 135 20 59.97 50.00 3.00 1.00 CTGGGACGGTGACATTTTCT SEQUENCE SIZE: 159 INCLUDED REGION SIZE: 159
PRODUCT SIZE: 127, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00 1 CTCCTGAGTCCAAGCCCTTCrCACTCACCTCTTTCTTGAACTAATTTCTTcCTGTTTTTT
61 TCCAGTCCTCCCTTCTGTTCATGTCTCTCCTCTGCACACTTCCATTTTgTGGTTCAGAAA
121 ATGTCACCGTCCCAGTCACACTTGCCTTATGGCTGTTGT
■ 62 ) Whole sequence : : : rs857998-rsl7284497
AGAGACAAGTCCTGACCTTCTCTCCTCCAGCTCTCCCAGgAGATAGGCAAGCCCCTAACTCCCTAACTAAGCCCTTCAGAC CTGAAATCCATTGAGTGGCTTCTTT (SEQ ID NO: 352)
OLIGO start len tm cτc% any 3J_ seq (SEQ ID NOs : 353 , 354)
LEFT PRIMEE 15 18 59.35 61 . 11 4. 00 0 .00 GCAAACTGCCCAGAGACC
RIGHT PRIMER ' 147 20 60. 57 55 . 00 2 . 00 2 .00 TTAGGGAGTTAGGGGCTTGC SEQUENCE SI2E : 189
INCLUDED REGION SIZE: 189
PRODUCT SIZE : 133 , PAIR ANY COMPL : 5 . 00 , PAIR 3 ' COMPL: 2.00 1 TGGAGAAAGTTGTTGCAAACTGCCCAGAGACCCTGGGAGTCACTCCAGTTTTCTGAAACC
»»»»»»»»»
61 CAGATATTTCAGtGCCTCAGGAGAGACAAGTCCTGACCTTCTCTCCTCCAGCTCTCCCAG
121 gAGATAGGCAAGCCCCTAACTCCCTAACTAAGCCCTTCAGACCTGAAATCCATTGAGTGG ««««««««««
181 CTTCTTTAC
9th group
63) Whole sequence : : : rs2836550-rs2212S96
CCCAGGAAGAGTGGAAAGATTAACCTTTGTGAGCCAAACCaGTGACACTTGATTACTTGA CAGAACTAATCCTTCTGTCCTGATGACAGAACTTCAACTACACAGGTACATGCAAGCTAA TATCTGTTGTAA (SEQ ID NO: 179) OLIGO start len tm qc% any 3J. seq (SEQ ID NOs: 180, 181)
LEFT PRIMER 1 21 59.56 47.62 3.00 2.00 CCCAGGAAGAGTGGAAAGATT
RIGHT PRIMER 120 21 56.03 42.86 6.00 1.00 TTAGCTTGCATGTACCTGTGT SEQUENCE SIZE: 132 INCLUDED REGION SIZE: 132
PRODUCT SIZE: 120, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 3.00
1 CCCAGGAAGAGTGGAAAGATTAACCTTTGTGAGCCAAACCaGTGACACTTGATTACTTGA 61 CAGAACTAATCCTTCTGTCCTGATGACAGAAcTTCAACTACACAGGTACATGCAAGCTAA
121 TATCTGTTGTAA
64) Whole sequence ::: rs2836660-rs2836661 GCCTGGCAAGCTAGATGGGGTGAATTTTCACCTGCCACAGcCGCAAGTCAAAGCCACCGG CTTCTCTCTTCTCCCTCCCATTGCTCCTGACAGCCAGGGTTAATATTTTGCCTCATGTAA ACAGGGAGGCAtCCACCCGAGAATCTCCCCTCAGCCCACATAAGC (SEQ ID NO: 182)
OLIGO start Ien tm gc% any 3' seq (SEQ ID NOs: 183, 184) LEFT PRIMER 9 20 55.41 40.00 4.00 2.00 AGCTAGATGGGGTGAATTTT
RIGHT PRIMER 158 18 61.14 61.11 3.00 3.00 TGGGCTGAGGGGAGATTC SEQUENCE SIZE: 165 INCLUDED REGION SIZE: 165 PRODUCT SIZE: 150, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 3.00
1 GCCTGGCAAGCTAGATGGGGTGAATTTTCACCTGCCACAGcCGCAAGTCAAAGCCACCGG
61 CTTCTCTCTTCTCCCTCCCATTGCTCCTGACAGCCAGGGTTAATATTTTGCCTCATGTAA
121 ACAGGGAGGCAtCCACCCGAGAATCTCCCCTCAGCCCACATAAGC
«««««««««
65) Whole sequence ::: rs465612-rs8131220 atcaagctaattaatgttatctatcacttcAcatagttcaacctttttttgtggtgagag tactgaagatctactctcttagcaattttcaaatctaaaatacattattattaacacagt cactgtgccGtacgttagctctgaggaccttattcatttt (SEQ ID NO: 185)
OLIGO start leti tm qc% any 3' seq (SEQ ID NOs: 186, 187>
LEFT PRIMER 1 22 47.51 22.73 6.00 4.00 atcaagctaattaatgttatct RIGHT PRIMER 158 20 50.92 40.00 5.00 5.00 aatgaataaggtcctcagag SEQUENCE SIZE: 160
INCLUDED REGION SIZE: 160
PRODUCT SIZE: 158, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00 1 atcaagctaattaatgttatctatcacttcAcatagttcaacctttttttgtggtgagag »»»»»»»»»»»
61 tactgaagatctactctcttagcaattttcaaatctaaaatacattattattaacacagt
121 cactgtgccGtacgttagctctgaggaccttattcatttt
««««««««««
66) Whole sequence : : : rs99800 TTTAATCTGATCATTGCCCTATGAGGTAGGgAGTATTCTGATTCCCATTTTATAAATAAG GAACCCGAGGCTTAGAGAGCATCaGTGACTTGTTCAAGGTCACCCACAGCTGTCAAGTGA > CAGA (SEQ ID NO: 188)
OLIGO start leπ cm gcfe any 3' seq (SEQ ID NOs: 189, 190)
LEFT PRIMER 1 21 55.02 33.33 6.00 2.00 TTTAATCTGATCATTGCCCTA
RIGHT PRIMER 111 18 57.61 55.56 5.00 1.00 AGCTGTGGGTGACCTTGA SEQUENCE SIZE: 124 INCLDDED REGION SIZE: 124 . . " • .- • .•
PRODUCT SIZE: 111, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00
1 TTTAATCTGATCATTGCCCTATGAGGTAGGgAGTATTCTGATTCCCATTTTATAAATAAG »>»»»»»»»»»
61 GAACCCGAGGCTTAGAGAGCATCaGTGACTTGTTCAAGGTCACCCACAGCTGTCAAGTGA
121 CAGA
10th group 67) Whole sequence ::: rs418359-rs2836926 tgtcccaccattgtgtattaggtttgtagagCgtagacaacttgcctttttagtttgtag gtttctgtatcaagagaagatgtgtgtGggcctaacctagattacaggatcctggacttc aagtctga (SEQ ID NO: 191)
OLIGO start len tm qc% any 3J. seg (SEQ ID NOs: 192, 193)
LEFT PRIMER 1 20 54.64 40.00 6.00 3.00 tgtcccaccattgtgtatta
RIGHT PRIMER 128 20 54.70 45.00 9.00 3.00 tcagacttgaagtccaggat SEQUENCE SIZE: 128 INCLUDED REGION SIZE: 128
PRODUCT SIZE: 128, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00
1 tgtcccaccattgtgtattaggtttgtagagCgtagacaacttgcctttttagtttgtag »»»»»»»»»»
61 gtttctgtatcaagagaagatgtgtgtGggcctaacctagattacaggatcctggacttc
121 aagtctga ««««
68) Whole sequence ::: rsll701943-rs4816634 tcatttgctaaggtcggatagctcctaattggcaaagtcaCgatgggatcccagggattc tgaggatgaagcctgtgtttaataactAttatgccaAGTGAGCATTTTCAAATATATGAG AGAAATTA (SEQ ID NO: 194)
OLIGO start leπ tin gc% any 3' seq (SEQ ID NOS: 195, 196) LEFT PRIMER 2 19 53.86 42.11 4.00 2.00 catttgctaaggtcggata RIGHT PRIMER 114 20 51.56 30.00 6.00 2.00 TATTTGAAAATGCTCACTtg SEQUENCE SIZE: 128 INCLUDED REGION SIZE: 128
PRODUCT SIZE: 113, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 0.00
1 tcatttgctaaggtcggatagctcctaattggcaaagtcaCgatgggatcccagggattc
61 tgaggatgaagcctgtgtttaataactAttatgccaAGTGAGCATTTTCAAATATATGAG
121 AGAAATTA
69) Whole sequence ::: rs7278447-rs7278858
CATTGCTTCAGGGGTGTTAGTTTTGTGTTCaCAACTAGATTATAAACTCCTCTTGCATTC CTGATGGCAGTGACTTGAAGGCAtttatttgaagaataatagacatacagaaaggggcac atgtcataaaggtacagctggacgacttttcacaaagtg <SEQ ID NO: 197)
OLIGO start left tm gc% any 3' seq (SEQ ID NOs: 198, 199)
LEFT PRIMER 5 20 55.96 45.00 2.00 0.00 GCTTCAGGGGTGTTAGTTTT RIGHT PRIMER 157 20 55.97 45.00 5.00 1.00 ctttgtgaaaagtcgtccag SEQUENCE SIZE: 159 INCLUDED REGION SIZE: 159
PRODUCT SIZE: 153, PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 0.00
1 CATTGCTTCAGGGGTGTTAGTTTTGTGTTCaCAACTAGATTATAAACTCCTCTTGCATTC »»»»»»»»»»
61 CTGATGGCAGTGACTTGAAGGCAtttatttgaagaataatagacatacagaaaggggcac
121 atgtcataaaggtacagctggacgacttttcacaaagtg
70) Whole sequence ::: rs385787-rs367001
GAGAGGATGGTGCCATCATGGAAAGCATGGGGCAGTCATGGAGATGACGGaGTAGCTCAT GGAGAAgATAATGCCATCATGGAAGGCATAGTGCAGTCATGGAGATGATGGTGCAGC (SEQ ID NO: 200) OLIGO start len tm qc% any 3J_ se_£ (SEQ ID NOs: 201, 202)
LEFT PRIMER 13 18 58.34 50.00 7.00 3.00 CCATCATGGAAAGCATGG
RIGHT PRIMER 108 20 55.09 45.00 4.00 2.00 TCATCTCCATGACTGCACTA SEQUENCE SIZE: 117 INCLUDED REGION SIZE: 117
PRODUCT SIZE: 96, PAIRANY COMPL: 7.00, PAIR 3" COMPL: 3.00
1 GAGAGGATGGTGCCATCATGGAAAGCATGGGGCAGTCATGGAGATGACGGaGTAGCTCAT 61 GGAGAAgATAATGCCATCATGGAAGGCATAGTGCAGTCATGGAGATGATGGTGCAGC
5 71) Whole sequence ::: rs367001-rs386095
ATGGGGCAGTCATGGAGATGACGGAGTAGCTCATGGAGAAaATAATGCCATCATGGAAGG CATAGTGCAGTCATGGAGATGATGGTGCAGCTCATGGAGAAGATGGTGCCATCATGgAAG GCATGGTGCAATCATGGAGTAGACAGTGCAGCTGGGCCaagattctc (SEQ ID NO: 203)
10
OLIGO start len tm get any 3' seq (SEQ ID NOs: 204, 205)
LEFT PRIMER 15 20 54.39 50.00 4.00 3.00 GAGATGACGGAGTAGCTCAT RIGHT PRIMER 156 18 55.17 61.11 6.00 2.00 CCCAGCTGCACTGTCTAC SEQUENCE SIZE: 167 15 INCLODED REGION SIZE: 167
PRODUCT SIZE: 142, PAIR ANΪ COMPL: 6.00, PAIR 3" COMPL: 2.00
1 ATGGGGCAGTCATGGAGATGACGGAGTAGCTCATGGAGAAaATAATGCCATCATGGAAGG
?Ω
61 CATAGTGCAGTCATGGAGATGATGGTGCAGCTCATGGAGAAGATGGTGCCATCATGgAAG
25 121 GCATGGTGCAATCATGGAGTAGACAGTGCAGCTGGGCCaagattCtC
«««««««««
72) Whole sequence ::: rs2837296-rs2837297
30 GATGTGCCTCTCTTGTTCCAATCACAGGACAGGGGTATAACTAGGGGCACTGTCTATACT GGCTGCACTCTGGCCAGTGCTGTCCCAgGTAGATTCATCAGGGTCTAGAGCTTCAGCTAA CAGCATGA (SEQ ID NO: 206)
OLIGO start len tm get any 3J_ aeq (SEQ ID NOs: 207, 208)
35 LEFT PRIMER 11 20 56.00 45.00 4.00 1.00 TCTTGTTCCAATCACAGGAC
RIGHT PRIMER 126 20 54.59 45.00 6.00 3.00 'ATGCTGTTAGCTGAAGCTCT SEQUENCE SIZE: 128 INCLUDED REGION SIZE: 128
40 PRODUCT SIZE: 116, PAIR ANΪ COMPL: 4.00, PAIR 3' COMPL: 1.00
1 GATGTGCCTCTCTTGTTCCAATCACAGGACAGGGGTATAAcTAGGGGCACTGTCTATACT
45 61 GGCTGCACTCTGGCCAGTGCTGTCCCAgGTAGATTCATCAGGGTCTAGAGCTTCAGCTAA
«««««««
121 CAGCATGA
««« . .
50
73) Whole sequence ::: rs4239808-rs2410205 AGGGCCATGGGATGATGCAGGTGGAGACTGGAGTGCTACAGCTGCAAGCAAATACATTTCTGTGCTGTGAAGCCACCCATT TTTTAATGATGACTGTTTTATT (SEQ ID NO: 355)
OLIGO start len tm gc% any 3J, sea tSE0- ID NOs: 356' 357J LEFT PRIMER 19 20 57.45 55.00 4.00 2.00 AGGTGGAGACTGGAGTGCTA
RIGHT PRIMER 145 22 56.58 31.82 2.00 0.00 AGAAACAAAAATACAGGCAACA SEQUENCE SIZE: 184 INCLUDED REGION SIZE: 184 PRODUCT SIZE: 127, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00
1 AGGGCCATGGGATGATGCAGGTGGAGACTGGAGTGCTACAGCTGCAAGCAAATACATTTC
»»»»»»»»»» 61 TGTGCTGTGAAGCCAcCCATTTGGTGGTACTACGTTAAAACAGCTCTAGGAAATTAAtAC . .
121 AGATGTTGCCTGTATTTTTGTTTCTCATATTACTACTCATTGTTTTAATGATGACTGTTT ««««««««««« .
181 TATT-
74) Whole sequence ::: rs2837381-rs4816672
TTTTATTCATTAAGTTGAAAGCTCCTAAAGCAGAGGGACCaTATTTTTATGTCCCAACTC TCCTTAAGgCCTTGCCTATGATAGCACATCTCTTCAATAGAATTGTCCT (SEQ ID NO: 209)
OLIGO start len • tin gc% any V. s_eg (SEQ ID NOs: 210, 211) LEFT PRIMER 16 20 55.17 45.00 4.00 0.00 TGAAAGCTCCTAAAGCAGAG
RIGHT PRIMER 97 20 50.59 35.00 4.00 3.00 TTGAAGAGATGTGCTATCAT SEQUENCE SIZE: 109 INCLUDED REGION SIZE: 109 PRODUCT SIZE: 82, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00
1 TTTTATTCATTAAGTTGAAAGCTCCTAAAGCAGAGGGACCaTATTTTTATGTCCCAACTC
61 TCCTTAAGgCCTTGCCTATGATAGCACATCTCTTCAATAGAATTGTCCT
««««««««««
11 th group 75) Whole sequence ::: rsl3047873-rs2837697
AAAGACCAGCTTTTAGCTGAACATCAGGGCTGCCTTCAGAGTTTAATTACCGCCCTCCCC ATGGGGCCAAATGAGCCATCGACTCCTCCCAAGGGGGTTCgGCTTGGTACTGATCTTTAA GTAAGTaAACGCTAAACCAGCTCATCTTAAAGCGCCCACATCTGATTTCCTGCTCTGCTG CAAGACAGTAGGTGACTGGTAATGACC (SEQ ID NO: 214)
OLIGO start len tm gc% any 3J_ s^a (SEQ ID NOs: 215, 216)
■ LEFT PRIMER 26 . 20 59.08 50.00 5.00 2.00 AGGGCTGCCTTCAGAGTTTA RIGHT PRIMER 155 20 59.62 50.00 5.00 2.00 GCGCTTTAAGATGAGCTGGT SEQUENCE SIZE: 207 INCLUDED REGION SIZE: 207
PRODUCT SIZE: 130, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00
1 AAAGACCAGCTTTTAGCTGAACATCAGGGCTGCCTTCAGAGTTTAATTACCGCCCTCCCC
»»»»»»»»»»
61 ATGGGGCCAAATGAGCCATCGACTCCTCCCAAGGGGGTTCgGCTTGGTACTGATCTTTAA
121 GTAAGTaAACGCTAAACCAGCTCATCTTAAAGCGCCCACATCTGATTTCCTGCTCTGCTG
181 CAAGACAGTAGGTGACTGGTAATGACC
76) Whole sequence : : : r3455999-rs9305700 ACTCTGCTCCCAGTGTGAACATGGGGAAAGTTGATTAAACTCTCTGACTTCAGATTCCTC aTGTAAAATGTGGGGAAACAGCTCTGACTTAATGGTGTCACTGTGAGGAGTAAATGAGGT AgCATATTTAAAGGATTTTGTATAGTGCTGGTGACAGTAACCAGCCAATAGATGATATAG GTAGTAATAGCA (SEQ ID NO: 217) OLIGO start Ien tm gc% any 3' seq (SEQ ID NOs: 218, 219)
LEFT PRIMER 16 20 57.84 40.00 4.00 2.00 TGAACATGGGGAAAGTTGAT RIGHT PRIMER 154 22 56.81 40.91 4.00 0.00 TCACCAGCACTATACAAAATCC SEQUENCE SIZE: 192 INCLUDED REGION SIZE: 192
PRODUCT SIZE: 139, PAIR- ANY COMPL: 4.00, PAIR 3' COMPL: 2.00
1 ACTCTGCTCCCAGTGTGAACATGGGGAAAGTTGATTAAACTCTCTGACTTCAGATTCCTC
»»»»»»»»»»
61 aTGTAAAATGTGGGGAAACAGCTCTGACTTAATGGTGTCACTGTGAGGAGTAAATGAGGT
121 AgCATATTTAAAGGATTTTGTATAGTGCTGGTGACAGTAACCAGCCAATAGATGATATAG
181 CTAGTAATAGCA
77) Whole sequence ::: rs9976207-rs455473 cttcactgaccacttccttaactgtccactccgaaacaccCcttcttcctgttcttccaa tacaccaaactctttcttgcctctgtgtgcttgcccatgctgttccttctggcttcttcc ttcACATTCAAGTCTTGACTTAGATGTCACTTGCCAAGGGAGACCTTGGA (SEQ ID NO: 220)
OLIGO start leu tm gcft. any 3J. seg (SEQ ID NOs: 221, 222)
LEFT PRIMER 12 21 54.96 47.62 4.00 0.00 acttccttaactgtccactcc
RIGHT PRIMER 159 19 54.64 47.37 7.00 2.00 CCTTGGCAAGTGACATCTA
SEQUENCE SIZE: 170 INCLUDED REGION SIZE: 170
PRODUCT SIZE: 148, PAIR ANY COMPL: 5.00, PAIR 31 COMPL: 3.00 1 cttcactgaccacttccttaactgtccactccgaaacaccCcttcttcctgttcttccaa
61 tacaccaaactctttcttgcctctgtgtgcttgcccatgctgttccttctggcttcttcc •
121 ttcACATTCAAGTCTTGACTTAGATGTCACTTGCCAAGGGAGACCTTGGA
78 ) Whole sequence : : : rs2837807-rs2837808
AAACATCCCAATAGACAAAACTCCAAGAAGAGTCAAAACAAGAATAAAGTaCAGGTCATC TTTTCTTTTGCACTCCTGACAGCACTTTGTACATGGTAATAATAATCTACCAATTAACTA . CATAAGCCACATGGTTTTATcATAGTGTGAAGCTTTGTATCCAGAAAGGAGAGAAGGCTCC (SEQ ID NO: 223)
OLIGO start len tm qc% any 3_^ seq (SEQ ID ROs: 224, 225)
LEFT PRIMER 23 22 56.31 36.36 3.00 0.00 CCAAGAAGAGTCAAAACAAGAA
RIGHT PRIMER 172 21 56.19 42.86 4.00 2.00 TCTCCTTTCTGGATACAAAGC SEQUENCE SIZE: 181 INCLUDED REGION SIZE: 181
PRODUCT SIZE: 150, PAIR ANY COMPL: 5.00, PAIR 31 COMPL: 1.00
1 AAACATCCCAATAGACAAAACTCCAAGAAGAGTCAAAACAAGAATAAAGTaCAGGTCATC »»»»»»»»»»»
61 TTTTCTTTTGCACTCCTGACAGCACTTTGTACATGGTAATAATAATCTACCAATTAACTA
121 CATAAGCCACATGGTTTTATcATAGTGTGAAGCTTTGTATCCAGAAAGGAGAGAAGGCTC
181 C
79) Whole sequence ::: rs9974587-rs2776356
GGCAGAGGCATGGGGTGCATAGGGATATGGGGTGGGCCAGTTTGCTCCTCAGACCAGAAG GGGTGCAGGAcTCCCCCCGATCAGGATCaTGGAGAAAGGTGTGGACAGAGGAAGGGAGGG AGGGAGAAATGGCAGCTGCCCTGCAGTGG (SEQ ID NO: 226)
OLIGO start len tm qcS any 3J_ seq (SEQ ID NOs: 227, 228)
LEFT PRIMER 42 20 60.52 55.00 3.00 2.00 TTGCTCCTCAGACCAGAAGG
RIGHT PRIMER 118 20 59.68 60.00 4.00 2.00 CTCCCTTCCTCTGTCCACAC SEQUENCE SIZE: 149
INCLUDED REGION SIZE: 149
PRODUCT SIZE: 77, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00 1 GGCAGAGGCATGGGGTGCATAGGGATATGGGGTGGGCCAGTTTGCTCCTCAGACCAGAAG
61 GGGTGCAGGAcTCCCCCCGATCAGGATCaTGGAGAAAGGTGTGGACAGAGGAAGGGAGGG > <«««««<:««««
121 AGGGAGAAATGGCAGCTGCCCTGCAGTGG
80) Whole sequence ::: rs2838089-rs2838090 cagggactaagtgtctctgacaatacattcagccactactAcagtatgaagccagcccct catccccaccttcagagacccctggtgcctcagattcctcggccattctggagctgctgt gCCCGAGGCTTGTGTAGTTGGAGATCATTTTGGCAGTCAGTGCTG (SEQ ID NO: 229)
OLIGO start len tm gc% any 3L ££3 (SE<2 ID NOs: 230' 231>
LEFT PRIMER 12 22 55.48 40.91 5.00 2.00 tgtctctgacaatacattcagc
RIGHT PRIMER 160 20 55.81 45.00 4.00 2.00 CTGACTGCCAAAATGATCTC SEQUENCE SIZE: 165
INCLUDED REGION SIZE: 165
PRODUCT SIZE: 149, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00 1 cagggactaagtgtctctgacaatacattcagccactactAcagtatgaagccagcccct
61 catccccaccttcagagacccctggtgcctcagattcctcggccattctggagctgctgt
121 gCCCGAGGCTTGTGTAGTTGGAGATCATTTTGGCAGTCAGTGCTG
««««««««««
12th group
81) Whole sequence : : : rs453592-rs380152
