Methods for Genotyping
First Claim
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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.
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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.
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Citations
20 Claims
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1. A method of genotyping a plurality of polymorphism in a nucleic acid sample comprising:
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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. - View Dependent Claims (2, 3, 4)
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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:
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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. - View Dependent Claims (6, 7, 8, 9)
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10. A method for genotyping a plurality of polymorphisms in a nucleic acid sample comprising:
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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. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification