Methods for Genotyping

• US 20070269817A1
• Filed: 12/21/2006
• Published: 11/22/2007
• Est. Priority Date: 06/17/2002
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Shapero, Michael
• Granted Patent: US 9,388,459 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 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention provides for methods for discriminating between alleles at polymorphic positions in a genome. In general the methods employ allele specific extension of oligonucleotides that are complementary to one of the alleles at the 3′ end of the oligonucleotide. The allele specific oligonucleotides are resistant to proof reading activity from a polymerase and may be extended in an allele specific manner by a DNA polymerase with a functional 3′ to 5′ exonuclease activity. The allele specific oligonucleotides may be attached to a solid support such as a chip or a bead.

Claims

1. A method of genotyping a plurality of polymorphism in a nucleic acid sample comprising:

a) mixing the nucleic acid sample with a plurality of pairs of capture probes wherein each pair of capture probes consists of a first and a second allele specific primer, wherein the first allele specific primer is complementary to a first allele of a selected polymorphism and includes the polymorphic position and the second allele specific primer is complementary to a second allele of the selected polymorphism and includes the polymorphic position, wherein said first and second allele specific primers are resistant to 3′

exonuclease activity;

b) subjecting the mixture from a) to a primer extension reaction, wherein said first and second allele specific primers are extended with a DNA polymerase comprising a proofreading activity to generate first and second allele specific extension products in the presence of the first and second alleles, respectively; and

c) detecting the presence of said first and second allele specific extension products, wherein the presence of a first allele specific extension product is indicative of the presence of the first allele of a selected polymorphism and presence of a second allele specific extension product is indicative of the presence of the second allele.

2. The method of claim 1 wherein said first and second allele specific primers comprise 1, 2 or 3 phosphorothioate linkages at the 3′

end.

3. The method of claim 1 wherein said first and second allele specific primers comprise a locked nucleic acid linkage at the 3′

end, wherein said linkage is between the terminal 3′

base and the penultimate base or between the penultimate base and the base immediately 5′

of the penultimate base.

4. The method of claim 1 wherein the nucleic acid sample is an amplified genomic DNA sample.

5. A method for genotyping a plurality of polymorphisms in a nucleic acid sample, where each polymorphism has a first and a second allele, comprising:

incubating the nucleic acid sample with a plurality of beads attached to allele specific capture probes to allow formation of complexes between target fragments and allele specific capture probes, wherein the allele specific capture probes comprise;

(i) a linker,

(ii) a cleavage region,

(iii) a tag region, wherein each different allele specific probe has a different sequence tag region and wherein the tag region is at least 15 bases; and

,

(iv) a target specific region that terminates at its 3′

end with a base that is complementary to a polymorphic base in the target;

extending the allele specific capture probes in the presence of labeled nucleotides using the target fragment as template to obtain labeled allele specific capture probes, wherein extension of the allele specific capture probes is blocked if there is a mismatch between the polymorphic position and the 3′

end of the allele specific capture probe;

separating the target fragment from the labeled allele specific capture probes;

cleaving at least a portion of the labeled allele specific capture probes from the beads;

detecting the released labeled allele specific capture probes by hybridization to an array of tag probes, wherein said tag probes of known sequence are present at known or determinable locations on said array and each tag probe is complementary to a different tag region present in the allele specific capture probes; and

determining the genotype of said plurality of polymorphisms by determining which alleles are present, wherein the presence of hybridized labeled allele specific capture probes is indicative of the presence of a particular allele in the nucleic acid sample.

6. The method of claim 5 wherein said step of cleaving comprises an enzymatic step.

7. The method of claim 6 wherein the cleavage region comprises one or more uracil bases and cleavage comprises incubation with a uracil DNA glycosylase to generate abasic sites and the abasic sites are cleaved with an AP endonuclease or with acid or heat treatment.

8. The method of claim 6 wherein the cleavage region comprises one or more inosines and cleavage comprises incubation with an Endonuclease V.

9. The method of claim 5 wherein said step of cleaving comprises photo cleavage of a light sensitive linkage in the capture probe.

10. A method for genotyping a plurality of polymorphisms in a nucleic acid sample comprising:

fragmenting the nucleic acid sample to generate fragments;

hybridizing a collection of capture probes to target fragments, wherein said capture probes are attached to a solid support at a 5′

end and each comprises;

(i) a spacer sequence near said 5′

end,

(ii) a dU region comprising a plurality of uracil residues,

(iii) a tag sequence of at least 15 bases that is unique for each species of capture probe,

(iv) a target specific sequence, and

(v) an allele specific nucleotide corresponding to one allele of a polymorphism in said plurality of polymorphisms, wherein the capture probes terminates at its 3′

end with said allele specific nucleotide;

extending said capture probes with a DNA polymerase to generate extended capture probes in an allele specific extension reaction;

washing the solid support to remove the target fragments;

cleaving the extended capture probes from the solid support by a method comprising photo cleavage or enzymatic cleavage;

hybridizing the extended capture probes to an array comprising a plurality of tag probe features, wherein each tag probe feature comprises a different tag probe and wherein said tag probes are complementary to the tag sequences of the capture probes; and

detecting the presence of capture probes wherein the presence of an extended capture probe complementary to a selected allele is indicative of the presence of that allele in the nucleic acid sample.

11. The method of claim 10 wherein the solid support comprises a plurality of beads.

12. The method of claim 11 wherein the beads are coated with anti-digoxigenin, and the capture probes comprise a digoxigenin label.

13. The method of claim 10 wherein the capture probes are exonuclease resistant at the 3′

end and the DNA polymerase has 3′

to 5′

exonuclease proof-reading activity.

14. The method of claim 13 wherein the DNA polymerase is selected from the group consisting of Taq Gold, Vent, Deep Vent, T4 DNA polymerase, E coli Klenow fragment, and T7 DNA polymerase.

15. The method of claim 10 wherein the cleavage from the solid support is enzymatic and comprises cleavage with an endonuclease.

16. The method of claim 10 wherein the cleavage from the solid support is enzymatic and comprises treatment with uracil DNA glycosylase and heat or acid treatment.

17. The method of claim 10, wherein the cleavage from the solid support is photo cleavage and comprises exposure to UV light with a wavelength between 200 and 400 nanometers.

18. The method of claim 10 wherein the dU region comprises a plurality of inosine residues and the cleavage step comprises cleavage with Endonuclease V.

19. The method of claim 18 wherein the dU region comprises UIUI.

20. The method of claim 10 wherein the 3′

end of the capture probes comprises 0, 1, and 3 phosphorothioate linkages.