CCTGTCTCCGTGCGTGAAAGCCGGCTCCAAAGTGCCTTCTGTCCTATCTGCCTTCCGCAC CTGGCTTTCCTGAAAGAAAGAAAACGCGTGGCTTATCTTTTCACGGCACGCCACCTTCAC TCTCaCTTTTTCTTTTCTAATAAATACCTCTGGATGGGTTAGTGGTAATCTCTCCTCAAAC (SEQ ID HO: 232) OLIGO start len tm get any 3J^ seq (SEQ ID NOs: 233, 234)
LEFT PRIMER 24 20 60.00 55.00 4.00 1.00 GCTCCAAAGTGCCTTCTGTC
RIGHT PRIMER 165 20 58.87 55.00 3.00 2.00 CCACTAACCCATCCAGAGGT SEQUENCE SIZE: 181
INCLUDED REGION SIZE: 181
PRODUCT SIZE: 142, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00 1 CCTGTCTCCGTGCGTGAAAGCCGGCTCCAAAGTGCCTTCTGTCCTATCTGCCTTCCGCAC
61 CTGGCTTTCCTGAAAGAAAGAAAACGCGTGGCTTATCTTTTCACGGCACGCCACCTTCAC 121 TCTCaCTTTTTCTTTTCTAATAAATACCTCTGGATGGGTTAGTGGTAATCTCTCCTCAAA
181 C
82) Whole sequence ::: rs442723-rs449888 GGGAGCACAACCTAGGCCCCTCCTGGGGAGGTGGTGGAGTCAGAATCACGTAAGAGaCAA AGTTCCAGTCCCTCAGTGCCGGCTCCATTGTCCCCTGGACTTCCCTTACAAACCACAGAT GCAAAGAGAGCACTTCTCgGAATCTCCACACAGCCACGGTGGAGCACTCAACCCACGCGA CCCTCGGGCGCAGGTGCT (SEQ ID HO: 235) OLIGO start Ien tm gc% any 3J_ seg (SEQ ID NOs: 236, 237)
LEFT PRIMER 23 20 65.82 65.00 3.00 1.00 CTGGGGAGGTGGTGGAGTCA
RIGHT PRIMER 169 20 66.12 65.00 7.00 1.00 GAGTGCTCCACCGTGGCTGT SEQDENCE SIZE: 198 INCLUDED REGION SIZE: 198
PRODUCT SIZE : 147, PAIR ANΪ COMPL : 7 . 00 , PAIR 3 1 COMPL: 1. 00
1 GGGAGCACAACCTAGGCCCC TCCTGGGGAGGTGGTGGAGTCAGAATCACGTAAGAGaCAA
»»»»»»»»»»
61 AGTTCCAGTCCCTCAGTGCCGGCTCCATTGTCCCCTGGACTTCCCTTACAAACCACAGAT
121 GCAAAGAGAGCACTTCTCgGAATCTCCACACAGCCACGGTGGAGCACTCAACCCACGCGA ««««««««««
181 CCCTCGGGCGCAGGTGCT
83) Whole sequence ::: rs375886-rs9976S60
CCTGAGAAGCTTCCAGCAAAGCACCAGCACGAACCGCCCCACCTCCCCACCTCCCCGCAA GCGTTGcCGGGACTGACAGATTACAGAGCTCTGgTCCCTCTGCACTCCTGCTCTGCCACC CCCAGGGTGTCAGAATGTGCCCCCCACACAGTTTCCAAAAG (SEQ ID NO: 238)
OLIGO start len tm gc% any 3' seg (SEQ ID HOs: 239, 240)
LEFT PRIMER 18 18 59.84 55.56 2.00 0.00 AAAGCACCAGCACGAACC RIGHT PRIMER 143 18 59.89 61.11 3.00 3.00 GGGGCACATTCTGACACC SEQUENCE SIZE: 161 INCLUDED REGION SIZE: 161
PRODUCT SIZE: 126, PAIR ANY COMPL: 3.00, PAIR 31 COMPL: 0.00
1 CCTGAGAAGCTTCCAGCAAAGCACCAGCACGAACCGCCCCACCTCCCCACCTCCCCGCAA »»»»»»»»»
61 GCGTTGcCGGGACTGACAGATTACAGAGCTCTGgTCCCTCTGCACTCCTGCTCTGCCACC 121 CCCAGGGTGTCAGAATGTGCCCCCCACACAGTTTCCAAAAG
84) Whole sequence ::: rs3819900-rs3819901
ATGGAGCTGCTGCGCCGGCCTGAGCTCTGATCCCTCCTCCGACCCAGCCTCACCCTGCaA GCAGCACCATGTGGGGCTCAGAATGGGGATCTTAAGGGACCCTcCCCACAACCTCCCGAT AAGCCTTTCCACGGAGGGCCCAAGCGGAGACAGGAGAACACT (SEQ ID NO: 241) OLIGO start len tm gc% any 3_^ seg (SEQ ID NOs: 242, 243)
LEFT PRIMER 20 '19 57.00 57.89 6.00 0.00 CTGAGCTCTGATCCCTCCT
RIGHT PRIMER 158 18 57.51 55.56 2.00 0.00 TTCTCCTGTCTCCGCTTG SEQUENCE SIZE: 162 INCLUDED REGION SIZE: 162
PRODOCT SIZE: 139, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00
1 ATGGAGCTGCTGCGCCGGCCTGAGCTCTGATCCCTCCTCCGACCCAGCCTCACCCTGCaA
61 GCAGCACCATGTGGGGCTCAGAATGGGGATCTTAAGGGACCCTCCCCACAACCTCCCGAT
121 AAGCCTTTCCACGGAGGGCCCAAGCGGAGACAGGAGAACACT
85) Whole sequence ::: rsl0451852-rsl0451853
ACTTTCAGAATGTGCTGCCTTCCACGTGTGAACCAGACTGAGCTCCTTTCTGCCACTGAT GTTGAATTGTCCATTTGCTCACaTCAGTGTCCACGTGGCAAATCCACAGGGCgTGGGTGG GATCCTGCAGTCTAGACAAAGCCAAGGAGCACCGCTGGAGGCCACGTTGGGCTTCCCAAT CCACATGCAAACCC (SEQ ID NO: 244)
OLIGO start len tm gc% any 3' seq (SEQ ID NOs: 245, 246) LEFT PRIMER 45 20 59.29 50.00 3.00 1.00 CCTTTCTGCCACTGATGTTG RIGHT PRIMER 190 19 60.46 47.37 4.00 0.00 TTGCATGTGGATTGGGAAG SEQUENCE SIZE: 194 INCLUDED REGION SIZE: 194 PRODUCT SIZE: 146, PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 0.00
1 ACTTTCAGAATGTGCTGCCTTCCACGTGTGAACCAGACTGAGCTCCTTTCTGCCACTGAT '
61 GTTGAATTGTCCATTTGCTCACaTCAGTGTCCACGTGGCAAATCCACAGGGCgTGGGTGG »»
121 GATCCTGCAGTCTAGACAAAGCCAAGGAGCACCGCTGGAGGCCACGTTGGGCTTCCCAAT
181 CCACATGCAAACCC «««««
86) Whole sequence : : : rs7278528-rsll701158 TCTCCAGCCAGCGTGTCACAAAGCCGCTCACCTGCTCGTGTGAGTGTCTGAATGCACGTG TTTGAGTGTCAGaGGCGTGTGAACCACAGCAACTCAATCTTGAATAGGGGCTGGGTAAAG TGAGGCTgAGACCTCCCGGGGCTGCATTCCCAGATGGTTAAGGCATTCTAAGTCACAAGA TGAGATAGGAAGTTCGCACAAGACACTGGTCAT (SEQ 10 NO: 247)
OLIGO start 3.en tm qc% any 3J_ seq {SEQ ID NOs: 248, 249)
LEFT PRIMER 28 20 60.53 55.00 4.00 0.00 TCACCTGCTCGTGTGAGTGT
RIGHT PRIMER 163 20 59.39 50.00 4.00 2.00 CCTTAACCATCTGGGAATGC SEQUENCE SIZE: 213
INCLUDED REGION SIZE: 213
PRODUCT SIZE: 136, PAIR ANY COMPL: 3.00, PAIR 31 COMPL: 0.00 1 TCTCCAGCCAGCGTGTCACAAAGCCGCTCACCTGCTCGTGTGAGTGTCTGAATGCACGTG
»»»»»»»»»»
61 TTTGAGTGTCAGaGGCGTGTGAACCACAGCAACTCAATCTTGAATAGGGGCTGGGTAAAG
121 TGAGGCTgAGACCTCCCGGGGCTGCATTCCCAGATGGTTAAGGCATTCTAAGTCACAAGA
181 TGAGATAGGAAGTTCGCACAAGACACTGGTCAT
87) Whole sequence ::: rs2839627-rsl70916
TTGAGTCCTCTTAAGTAGTTACTATAGTGGAGAACTTGAGTCATTCTTTGTAGCGTGCTT CGTAGAGCAGCGTGTTTGTTAGAAGGATTTGTTAATCCTGTATAGgGTCTTTACGAAGGC TGTTTTCATGGAAGCTTCTCTTTGTTGACTCC (SEQ ID NO: 250)
OLIGO start leπ tm gc% any 3' seq [SEQ ID NOs: 251, 252)
LEFT PRIMER 28 22 55.68 36.36 5.00 1.00 TGGAGAACTTGAGTCATTCTTT RIGHT PRIMER 152 19 52.33 47.37 3.00 2.00 GGAGTCAACAAAGAGAAGC SEQUENCE SIZE: 152 INCLUDED REGION SIZE: 152
PRODUCT SIZE: 125, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 3.00
1 TTGAGTCCTCTTAAGTAGTTACTATAGTGGAGAACTTGAGTCATTCTTTGTAGCGTGCTT
»»»»»»»»»»»
61 CGTAGAGCAGCGTGTTTGTTAGAAGGATTTGTTAATCCTGTATAGgGTCTTTACGAAGGC
121 TGTTTTCATGGAAGCTTCTCTTTGTTGACTCC
88) Whole sequence ::: rs2839628-rs234740
CATTCTCTCCAGCTGCAAACTTTCTTCAACTTTCCTAAATTCTTAcTAAATTCAGAGGAA TAGGATAAAGATCACTTAGAGAAAGGGTGCTTATGGACATAGCCTGAGTTTCCTTTAACC TCTCTgCAATGGGTGCTTTTAACTAGCTTCTACATGGCAAGCTGTTTCAGTTTG (SEQ ID NO: 253) OLIGO start len tm qc% any 3J_ seg (SEQ ID NOs: 254, 255)
LEFT PRIMER 20 21 50.06 28.57 3.00 2.00 CTTTCTTCAACTTTCCTAAAT RIGHT PRIMER 160 19 50.96 42.11 4.00 2.00 TTGCCATGTAGAAGCTAGT SEQaEHCE SIZE: 174 INCLUDED REGION SIZE: 174
PRODUCT SIZE: 141, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00
1 CATTCTCTCCAGCTGCAAACTTI1CTTCAACTTTCCTAAATTCTTACTAAATTCAGAGGAA
61 TAGGATAAAGATCACTTAGAGAAAGGGTGCTTATGGACATAGCCTGAGTTTCCTTTAACC
121 TCTCTgCAATGGGTGCTTTTAACTAGCTTCTACATGGCAAGCTGTTTCAGTTTG
89) Whole sequence ::: rs2838239-rs2838240
