Methods for genotyping polymorphisms in humans

• US 20060024715A1
• Filed: 07/05/2005
• Published: 02/02/2006
• Est. Priority Date: 07/02/2004
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Di, Xiaojun, Matsuzaki, Hajime, Hubbell, Earl A., Cawley, Simon, Mei, Rui, Webster, Teresa A., Chiles, Richard, Liu, Guoying, Yang, Geoffrey
• Granted Patent: US 7,361,468 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Methods, kits and arrays of nucleic acid probes for genotyping large numbers of human SNPs in parallel are provided. A set of more than 100,000 human SNPs, known to be biallelic in at least two populations is provided. Allele specific perfect match probes and genotyping probe sets are provided for each allele of each biallelic SNP in a set of human SNPs that is useful for genetic analysis within and across populations. Probe sets that include perfect match and mismatch probes are provided. The probe sets are suitable for inclusion in an array. The invention provides the SNP and surrounding sequence and provides the sequences in such a way as to make them available for a variety of analyses including genotyping. As such, the invention relates to diverse fields impacted by the nature of genetics, including biology, medicine, and medical diagnostics.

Claims

1. An array of oligonucleotides, the array comprising:

at least 10,000 different allele specific perfect match probes attached to a solid support;

wherein each allele specific perfect match probe comprises at least 20 contiguous nucleotides from a different sequence listed in SEQ ID Nos. 1-116,211;

wherein each of the allele specific perfect match probes includes the polymorphic position of the sequence and each probe is perfectly complementary to one of the two possible alleles, and wherein each different allele specific perfect match probes is attached to a solid support in a known or determinable location of the array.

2. The array of claim 1 wherein the array comprises at least 100,000 different allele specific perfect match probes.

3. The array of claim 1 wherein the array comprises a first allele specific perfect match probe for the first allele of each SNP represented by SEQ IS Nos. 1-116,211 and a second allele specific perfect match probe for the second allele of each SNP represented by SEQ ID Nos. 1-116,211.

4. The array of claim 1 wherein the array comprises at least one allele specific perfect match probe for each allele of each SNP represented by SEQ ID Nos. 1-116,211;

wherein each allele specific perfect match probe comprises at least 20 contiguous nucleotides from a sequence listed in SEQ ID Nos. 1-116,211, including the polymorphic position, and wherein each allele specific perfect match probe comprises sequence from a different sequence listed in SEQ ID NOS. 1-116,211.

5. The array of claim 1 wherein the array comprises at least 10,000 different genotyping probe sets, wherein each genotyping probe set comprises:

at least one allele specific perfect match probe that is perfectly complementary to a first allele of each SNP, at least one allele specific perfect match probe that is complementary to a second allele of each SNP, at least one mismatch probe for the first allele of each SNP, and at least one mismatch probe for the second allele of each SNP.

6. The array of claim 1 wherein the array comprises at least 10,000 different genotyping probe sets, wherein each genotyping probe set comprises:

a plurality of allele specific perfect match probes that are perfectly complementary to a first allele of each SNP, a plurality of allele specific perfect match probes that are complementary to a second allele of each SNP, a plurality of mismatch probes for the first allele of each SNP, and a plurality of mismatch probe for the second allele of each SNP.

7. The array of claim 6 wherein each of the allele specific perfect match probes for a selected allele of a selected SNP varies from every other allele specific perfect match probe for that allele of that SNP in the position of the polymorphic base relative to the center of the probe.

8. The array of claim 7 wherein the polymorphic base in a probe is in a position selected from −

7, −

6, −

5, −

4, −

3, −

2, −

1, 0, 1, 2, 3, 4, 5, 6, or 7 relative to the center, where 0 is the central position of the probe.

9. The array of claim 8 wherein the probes are 25 bases in length.

10. The array of claim 1 wherein each allele specific perfect match probe is attached to a different bead.

11. The array of claim 2 wherein each allele specific perfect match probe is attached to a different bead.

12. A kit for genotyping at least 1,000 of the SNPs represented by SEQ ID Nos. 1-116,211, the kit comprising:

a first plurality of oligonucleotides, wherein each oligonucleotide in the plurality of oligonucleotides comprises 15 to 50 bases of a sequence from SEQ ID Nos. 1-116,211 or the complement thereof and a second plurality of mismatch oligonucleotides corresponding to each oligonucleotide in said first plurality of oligonucleotides and wherein each different oligonucleotide is attached to a solid support in a known or determinable location.

13. A method of genotyping at least 5,000 single nucleotide polymorphisms in parallel comprising:

obtaining a nucleic acid sample;

amplifying fragments of DNA in the nucleic acid sample;

hybridizing the amplified sample to a genotyping array, wherein the genotyping array comprises at least 10,000 different allele specific perfect match probes each attached to a solid support;

wherein each allele specific perfect match probe comprises at least 20 contiguous nucleotides from a different sequence listed in SEQ ID Nos. 1-116,211;

wherein each of the allele specific perfect match probes includes the polymorphic position of the sequence and each probe is perfectly complementary to one of the two possible alleles; and

,

wherein each different allele specific perfect match probes is attached to a solid support in a known or determinable location of the array;

analyzing the resulting hybridization pattern; and

determining the genotype of the sample for each of the at least 1,000 single nucleotide polymorphisms.

14. The method of claim 13 wherein the solid support is a plurality of beads wherein each allele specific perfect match probe is attached to a different bead.

15. The method of claim 13 wherein the solid support comprises a plurality of separate supports.

16. The method of claim 13 wherein the step of amplifying fragments of DNA in the nucleic acid sample comprises fragmenting the sample with at least one restriction enzyme, ligating an adapter to at least some of the fragments to generate adapter ligated fragments, and amplifying at least some of the adapter ligated fragments by polymerase chain reaction using a primer to the adapter.

17. The method of claim 16 wherein the at least one restriction enzyme is selected from the group consisting of XbaI and HindIII.

18. The method of claim 13 wherein the step of amplifying fragments of DNA in the nucleic acid sample comprises:

(a) fragmenting a first aliquot of the sample with a first restriction enzyme to generate a first fragmented aliquot;

(b) fragmenting a second aliquot of the sample with a second restriction enzyme to generate a second fragmented aliquot;

(c) ligating a first adapter to at least some of the fragments in the first fragmented aliquot;

(d) ligating a second adapter to at least some of the fragments in the second fragmented aliquot;

(e) amplifying at least some of the fragments from (c); and

,

(f) amplifying at least some of the fragments from (d); and

wherein the product of step (e) is hybridized to a first solid support and the product of step (f) is hybridized to a second solid support.

19. The method of claim 18 wherein the first restriction enzyme is XbaI, the second restriction enzyme is HindIII and the first primer and the second primer have the same sequence.

20. The method of claim 13 wherein the genotyping array comprises at least 10,000 different genotyping probe sets, wherein each probe set comprises at least 1 probe quartet for each allele of each single nucleotide polymorphism to be genotyped.

21. The method of claim 13 wherein the steps of analyzing the resulting hybridization pattern;

and determining the genotype of the sample for each of the at least 1,000 single nucleotide polymorphisms are performed by a computer.

22. The method of claim 13 wherein the nucleic acid sample is from a human.

23. The method of claim 13 wherein the nucleic acid sample is from a non-human primate.