Methods for genotyping selected polymorphism

• US 20050142577A1
• Filed: 08/05/2004
• Published: 06/30/2005
• Est. Priority Date: 10/04/2002
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Foder, Stephen P.A., Jones, Keith W., Shapero, Michael H.
• Granted Patent: US 7,459,273 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Methods for genotyping polymorphisms using a locus specific primer that is complementary to a region near a selected polymorphism are described. Methods for synthesizing pools of locus specific primers that incorporate some degenerate positions are also disclosed. A plurality of different sequence capture probes are synthesized simultaneously using degenerate oligonucleotide synthesis. The sequence of the locus specific regions of the capture probes are related in that they have some bases that are identical in each sequence in the plurality of sequences and positions that vary from one locus specific region to another. The sequences are selected based on proximity to a polymorphism of interest and because they conform to a similar sequence pattern.

Claims

1. A method of genotyping a plurality of polymorphisms in an organism comprising:

(a) identifying a plurality of target sequences each comprising a polymorphic position, wherein each target sequence has the same common sequence within 1000 bases of the polymorphic position and wherein each target sequence has a consensus sequence immediately adjacent to the common sequence;

(b) obtaining a plurality of capture probes, comprising a first universal priming region, a region that is complementary to the common sequence and a degenerate region that is complementary to the consensus sequence;

(c) contacting a nucleic acid sample comprising genomic DNA from the organism with the plurality of capture probes;

(d) extending the capture probes through the polymorphic positions;

(e) amplifying the extended capture probes;

(f) hybridizing the amplified extended capture probes to an array of probes comprising allele specific probes for a plurality of the polymorphic positions; and

(g) analyzing the hybridization pattern to determine which alleles are present for at least two of the polymorphisms.

2. The method of claim 1 wherein prior to step (c) the nucleic acid sample is fragmented and an adaptor comprising a second universal priming region is ligated to the fragments.

3. The method of claim 2 wherein the nucleic acid sample is fragmented with a first restriction enzyme, wherein each target sequence contains a recognition site for said first restriction enzyme and wherein the polymorphic position in each target sequence is between the recognition site for said first restriction enzyme and the common sequence.

4. The method of claim 2 wherein the amplifying of step (e) is by PCR using a first and a second primer for the first and the second universal priming regions.

5. The method of claim 1 wherein the common sequence is a recognition site for a restriction enzyme.

6. The method of claim 5 wherein the restriction enzyme is a Type IIS restriction enzyme.

7. The method of claim 1 wherein the consensus sequence is 10-25 bases and at least 50% of the positions are identical in each target sequence.

8. The method of claim 1 wherein the amplified extended capture probes are labeled with a detectable label.

9. The method of claim 7 wherein there are at least 2 degenerate positions in the consensus sequence.

10. The method of claim 7 wherein there are at least 5 degenerate positions in the consensus sequence.

11. The method of claim 7 wherein there are at least 6 degenerate positions in the consensus sequence.

12. The method of claim 1 wherein there are at least 10 degenerate positions in the consensus sequence.

13. The method of claim 1 wherein the capture probes are synthesized as a single pool wherein a mixture of at least two different nucleotides are incorporated at each degenerate position.

14. A kit comprising at least 100 different species of capture probes for genotyping a plurality of polymorphic positions each present on a different target sequence:

wherein each target sequence comprises a common sequence that is within 1000 base pairs of the polymorphic position;

wherein each target sequence comprises a consensus sequence immediately adjacent to the common sequence wherein the consensus sequence comprises at least one base that is identical in all the target sequences and at least one base that is variable between target sequences;

wherein each capture probe comprises a first universal priming sequence and a target specific sequence that comprises a region that is complementary to the consensus sequence from one target sequence and a region that is complementary to the common sequence, and

wherein the capture probes are optionally pooled into 1 to 5 containers each containing a plurality of different species of capture probes.

15. The kit of claim 14 further comprising an array of allele specific probes wherein the array comprises at least one allele specific probe for each allele of each SNP in said plurality of polymorphisms.

16. The kit of claim 15 wherein the array further comprises control probes that are identical to the allele specific probes except for the presence of a single base mismatch at the central position of each probe.

17. The kit of claim 14 wherein the common sequence is between 4 and 10 base pairs.

18. The kit of claim 14 wherein the common sequence is a recognition site for a restriction enzyme.

19. The kit of claim 14 wherein each polymorphism in the plurality is within 1000 base pairs of a recognition site for a selected restriction enzyme and wherein the polymorphic position is between said recognition site and said common and consensus sequences.