GGACATCTGGAACTGCACCAGCACAGAACCGACACGTTGTTAcTCATCGTCACTCGGCAG GGCTGAAGACCACCAGAACTCATGACAGGCAGACGTGCCTGGCCCAGTTGAGGATGTAGC tTCAGAGCCAAGCGCCAGTCCTGTTGGCCACGTGGGCTGGGGGCAGGATAGACCA (SEQ ID NO: 256)
OLIGO start len tm gc% any 3_^_ seq (SEQ ID NOs: 257, 258)
LEFT PRIMER 17 19 59.73 57.89 2.00 0.00 ACCAGCACAGAACCGACAC
RIGHT PRIMER 145 18 62.40 61.11 4.00 0.00 AACAGGACTGGCGCTTGG SEQUENCE SISE: 175 INCLUDED REGION SIZE: 175
PRODUCT SI2E: 129, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00
1 GGACATCTGGAACTGCACCAGCACAGAACCGACACGTTGTTAcTCATCGTCACTCGGCAG »>»»»»»»»»
61 GGCTGAAGACCACCAGAACTCATGACAGGCAGACGTGCCTGGCCCAGTTGAGGATGTAGC
121 tTCAGAGCCAAGCGCCAGTCCTGTTGGCCACGTGGGCTGGGGGCAGGATAGACCA
90) Whole sequence ::: rs630397-rsll089106 GGCTGGTTCTGCCCTTGGGAGGTGGTTCCTTTGGCTGGACCAGAATGTCTGaAGATGATC AGGAGAGGGCCAAGGGTTGGGGGGTGCCCCATGTGCACCCTGAGAATTGCACCAGGCACA GtGAGCAACTTCAGCCCTCCTTGTGCAGAGCTGCAGCGTACAGTGCCAGCCCTCGCTGGC CC (SEQ ID NO: 259) O OLLIIGGOO 3 3ttaarrtt lleenn ttmm g2 £clS _ aannyy. 3 31' seq (SEQ ID NOs: 260, 261)
LEFT PRIMER 14 20 61 .79 55 .00 3. 00 0 0..000 CTTGGGAGGTGGTTCCTTTG
RIGHT PRIMER 148 18 61 .15 61 .11 4. 00 1 1..000 CTGCACAAGGAGGGCTGA
SEQUENCE SIZE: 182
INCLUDED REGION SIZE: 182 PRODUCT SIZE: 135, PAIR ANY COMPL: 5.00, PAIR 31 COMPL: 0.00
1 GGCTGGTTCTGCCCTTGGGAGGTGGTTCCTTTGGCTGGACCAGAATGTCTGaAGATGATC
61 AGGAGAGGGCCAAGGGTTGGGGGGTGCCCCATGTGCACCCTGAGAATTGCACCAGGCACA
121 GtGAGCAACTTCAGCCCTCCTTGTGCAGAGCTGCAGCGTACAGTGCCAGCCCTCGCTGGC «««««««««
181 CC
91) Whole sequence ::: rs9637180-rs481767
GTTCTCACTTTACTGAGAAACCTGGCAGCTTCTCAGGCCACCGCCCAGGTCACCTGCTCA CCAGCAAcGTGAACCACAGGAACtGAGGCTGTGCGGGAGGCGGCTCTGCTCTGTGCTGGG CCCCCCTCCTCCTCACTCACCCTCTTCAGTCAAAG <SEQ ID NO: 262)
OLIGO start leπ tm qci any 3' seq (SEQ ID NOs: 263, 264)
LEFT PRIMER 11 20 57.70 50.00 5.00 5.00 TACTGAGAAACCTGGCAGCT RIGHT PRIMER 155 20 54.98 50.00 3.00 0.00 CTTTGACTGAAGAGGGTGAG SEQUENCE SIZE: 155
INCLUDED REGION SIZE: 155
PRODUCT SIZE: 145, PAIR ANY COMPL: 3.00, PAIR 31 COMPL: 2.00 1 GTTCTCACTTTACTGAGAAACCTGGCAGCTTCTCAGGCCACCGCCCAGGTCACCTGCTCA
»»»»»»»»»»
61 CCAGCAACGTGAACCACAGGAACtGAGGCTGTGCGGGAGGCGGCTCTGCTCTGTGCTGGG
121 CCCCCCTCCTCCTCACTCACCCTCTTCAGTCAAAG ««««««««««
92) Whole sequence ::: rsl62360-rsl62359
TTAGTATTATTATTTTCATATATATTTTTTATAATAATCATATATTCAATTTTATCATCA AGAAAAAAGTTTTAAAATTCaAAATCCTTTCATGTGCACTGTTTTAAACTtAGGTAGAAG AAAAAAAGTCACTGAAAATCCAAGATGTAATAAACAGGCCCAACAAAGGCCAACAAACTT (SEQ ID NO: 265) OLIGO start len tm gel any 3' seq (SEQ ID NOs: 266, 267)
LEFT PRIMER 45 20 48.37 20.00 5.00 3.00 TTCAATTTTATCATCAAGAA RIGHT PRIMER 163 20 55.18 40.00 4.00 1.00 TTGGGCCTGTTTATTACATC SEQUENCE SIZE: 180 INCLUDED REGION SIZE: 180
PRODUCT SIZE: 119, PAIR ANY COMPL: 4.00, PAIR 3" COMPL: 1.00
1 TTAGTATTATTATTTTCATATATATTTTTTATAATAATCATATATTCAATTTTATCATCA
»»»»»»»» 61 AGAAAAAAGTTTTAAAATTCaAAATCCTTTCATGTGCACTGTTTTAAACTtAGGTAGAAG »» 121 AAAAAAAGTCACTGAAAATCCAAGATGTAATAAACAGGCCCAACAAAGGCCAACAAACTT
93) Whole sequence ::: rsl62356-rsl62355 AGGGAACATGGCCTTGCCCACACAGATTTCAGACATCTGGCTCCAGAACTGTGGGAGGAC ACATTTCTGTTGTTTAGAACTGCaTGTTTTTTATACTTTGTTATGGCTGCCCTAGGcAAC TAATACAGATATTATTTTCCACTTCTGAACTTAGCAAAATATTTTTAAAATGAAAATTCT TAAATGTTGGCACAGT (SEQ ID HO: 268) OLIGO start len tm qc% any 3J_ seq (SEQ ID NOs: 269, 270)
LEFT PRIMER 14 20 60.24 45.00 3.00 3.00 TTGCCCACACAGATTTCAGA
RIGHT PRIMER 156 22 56.88 36.36 5.00 0.00 TGCTAAGTTCAGAAGTGGAAAA SEQUENCE SIZE: 196 INCLUDED REGION SIZE: 196
PRODUCT SIZE: 143, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00
1 AGGGAACATGGCCTTGCCCACACAGATTTCAGACATCTGGCTCCAGAACTGTGGGAGGAC
61 ACATTTCTGTTGTTTAGAACTGCaTGTTTTTTATACTTTGTTATGGCTGCCCTAGGcAAC
121 TAATACAGATATTATTTTCCACTTCTGAACTTAGCAAAATATTTTTAAAATGAAAATTCT
181 TAAATGTTGGCACAGT
94) Whole sequence ::: rs91424-rs463738
CTGGATAAAGGATGCTACACGTCCCTGGTGGGACAGAGCAGGACGGCAGGGGATTTCATT AcGCCAcTCAGAATGGCAGGCAATTGAAAAAACTTATAAATTGTTTATTTCCAGAATTTT <SEQ ID NO: 271) OLIGO start lβn tm gci any 3' seq (SEQ ID NOs: 272, 273)
LEFT PRIMER 3 20 54.33 45.00 4.00 4.00 GGATAAAGGATGCTACACGT
RIGHT PRIMER 120 20 49.40 20.00 4.00 0.00 AAAATTCTGGAAATAAACAA SEQUENCE SIZE: 120 INCLUDED REGION SIZE: 120
PRODUCT SIZE: 118, PAIR ANY COMPL: 3.00, PAIR 31 COMPL: 1.00
1 CTGGATAAAGGATGCTACACGTCCCTGGTGGGACAGAGCAGGACGGCAGGGGATTTCATT
61 AcGCCAcTCAGAATGGCAGGCAATTGAAAAAACTTATAAATTGTTTATTTCCAGAATTTT
««««««««««
95) Whole sequence : : : rs2838318-rs2838319 TGTCAGTGGTGTAATCCGACTGTGAAAGATCAGTCTAACAAAACAGCGGGGAGAGAGAGG GCTGAATCAGAGCaACTAGGTCCAAAGCCGAGGGAACCACCAACAGATCCCCTGGTGACC CAACAAGAAATGCTCACAGTCTGGACCCAgTCAGAGTCTGCAGGACACAGCAGACATTCT GGAAGTTACAACAGCCAGGAGCAAGAGGACGCATGGCCTGACTG (SEQ ID NO: 274)
OLIGO start Ien tm gc% any 3' seq (SEQ ID NOs: 275, 276)
LEFT PRIMER 49 20 60.30 60.00 3.00 3.00 GGGAGAGAGAGGGCTGAATC RIGHT PRIMER 202 21 59.00 52.38 4.00 2.00 GCTCCTGGCTGTTGTAACTTC SEQUENCE SIZE: 224
INCLUDED REGION SIZE: 224
PRODUCT SIZE: 154, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00 1 TGTCAGTGGTGTAATCCGACTGTGAAAGATCAGTCTAACAAAACAGCGGGGAGAGAGAGG
»»»»»»
61 GCTGAATCAGAGCaACTAGGTCCAAAGCCGAGGGAACCACCAACAGATCCCCTGGTGACC »»»»
121 CAACAAGAAATGCTCACAGTCTGGACCCAgTCAGAGTCTGCAGGACACAGCAGACATTCT
181 GGAAGTTACAACAGCCAGGAGCAAGAGGACGCATGGCCTGACTG
96) Whole sequence ::: rs915770-rs731935
CGCCAGAGCACCCCTTCTCAGAACAGAAAGCGTCTCTACAaAGTGATCCGGAAGTGAGTG TGTGAGGGCGCTGCGTCCTCCCTGCTCCCCTTGGAGTTGCCCTTTCTTGCTCAGATCTGG
GTGCCTTgGCCTTGTCCTGGGCCCTTCCGCAGCCCCCGGGGTGATCCCCGCTAG (SEQ ID HO: 277)
OLIGO start len tm gc% any 3j_ sec? (SEQ ID NOs: 278, 279)
LEFT PRIMER 3 19 60.95 63.16 3.00 3.00 CCAGAGCACCCCTTCTCAG RIGHT PRIMER 148 18 62.95 66.67 6.00 0.00 GGAAGGGCCCAGGACAAG SEQUENCE SIZE: 174 INCLUDED REGION SIZE: 174
PRODUCT SIZE: 146, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 2.00
1 CGCCAGAGCACCCCTTCTCAGAACAGAAAGCGTCTCTACAaAGTGATCCGGAAGTGAGTG
61 TGTGAGGGCGCTGCGTCCTCCCTGCTCCCCTTGGAGTTGCCCTTTCTTGCTCAGATCTGG
121 GTGCCTTgGCCTTGTCCTGGGCCCTTCCGCAGCCCCCGGGGTGATCCCCGCTAG ««««««««« " Final Set
97) Whole sequence ::: rsl573338-rsl573339 TATCTTACGGATTTGTCAACATCATTTGAGAAGAAGTCCATAGGCTCAGCAGATTTTTAT GCCAGGTGGGCCATGGCATAAAAATGTGAAGAATGTGCTCaCTTAGACAATACcTGTGCT AAAATTGGAACAATACAGAGAAGATTAGCAAATTAAAACAATGTTAGGAAGTCAGTGTGG TGAGGTACGGTGCCTCATGCC (SEQ ID NO: 280) OLIGO start len tm gc% any 3' seq (SEQ ID NOs: 281, 282)
LEFT PRIMER 47 21 59.24 42.86 3.00 1.00 CAGCAGATTTTTATGCCAGGT RIGHT PRIMER 192 20 60.06 60.00 4.00 3.00 CACCGTACCTCACCACACTG SEQUENCE SIZE: 201 INCLUDED REGION SIZE: 201
PRODUCT SIZE: 146, PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 3.00
1 TATCTTACGGATTTGTCAACATCATTTGAGAAGAAGTCCATAGGCTCAGCAGATTTTTAT
»»»»»»»
61 GCCAGGTGGGCCATGGCATAAAAATGTGAAGAATGTGCTCaCTTAGACAATACcTGTGCT
121 AAAATTGGAACAATACAGAGAAGATTAGCAAATTAAAACAATGTTAGGAAGTCAGTGTGG ««««
181 TGAGGTACGGTGCCTCATGCC «««««« 98) Whole sequence ::: rs3788094-rs3788095
AGGCAGGGCCCTCCTTGCCACATGTAAAGCTGCACAGAGCGGTCACTATATGTGTTTCCA TATTTGCAATCCAACCACCACCAACTGAGTGTGCGTCCTGaTCAGCCGAGCCTGCCCACG GTGGCCACAGGCCCTCTACATTCTAATCTCGAGAGCCTGAGCATGTACAAATTAAACgAA GCAAAACGACACCACCCAGTTCTGGCCGTACTATAGGAGGTTTCCAGGAAGGGTTTGTGA ACATAAACATAAGCTAGGTAACACTCCTTTCTGAA (SEQ ID NO: 283)
OLIGO start len tm gc3 any 3' seq (SEQ ID NOs: 284, 285) LEFT PRIMER 73 20 57.88 50.00 5.00 3.00 AACCACCACCAACTGAGTGT RIGHT PRIMER 220 20 56.94 55.00 6.00 2.00 CCTCCTATAGTACGGCCAGA SEQUENCE SIZE: 275 INCLUDED REGION SIZE: 275 PRODUCT SIZE: 148, PAIR ANY COMPL: 3.00, PAIR 31 COMPL: 1.00
1 AGGCAGGGCCCTCCTTGCCACATGTAAAGCTGCACAGAGCGGTCACTATATGTGTTTCCA
61 TATTTGCAATCCAACCACCACCAACTGAGTGTGCGTCCTGaTCAGCCGAGCCTGCCCACG
»»»»»»»»»»
121 GTGGCCACAGGCCCTCTACATTCTAATCTCGAGAGCCTGAGCATGTACAAATTAAACgAA
181 GCAAAACGACACCACCCAGTTCTGGCCGTACTATAGGAGGTTTCCAGGAAGGGTTTGTGA
««««««««««
241 ACATAAACATAAGCTAGGTAACACTCCTTTCTGAA 99)Whole sequence : : : rs756554-rs756555 TCAGAGCATCGCCTCAGTGGCCATCAATAGCTCGGGGGACTGGATTGCTTTTGGCTGTTC AGGTTTGTCCCCaGCCTGGGTGGTAGAGATGGACTCCCCATTAGGGACCAGTGCTGCCCG GCTACAGGCtTACTTGACAGCCACCCACTGGGGGTGCCCTCCCCTCCCCCAGTTGTCTTC CATGGGGTGCCCTCTCCCCCAGCCGCCTTTCAGAAGGGGCCCTCCCCTCC (SEQ ID NO: 286) OLIGO start len tm gc% any 3_^ seq (SEQ ID NOs: 287, 288)
LEFT PRIMER 41 20 61.15 45.00 2.00 0.00 TGGATTGCTTTTGGCTGTTC
RIGHT PRIMER 189 20 61.37 55.00 6.00 2.00 CACCCCATGGAAGACAACTG SEQUENCE SIZE: 230 INCLUDED REGION SIZE: .230
PRODUCT SIZE: 149, PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 2.00
1 TCAGAGCATCGCCTCAGTGGCCATCAATAGCTCGGGGGACTGGATTGCTTTTGGCTGTTC
61 AGGTTTGTCCCCaGCCTGGGTGGTAGAGATGGACTCCCCATTAGGGACCAGTGCTGCCCG
121 GCTACAGGCtTACTTGACAGCCACCCACTGGGGGTGCCCTCCCCTCCCCCAGTTGTCTTC . «««<««
181 CATGGGGTGCCCTCTCCCCCAGCCGCCTTTCAGAAGGGGCCCTCCCCTCC
100 )Whole sequence ::: rs4350841-rs2838545
CTCATGCTTACATCCTTAGCTGATCATTAAACTTTGTGACCATTTCATGCTCACTGCTTT CTTGCCcGGGAGCTAATGGTGAGGAAAGGTCACTGGGAACCAGCGCACCAACCTCAGACA TcGATTTTGTTCCAGCCTTTTTTCCTGGGCAGGGGTGGCTATCACCTGCTGGTAGGCAGC GGCAGGCCCACTGTCCTGC (SEQ ID NO: 289)
OLIGO start len tm gc% any 3J_ seq (SEQ ID NOs: 290, 291)
LEFT PRIMER 27 21 53.45 28.57 5.00 2.00 TTAAACTTTGTGACCATTTCA
RIGHT PRIMER 174 18 54.55 55.56 6.00 2.00 TACCAGCAGGTGATAGCC SEQUENCE SIZE: 199
INCLUDED REGION SIZE: 199
PRODUCT SIZE: 148, PAIR ANX COMPL: 3.00, PAIR 31 COMPL: 0.00 1 CTCATGCTTACATCCTTAGCTGATCATTAAACTTTGTGACCATTTCATGCTCACTGCTTT
61 CTTGCCCGGGAGCTAATGGTGAGGAAAGGTCACTGGGAACCAGCGCACCAACCTCAGACA '
121 TCGATTTTGTTCCAGCCTTTTTTCCTGGGCAGGGGTGGCTATCACCTGCTGGTAGGCAGC
«««««««««
181 GGCAGGCCCACTGTCCTGC 101 ) Whole sequence ::: rs2838551-rs2838552 TGACAGAAAAGTCTCAGAGCAGTGCCTTCTGAGCTCTTCTACACCAAGCAGGCAGAATGT TCACTGCTAATGAGgCTGGAGCTGGTCCCCAGCAGTGGTAGGAAGCTTCCAaCAGGCTCA GGCTGTGGGTGCTTGCAGGGGCACAGTGTGACGGCCACGGGCCTCAGAGCTCTGGTGGGC T (SEQ ID NO: 292) OLIGO start len tin gc% any 3J_ seq (SEQ ID NOs: 293, 294)
LEFT PRIMER 2 20 53.05 «5.00 5.00 3.00 GACAGAAAAGTCTCAGAGCA
RIGHT PRIMER 135 18 62.10 61.11 5.00 3.00 CAAGCACCCACAGCCTGA SEQUENCE SIZE: 181 INCLyDED REGION SIZE: 181
PRODUCT SIZE: 134, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 3.00
1 TGACAGAAAAGTCTCAGAGCAGTGCCTTCTGAGCTCTTCTACACCAAGCAGGCAGAATGT
61 TCACTGCTAATGAGgCTGGAGCTGGTCCCCAGCAGTGGTAGGAAGCTTCCAaCAGGCTCA
<«
121 GGCTGTGGGTGCTTGCAGGGGCACAGTGTGACGGCCACGGGCCTCAGAGCTCTGGTGGGC «««««<««
181 T
102 )Whole sequence ::: rs8134902-rs8133874
ACATCTTTCTCAAATAAAGATAACAGCGATGTATTTTCACAAAAGCAAGAGCTTAGAAAG TACTcCACCCAGGTATCCCTCTTGGAAAAAATaCTTAAGGAAATATGACAAATGGCAAAG TGATTGTTATGGATGGAATGTTTGTATCCTCCCAAAATTCACATGTTGAGACCCTAATTC CAATATG (SEQ ID NO: 295)
OLIGO start len tπt g-c% any 3 ' seg (SEQ ID NOs: 296, 297)
LEFT PRIMER 33 20 54.84 35.00 5.00 2.00 ATTTTCACAAAAGCAAGAGC RIGHT PRIMER 155 20 54.97 40.00 3.00 0.00 TTGGGAGGATACAAACATTC SEQUENCE SIZE: 187
INCLUDED REGION SIZE: 187
PRODUCT SIZE: 123, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00 1 ACATCTTTCTCAAATAftAGATAACAGCGATGTATTTTCACAAAAGCAAGAGCTTAGAAAG
61 TACTcCACCCAGGTATCCCTCTTGGAAAAAATaCTTAAGGAAATATGACAAATGGCAAAG
121 TGATTGTTATGGATGGAATGTTTGTATCCTCCCAAAATTCACATGTTGAGACCCTAATTC ««««««««««
181 CAATATG 103 )Whole sequence ::: rs425667-rs382478 AGGGGCATTCTACAAAACACCCAACCGGTCAAGGTCGCTGAGGCCAAGGAGAGATTGGGC AACCGTCACAAACCAGAGAAGCCGAGGAGACCTTTCAGCCAACGCCATGTGGGGTCCTGA GCAGGACCCACCGGAAGTTGGTGCAGCTGCCTAAAGACCGTCCTGGCTGAGAAGAAACAG (SEQ ID NO: 298)
OLIGO ' start lea tm qc% any 3' 5eq (SEQ ID NOs: 299, 300> LEFT PRIMER 46 18 55.06 50.00 4.00 2.00 AAGGAGAGATTGGGCAAC
RIGHT PRIMER 178 19 54.85 52.63 3.00 1.00 GTTTCTTCTCAGCCAGGAC SEQUENCE SIZE: 180 INCLUDED REGION SIZE: 180 PRODUCT SIZE: 133, PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 0.00
1 AGGGGCATTCTACAAAACACCCAACCGGTCAAGGTCGCTGAGGCCAAGGAGAGATTGGGC
61 AACCGTCACAAACCAGAGAAGcCGAGGAGAcCTTTCAGCCAACGCCATGTGGGGTCCTGA >»
121 GCAGGACCCACCGGAAGTTGGTGCAGCTGCCTAAAGACCGTCCTGGCTGAGAAGAAACAG
104 )Whole sequence ::: rs2838650-rs2838651
TGGCCCTGACCTGCCAGAGCTGTTGGCCTCCAGCTGGCGGGTAAAACCCACGGCCTTCTC
AGAACAGGTTTCTCAACACATGAGACSGAACACACCAGACTTCCaAGGGGAACACCTGGA TGGAGCTGGTTACCCAGATcGTTCAACACCGAGGGGCAGCGGCTTGAGGGTCTTTCCACG
AAGGCTTGGATTAACAAGAGGAGCASRGGTCTCTCCAGGATGGGCCCA (SEQ ID NO: 301)