20. The kit of claim 19 wherein said selected restriction enzyme is Not 1.

21. The kit of claim 19 further comprising an adaptor sequence comprising a single strand overhang that is complementary to the overhang generated by digestion with said selected restriction enzyme.

22. The kit of claim 14 wherein said plurality of different species of capture probes are attached to a solid support selected from the group consisting of arrays, beads, microparticles, microtitre dishes and gels.

23. The kit of claim 22 wherein the capture probes are attached to the solid support through a covalent interaction and wherein optionally each species of capture probe is attached to the solid support in a discrete location.

24. The kit of claim 22 wherein each capture probe further comprises a tag sequence that is unique for each species of capture probe and the capture probes are attached to the solid support by hybridization to a collection of tag probes that are covalently attached to the solid support.

25. The kit of claim 14 wherein the capture probes are synthesized in one or more pools by a series of separate and pooled monomer additions.

26. The kit of claim 21 further comprising a first primer for the adaptor and a second primer for the universal priming sequence and optionally comprising a polymerase, dNTPs, said selected restriction enzyme, and buffer.

27. A method for amplifying a collection of target sequences from a nucleic acid sample using the kit of claim 26, said method comprising:

fragmenting the nucleic acid sample with said selected restriction enzyme;

ligating said adaptor to the fragments;

contacting the adaptor-ligated fragments with the at least 100 different species of capture probes in a single reaction;

extending at least some of the capture probes; and

amplifying the extended capture probes with said first and second primers.

28. The method of claim 27, wherein said plurality of different species of capture probes are synthesized on a solid support and the solid support is selected from the group consisting of arrays, beads, microparticles, microtitre dishes and gels;

wherein the step of extending at least some of the capture probes is done while the capture probes are attached to the solid support; and

further comprising releasing the extended capture probes from the solid support prior to amplifying the extended capture probes.

29. The method of claim 28, wherein, prior to releasing the extended capture probes from the solid support, nucleic acids that are not covalently attached to the solid support are removed.

30. The method of claim 27, wherein labeled nucleotides are incorporated into the extended capture probes and extended capture probes are isolated by affinity chromatography.

31. The method of claim 30, wherein said labeled nucleotides are labeled with biotin and, wherein avidin, streptavidin or an anti-biotin antibody is used to isolate extended capture probes.

32. The method of claim 27, wherein prior to amplification the extended capture probes are made double stranded and single stranded nucleic acid in the sample is digested with a nuclease.

33. The method of claim 27, wherein there are 1,000 to 5,000 different target sequences in the collection of target sequences.

34. The method of claim 27, wherein there are 2,000 to 10,000 different target sequences in the collection of target sequences.

35. The method of claim 27, wherein there are 10,000 to 1,000,000 different target sequences in the collection of target sequences.

36. A method of genotyping a plurality of polymorphisms in an individual comprising:

(a) selecting a plurality of polymorphisms to genotype wherein each polymorphism selected is within 1,000 base pairs of a recognition site for a first restriction enzyme;

(b) obtaining a plurality of at least 1000 capture probes wherein each capture probe comprises a common 5′

sequence and a different 3′

locus specific region that is complementary to a region that is within 1000 base pairs of one of the polymorphisms in the plurality of polymorphisms, and wherein the polymorphism is between said region and the recognition site for the first restriction enzyme;

(c) obtaining a biological sample comprising genomic DNA from the individual;

(d) contacting the biological sample with the plurality of oligonucleotides;

(e) extending at least some of the plurality of oligonucleotides;

(f) contacting the sample with the first restriction enzyme;

(g) ligating an adaptor to the fragments wherein the adaptor has an overhang that is complementary to the overhang generated by cleavage with the first restriction enzyme;

(h) amplifying fragments using a first adaptor primer and a second common primer;

(i) labeling the amplified fragments;

(j) hybridizing the amplified fragments to an array of probes comprising probes that are allele specific for each allele of each polymorphism in the plurality of polymorphisms; and

(k) analyzing the resulting hybridization pattern to determine which alleles of each of the polymorphisms in the plurality of polymorphisms is present.

37. The method of claim 36 wherein the obtaining of step (b) is by synthesizing the oligonucleotides in a pool wherein at least one base is a degenerate base.

38. The method of claim 37 wherein between 4 and 10 bases in the capture probes are degenerate.

39. The method of claim 37 wherein the oligonucleotides are synthesized in a series of separate and pooled steps.

40. The method of claim 37 wherein said oligonucleotides are synthesized on a solid support.

41. The method of claim 36 wherein said oligonucleotides are attached to a solid support and the 3′

end of the oligonucleotides is available for extension and wherein the extension step takes place while the oligonucleotides are attached to the solid support.