OLIGO start len tm qc6 any 3' seq (SEQ ID HOs: 302, 303)
LEFT PRIMER 79 20 54.89 50.00 4.00 1.00 CATGAGACAGAACACACCAG RIGHT PRIMER 'l99 20 54.61 40.00 5.00 3.00 TCTTGTTAATCCAAGCCTTC SEQUENCE SIZE: 228 INCLUDED REGION SIZE: 228
PRODUCT SIZE: 121, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00
1 TGGCCCTGACCTGCCAGAGCTGTTGGCCTCCAGCTGGCGGGTAAAACCCACGGCCTTCTC
61 AGAACAGGTTTCTCAACACATGAGACAGAACACACCAGACTTCCaAGGGGAACACCTGGA »»»»»»»»»»
121 TGGAGCTGGTTACCCAGATcGTTCAACACCGAGGGGCAGCGGCTTGAGGGTCTTTCCACG
< 181 AAGGCTTGGATTAACAAGAGGAGCASRGGTCTCTCCAGGATGGGCCCA
105 ) Whole sequence : : : rs2838654-rsl296489 CCACCCAGTGTCACGTCACGGCCCCGGCACGCCATCCACGGACCCTGGATGGAGCCCAGC TGCCTCCaGGAGCGCAGTTTAACTACAAAGGAGCCCTGGCTGCCCGCCCCGCCCAGACGC ACTGACCTGTTGTTCTCTGTGGCTGCTGATGGCCCaTCCCCAACCACTGGTGACTCTTCC CTGGGGCCCCAAGCTCAGCCCCTAACCCCCTGTTGCTGGAAGT (SEQ ID NO: 304)
OLIGO start len Cm gcS any 3' seq (SEQ .ID NOs: 305, 306)
LEFT PRIMER 37 18 62.56 66.67 5.00 2.00 CACGGACCCTGGATGGAG RIGHT PRIMER 183 18 53.14 55.56 3.00 2.00 CAGGGAAGAGTCACCAGT SEQQENCE SIZE: 223 INCLUDED REGION SIZE: 223
PRODUCT SIZE: 147, PAIR ANY COMPL: 5.00, PAIR 31 COMPL: 2.00
1 CCACCCAGTGTCACGTCACGGCCCCGGCACGCCATCCACGGACCCTGGATGGAGCCCAGC »»>»»»»»?•»
61 TGCCTCCaGGAGCGCAGTTTAACTACAAAGGAGCCCTGGCTGCCCGCCCCGCCCAGACGC
121 ACTGACCTGTTGTTCTCTGTGGCTGCTGATGGCCCaTCCCCAACCACTGGTGACTCTTCC
181 CTGGGGCCCCAAGCTCAGCCCCTAACCCCCTGTTGCTGGAAGT
<«
106 )Whole sequence ::: rs2838659-rsll08261
CAGAGGACTGGGCTGCGGGGTCAGGAATGGGCACACTTCCTAACTGCAGGACACTCTAAG GGCTTTGGTCATGCACACgCAGCCAAGAGAAGGTGTCGCTGaCACACAGCCTTCCAGGAG CGGACTTGGAGACCTCGCCAAGGACCAGGACTCCCCAGCACTCACACTCCCTTAGGCGCT GAAGTC (SEQ ID NO: 307)
OLIGO start len tm gc% any 3' seq (SEQ ID NOs: 308, 309)
LEFT PRIMER 53 20 55.48 45.00 4.00 2.00 ACTCTAAGGGCTTTGGTCAT RIGHT PRIMER 175 20 56.02 55.00 3.00 1.00 CTAAGGGAGTGTGAGTGCTG SEQUENCE SIZE: 186 INCLUDED REGION SIZE: 186
PRODUCT SIZE: 123, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00
1 CAGAGGACTGGGCTGCGGGGTCAGGAATGGGCACACTTCCTAACTGCAGGACACTCTAAG
»»»»
61 GGCTTTGGTCATGCACACgCAGCCAAGAGAAGGTGTCGCTGaCACACAGCCTTCCAGGAG »»»»»»
121 CGGACTTGGAGACCTCGCCAAGGACCAGGACTCCCCAGCACTCACACTCCCTTAGGCGCT
«««««««««« 181 GAAGTC
107 )Whole sequence ::: rs585587-rs585601 GAAGAGGACAACACGGGGCTGTCTGCAGAGCACCTGCCACGCGCCAGGCTCTGTGTCCAC AAGCACGGCGGCTGCTCCCACATGACaGAGCTCGTGcGGCAGCTCCAGGACTGTCTGGTG CCAGAGCCCCAGCTCTCCGCCAGCCCCAGGCCACTGTGCGAGGCCCTCAGTGAAGAGGGG GCCGT (SEQ ID NO: 310)
OLIGO start len tm qc% any 3J_ seq (SEQ ID NOs: 311, 312)
LEFT PRIMER 42 18 64.78 66.67 5.00 2.00 CGCCAGGCTCTGTGTCCA
RIGHT PRIMER 183 18 60.76 66.67 5.00 3.00 GGCCCCCTCTTCACTGAG SEQUENCE SIZE: 185 INCLUDED REGION SIZE: 185
PRODUCT SIZE: 142, PAIR ANX COMPL: 5.00, PAIR 3' COMPL: 3.00
1 GAAGAGGACAACACGGGGCTGTCTGCAGAGCACCTGCCACGCGCCAGGCTCTGTGTCCAC »»»»»»»»»
61 AAGCACGGCGGCTGCTCCCACATGACaGAGCTCGTGcGGCAGCTCCAGGACTGTCTGGTG
121 CCAGAGCCCCAGCTCTCCGCCAGCCCCAGGCCACTGTGCGAGGCCCTCAGTGAAGAGGGG
181 GCCGT
<«
108 JWhole sequence ::: rs9981033-rs4818998
TCTAAATAATGTTAATGATCAAATTTAGTCAGATCTCAATCTTCATATGTTAGTTGCCTT CTTAaTAAATATTCTGTTTTCTTTATCGTTCTTTATTTGTATCTCcACCTTCATTTCTGA TTAAATTAAGAAGTTTTGTCTCTTCCATTTAATAATTAATGTATTTAATAACC (SEQ ID NO: 313)
OLIGO start len tm gc% any 3' seq (SEQ ID NOs: 314, 315)
LEFT PRIMER 24 22 51.86 31.82 6.00 2.00 TTTAGTCAGATCTCAATCTTCA RIGHT PRIMER 149 22 54.02 31.82 4.00 3.00 AATGGAAGAGACAAAACTTCTT SEQUENCE SIZE: 173
INCLUDED REGION SIZE: 173
PRODUCT SIZE: 126, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00 1 TCTAAATAATGTTAATGATCAAATTTAGTCAGATCTCAATCTTCATATGTTAGTTGCCTT
61 CTTAaTAAATATTCTGTTTTCTTTATCGTTCTTTATTTGTATCTCCACCTTCATTTCTGA
121 TTAAATTAAGAAGTTTTGTCTCTTCCATTTAATAATTAATGTATTTAATAACC
109 JWholβ sequence ::: rs2838802-rs2838803
CACACTCCACACTGGCCCCACGCGGGTGGCGAAGGACTCAGCCAGAGCCTGGCAGGATCC TGGGGTGTCTaTTTCCAAGGAATGTTCTGGAAGAAACATACACACATACTTGTTTGCCAG ATTTACCTGTGTGGTCTTCCAGATGAGAAGCAGCCTGTGTCACTCCATAAGGGAGAGTGC GTGCAGCATTGAGA (SEQ ID NO: 316) OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 317, 318)
LEFT PRIMER 31 18 55.96 61.11 5.00 3.00 GAAGGACTCAGCCAGAGC RIGHT PRIMER 177 20 55.20 50.00 7.00 3.00 CTCTCCCTTATGGAGTGACA SEQOENCE SIZE: 194
INCLUDED REGION SIZE: 194
PRODUCT SIZE: 147, PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 0.00 1 CACACTCCACACTGGCCCCACGCGGGTGGCGAAGGACTCAGCCAGAGCCTGGCAGGATCC
61 TGGGGTGTCTaTTTCCAAGGAATGTTCTGGAAGAAACATACACACATACTTGTTTGCCAG
121 ATTTACCTGTGTGGTCTTCCAGATGAGAAGCAGCCTGTGTCACTCCATAAGGGAGAGTGC
181 GTGCAGCATTGAGA
110 ) Whole sequence ::: rs2183596-rs2150452
AAGAAACTCCCAAGGAACGCATTGTCCCAAGTTGCTGCACCAGTCAGTGTACATTCCCAC AAaCAGTGCATGAGAGTTCCTGTTGCTTGTGAAATAAATGGTCAGCATTCAGTGTTGTCA GCTTTTAAAATTTTCTCCTTTCTAGTGGGCATGTAATGGTcTCACATTATAGTTTTAATT TGCATTTTCCTGGTGACATGTGATACGGAACCTTCCTCCCATGCT (SEQ ID NO: 319)
OLIGO start len tm _ Sel _ an 3 3'' seq (SEQ ID NOs: 320, 321) L LEEFFTT PPRRIIMMEERR 3 399 1 199 5 500. .1199 4 477. .3377 6 6..0 000 2 2..000 ACCAGTCAGTGTACATTCC
RIGHT PRIMER 190 19 50 .12 26 .32 4. 00 0 0..000 GGAAAATGCAAATTAAAAC
SEQUENCE SIZE: 225
INCLUDED REGION SIZE: 225
PRODUCT SIZE: 152, PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 1. 00
1 AAGAAACTCCCAAGGAACGCATTGTCCCAAGTTGCTGCACCAGTCAGTGTACATTCCCAC
61 AAaCAGTGCATGAGAGTTCCTGTTGCTTGTGAAATAAATGGTCAGCATTCAGTGTTGTCA
121 GCTTTTAAAATTTTCTCCTTTCTAGTGGGCATGTAATGGTCTCACATTATAGTTTTAATT
181 TGCATTTTCCTGGTGACATGTGATACGGAACCTTCCTCCCATGCT
«««««
111 JWhole sequence ::: rs4599218-rs9978646
GTGCAATTTAATTACAAACGCTTAAATGGGGAGGTCAGGGGCAGAGGGATGATGTCACAA ACACACCCAcGTGTGCTTGGTGCAAAACAGTAAAACAAACAGCAAGAAGgTCCATGAAGG AAAGATCGCCTCTGTCAGTGGGAGTAATGAGAGTGGCTGATGGACAGGTG (SEQ ID NO: 322) OLIGO start len tm qe% any 3' seq (SBQ ID NOs: 323, 324)
LEFT PRIMER 19 20 61.86 55.00 4.00 1.00 CGCTTAAATGGGGAGGTCAG RIGHT PRIMER 168 20 60.83 60.00 3.00 0.00 CCTGTCCATCAGCCACTCTC SEQUENCE SIZE: 170 INCLUDED REGION SIZE: 170
PRODUCT SIZE: 150, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00
1 GTGCAATTTAATTACAAACGCTTAAATGGGGAGGTCAGGGGCAGAGGGATGATGTCACAA »»»»»»»»»»
61 ACACACCCAcGTGTGCTTGGTGCAAAACAGTAAAACAAACAGCAAGAAGgTCCATGAAGG
121 AAAGATCGCCTCTGTCAGTGGGAGTAATGAGAGTGGCTGATGGACAGGTG
««««««««««
112 >Whole sequence ::: rsll702503-rs3827270 ACGCCAAGCAGGAGATGCCAGACACAGAGTCCATCCTGAGAGAGTCTGTTCCTGTCCAAG CTCAGAAACACAGGAAGCcACCTGTGCTGTAGCAGCACaCGGAGATGCATCCTTTCTGGT CCACCCCACGGCCCTCATTGCAGTCAGGGATCCTCTCCCAGAAAGTCCCTGCTGCCAGCC CCTGCCCTT (SEQ ID NO: 325) OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 326, 327)
LEFT PRIMER 7 20 62.02 55.00 3.00 0.00 AGCAGGAGATGCCAGACACA
RIGHT PRIMER 125 20 63.37 55.00 5.00 4.00 GGTGGACCAGAAAGGATGCA SEQUENCE SIZE: 189 INCLUDED REGION SIZE: 189
PRODUCT SIZE: 119, PAIR ANY COMPL: 3.00, PAIR 31 COMPL: 0.00
1 ACGCCAAGCAGGAGATGCCAGACACAGAGTCCATCCTGAGAGAGTCTGTTCCTGTCCAAG
61 CTCAGAAACACAGGAAGCcACCTGTGCTGTAGCAGCACaCGGAGATGCATCCTTTCTGGT
121 CCACCCCACGGCCCTCATTGCAGTCAGGGATCCTCTCCCAGAAAGTCCCTGCTGCCAGCC ««<
181 CCTGCCCTT
113 ) Whole sequence ::: rs2839084-rs9984302
CATGAGAAAGACTTTGTTCCCATGAGAACAACAAGAGAAACTCAAACAAAATTAAAATTG TACTTTTCTAAAAGACcGGGGTGGGGGTCGTGGTCAGGCAGCaGCATGAAGAAAGCCTTG AGAACTGAATTCCAGAAAGAAACAAGCATAGGCAAGAAAGAGAGATGACA (SEQ ID NO: 328)
OLIGO start len tm gc% any 3_1 seer (SEQ ID NOs: 329, 330)
LEFT PRIMER 19 22 59.21 40.91 4.00 0.00 CCCATGAGAACAACAAGAGAAA RIGHT PRIMER 162 20 55.46 45.00 4.00 2.00 CTCTTTCTTGCCTATGCTTG SEQUENCE SIZE: 170 INCLUDED REGION SIZE: 170
PRODUCT SIZE: 144, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 3.00
1 CATGAGAAAGACTTTGTTCCCATGAGAACAACAAGAGAAACTCAAACAAAATTAAAATTG »»»»»»»»»»»
61 TACTTTTCTAAAAGACcGGGGTGGGGGTCGTGGTCAGGCAGCaGCATGAAGAAAGCCTTG
121 AGAACTGAATTCCAGAAAGAAACAAGCATAGGCAAGAAAGAGAGATGACA
«««««««««« 114 )Whole sequence ::: rs22490S7-rs2249060
AAGATTTAGAACAGCTGAAGCAGCGAGAAAAAACCCAGCATGAGTCaGAACTGGAGCAAC TGAGGATTTATTTTGAAAAGAAGTTAAGGGATGCTGAGAAAACTTACCAAGAAGACCTAA cCCTGTTACAGCAGAGGCTGCAGGGGGCGAGGGAAGATGCTCTTCTG (SEQ ID NO: 331)
OLIGO start len tin gel any 3' seq (SEQ ID NOs: 332, 333)
LEFT PRIMER 12 21 63.07 47.62 6.00 0.00 CAGCTGAAGCAGCGAGAAAAA RIGHT PRIMER 146 19 6S.33 68.42 6.00 3.00 CCCCTGCAGCCTCTGCTGT SEQUENCE SIZE: 167 INCLUDED REGION SIZE: 167
PRODUCT SIZE: 135, PAIR ANY COMPL: 7.00, PAIR 31 COMPL: 1.00
1 AAGATTTAGAACAGCTGAAGCAGCGAGAAAAAACCCAGCATGAGTCaGAACTGGAGCAAC
61 TGAGGATTTATTTTGAAAAGAAGTTAAGGGATGCTGAGAAAACTTACCAAGAAGACCTAA
121 cCCTGTTACAGCAGAGGCTGCAGGGGGCGAGGGAAGATGCTCTTCTG
115 ) Whole sequence ::: rs2839226-rs2839227 GGGAAACTGACTTGGCTTTTGCAAGGGTCATTGCTTCCTGATGCATGTTTAACTGTCCTG TGTTCACTTTGTTGCcGCAGGTTTTTAGAGGAACGTAAAGAGATCaCCGAGAAATTCAGT GCGGAACAAGATGCCTTCCTGCAGGAGGCCCAGGAGCAGCATGCCCGTGAGCTG (SEQ ID NO: 334)
OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 335, 336) LEFT PRIMER 1 22 64.29 50.00 3.00 2.00 GGGAAACTGACTTGGCTTTTGC
RIGHT PRIMER 135 20 64.63 55.00 3.00 2.00 GGCATCTTGTTCCGCACTGA SEQUENCE SIZE: 174 INCLUDED REGION SIZE: 174 PRODUCT SIZE: 135, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00
1 GGGAAACTGACTTGGCTTTTGCAAGGGTCATTGCTTCCTGATGCATGTTTAACTGTCCTG 61 TGTTCACTTTGTTGCcGCAGGTTTTTAGAGGAACGTAAAGAGATCaCCGAGAAaTTCAGT
<««
121 GCGGAACAAGATGCCTTCCTGCAGGAGGCCCAGGAGCAGCATGCCCGTGAGCTG «<««««««
116 )Whole sequence ::: rsl0854482-rs2839261
CCCTGCACACTGACCTGCATGCCCTCGTCACCTGCACTCTGCATGCTCACCATCTGACGG ACTCCTGCGAcGGGCATGGGAAGGTCGCCGCCGCCGGCAGCCtTGCGAGCACTTTGGATG
TGTGCACCCGGCATGCCAGGCCCGAGTCAACAGACTGGCCGACCTTGGCGTCCTG (SEQ ID NO: 337)
OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 338, 339)
LEFT PRIMER 21 20 65.22 65.00 4.00 0.00 GCCCTCGTCACCTGCACTCT RIGHT PRIMER 168 20 64.77 60.00 5.00 1.00 CCAAGGTCGGCCAGTCTGTT SEQUENCE SIZE: 175 INCLUDED REGION SIZE: 175
PRODUCT SIZE: 148, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00
1 CCCTGCACACTGACCTGCATGCCCTCGTCACCTGCACTCTGCATGCTCACCATCTGACGG
61 ACTCCTGCGAcGGGCATGGGAAGGTCGCCGCCGCCGGCAGCCtTGCGAGCACTTTGGATG
121 TGTGCACCCGGCATGCCAGGCCCGAGTCAACAGACTGGCCGACCTTGGCGTCCTG
117 )Whole sequence ::: rs2032111-rs718496
TTTATTGCTGAGTGGTATTCCATTTTATGGGTCCATTATAGTTTATTTGTCCAGACACTT CATGGAAaGACATCAGTGTTTCCtGTTTTTCAATCATAAATTGATGTTTAATTTTAAAAT TTTGGAATTGTAGAAGAAATGCAATTCTTTTTTCC (SEQ ID NO: 340)
OLIGO start len tm qc% any 3± seq (SEQ ID NOs: 341, 342)
LEFT PRIMER 28 22 53.65 31.82 4.00 3.00 TGGGTCCATTATAGTTTATTTG
RIGHT PRIMER 143 22 57.46 31.82 4.00 2.00 TGCATTTCTTCTACAATTCCAA SEQUENCE SIZE: 155 INCLUDED REGION SIZE: 155
PRODUCT SIZE: 116, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 3.00
1 TTTATTGCTGAGTGGTATTCCATTTTATGGGTCCATTATAGTTTATTTGTCCAGACACTT
61 CATGGAAaGACATCAGTGTTTCCtGTTTTTCAATCATAAATTGATGTTTAATTTTAAAAT
121 TTTGGAATTGTAGAAGAAATGCAATTCTTTTTTCC
118 JWhole sequence ::: rs2070434-rs2070435 CTTTGGTGCAGAATCATGCTGCAGGCAAGGTGGGCCCACCTCCCTGGAATTTCATCCCCC CCGTCAGTTAAACCCATGGTGGTTTTATTTTCTAGGCCACCTGATCTGGGAGGACCACCT CCAAGAAAAGCAGTCCTATCGATGAACGGTCTAAGTTATGGTGTTATCAGAGTGGATACT GAAGAAAAGTTGTCAGTCCTTACTGTTC (SEQ ID NO: 343)
OLIGO start len tm qc% any 3' seq (SEQ ID NOs: 344, 345)
LEFT PRIMER 33 20 66.57 60.00 4.00 3.00 GGCCCACCTCCCTGGAATTT RIGHT PRIMER 176 22 54.26 40.91 4.00 0.00 TCCACTCTGATAACACCATAAC SEQUENCE SIZE: 208 ■ XNCLODED REGION SIZE: 208
PRODUCT SIZE: 144, PAIR ANY COMPL: 4.00, PAIR 31 COMPL: 1.00
1 CTTTGGTGCAGAATCATGCTGCAGGCAAGGTGGGCCCACCTCCCTGGAATTTCATCCCCC
61 cCGTCAGTTAAACCCATGGTGGTTTTATTTTCTAGGCCACCTGATCTGGGAGGACCACCT
121 CCAAGAAAAGCAGTCCTaTCGATGAACGGTCTAAGTTATGGTGTTATCAGAGTGGATACT
181 GAAGAAAAGTTGTCAGTCCTTACTGTTC
All publications, patents and patent applications cited herein are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. AU methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Claims What is claimed is:
1. A method for determining whether a fetus has at least one chromosomal abnormality, comprising:
' using tandem single nucleotide polymorphisms to compare fetal DNA to maternal DNA so as to determine whether the fetus has at least one chromosomal abnormality.
2. The method of claim I9 wherein the fetal DNA is obtained from maternal blood, maternal urine, maternal sweat, maternal cells, or cell free DNA from the mother.
3. The method of claim 1 or 2, wherein the fetal DNA is cell-free fetal DNA.
4. The method of any one of claims 1-3, wherein the maternal DNA is obtained from maternal blood.
5. The method of any one of claims 1-3, wherein the maternal DNA is obtained from a buccal swab.
6. The method of any one of claims 1-5, wherein the comparison step comprises using high-fidelity PCR (HiFi-PCR) and constant denaturant capillary electrophoresis (CDCE) to compare the fetal DNA to maternal DNA.
7. The method of any one of claims 1-6, wherein the comparison step comprises using at least 96 tandem single nucleotide polymorphisms.
.
8. The method of any one of claims 1-7, wherein the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 250 basepairs apart.
9. The method of claim 8, wherein the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 200 basepairs apart.
10. The method of claim 9, wherein the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 150 basepairs apart.
11. The method of claim 10, wherein the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 100 basepairs apart.
12. The method of claim 11, wherein the single nucleotide polymorphisms in each tandem single nucleotide polymorphism are each at most about 50 basepairs apart.
13. The method of any one of claims 1-12, wherein at least one tandem single nucleotide polymorphism is located on the p arm or the q arm of chromosome 21.
14. The method of any one of claims 1-13, wherein at least one tandem single nucleotide polymorphism is located on the q arm of chromosome 21 and at least one tandem single nucleotide polymorphism is located p arm of chromosome 21.
15. The method of any one of claims 1-14, wherein the chrom osom al abnormality is chromosomal aneuploidy.
16. The method of claim 15, wherein the chromosomal abnormality is trisomy 13, 18 or 21.
17. The method of claim 16, wherein the chromosomal abnormality is trisomy 21.
18. The method of any one of claims 1-14, wherein the chromosomal abnormality is a deletion or insertion mutation.
19. The method of claim 18, wherein the chromosomal abnormality is a deletion or insertion mutation of more than about 3 megabasepairs.
20. The method of claim 18, wherein the chromosomal abnormality is a deletion or insertion mutation of fewer than about 3 megabasepairs.
21. The method of any one of claims 1-14, wherein the chromosomal abnormality is a copy number polymorphism, a copy number variant, chromosome 22ql 1 deletion syndrome, 1 Iq deletion syndrome on chromosome 11 or 8ρ deletion syndrome on chromosome 8.
22. The method of any one of claims 1-21, wherein the fetus is a male fetus.
23. The method of any one of claims 1-21, wherein the fetus is a female fetus.
24. The method of any one of claims 1-23, wherein the fetus is a mammal.
25. The method of any one of claims 1-24, wherein the fetus is a human.
26. The method of any one of claims 1 -24, wherein the fetus is a non-human mammal.
27. The method of any one of claims 1 -26, wherein the fetus has been determined to be at an elevated risk for having a chromosomal abnormality.
28. The method of any one of claims 1-27, further comprising using tandem single nucleotide polymorphisms to compare paternal DNA to the fetal and/or maternal DNA.
29. The method of any one of claims 1-28, wherein the comparison step further comprises the step of converting nucleic acid molecules to a homoduplex state.
30. A method for identifying chromosomes, comprising comparing tandem single nucleotide polymorphisms on the chromosomes so as to identify the chromosomes.
31. The method of claim 30 further comprising, prior to the comparison step, determining a set of tandem single nucleotide polymorphisms for a specific chromosome.
32. A system comprising packaging material and primers that specifically hybridize to each of the single nucleotide polymorphisms of at least one of tandem single nucleotide polymorphisms 1-118.
33. A system comprising packaging material and primers that specifically hybridize flanking sequences of at least one of the tandem single nucleotide polymorphisms 1-118.
34. A method for amplifying a single nucleotide polymorphism or a tandem single nucleotide polymorphism comprising using high-fidelity PCR to amplify the single nucleotide polymorphism or the tandem single nucleotide polymorphism.
35. The method of claim 34, wherein the single nucleotide polymorphism or a tandem single nucleotide polymorphism is comprised in a nucleic acid segment obtained from a maternal biological sample.
36. An isolated nucleic acid sequence comprising at least one of SEQ ED NOs 1-357.
37. An isolated nucleic acid sequence as described by claim 36 for use in medical treatment or diagnosis.
PCT/US2007/005399 2006-02-28 2007-02-28 Detecting fetal chromosomal abnormalities using tandem single nucleotide polymorphisms WO2007100911A2 (en)

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AU2007220991A AU2007220991C1 (en) 2006-02-28 2007-02-28 Detecting fetal chromosomal abnormalities using tandem single nucleotide polymorphisms
CA2647793A CA2647793C (en) 2006-02-28 2007-02-28 Detecting fetal chromosomal abnormalities using tandem single nucleotide polymorphisms
DK07752121.9T DK1996728T3 (en) 2006-02-28 2007-02-28 Detection of fetal chromosomal abnormalities using tandem single nucleotide polymorphisms
DE602007014335T DE602007014335D1 (en) 2006-02-28 2007-02-28 DETECTION OF CHROMOSOME ABNORMALITIES IN FATUS USING TANDEM INDIVIDUAL LIQUID POLYMORPHISMS
AT07752121T ATE508209T1 (en) 2006-02-28 2007-02-28 DETECTION OF CHROMOSOME ABNORMALITIES IN THE FETUS USING TANDEM SINGLE NUCLEOTIDE POLYMORPHISMS
SI200730667T SI1996728T1 (en) 2006-02-28 2007-02-28 Detecting fetal chromosomal abnormalities using tandem single nucleotide polymorphisms
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