Enzymatic Methods for Genotyping on Arrays
Disclosed are methods for enzymatic genotyping of polymorphisms on solid supports. In one aspect the method includes hydrolysis of a nucleotide comprising a label on an array-bound probe by a 5′ to 3′ exonuclease activity specific for single-stranded DNA. If there is target-probe sequence mismatch at the polymorphic position (the labeled nucleotide in the probe), the labeled nucleotide is hydrolyzed from the probe by the exonuclease. The presence of a detectable signal on the array is indicative of the identity of the nucleotide at the polymorphic position in the target. In another aspect, the queried position on the probe may be a labeled ribonucleotide, and if there is a sequence mismatch at the polymorphic position on the probe, the labeled ribonucleotide will be hydrolyzed from the nucleic acid by the activity of an exoribonuclease enzyme specific for single-stranded sequences.
1 . A method for genotyping a target polymorphism in a nucleic acid sample comprising:
a) providing an array comprising a plurality of features
wherein each feature comprises a plurality of probes having the same sequence;
wherein the probes in a feature are perfectly complementary to the sequence that is immediately adjacent to a target polymorphism in a target sequence;
wherein a first subset of the features comprise probes that are perfectly complementary to a first allele of the target polymorphism but not to a second allele of the target polymorphism;
wherein a second subset of the features comprises probes that are perfectly complementary to the second allele of the target polymorphism;
wherein the probe position corresponding to the target polymorphism is modified to include a detectable label; and
wherein the probe further comprises a noncomplementary polynucleotide sequence of at least 7 nucleotides immediately 3′ to the probe position corresponding to the target polymorphism;
b) hybridizing the nucleic acid sample to the array to allow formation of probe-target complexes;
c) adding an effective amount of an exonuclease enzyme possessing a 5′ to 3′ exonuclease activity that is specific for single-stranded DNA;
d) determining the genotype of a polymorphism by detection of the presence of the label either:
i) on the probe complementary to the first allele of a target polymorphism,
ii) on the probe complementary to the second allele of a target polymorphism, or
iii) on both probes complementary to the first and second alleles of a target polymorphism, respectively.
2 . The method according to claim 1 , wherein the label is biotin.
3 . The method according to claim 1 , wherein the noncomplementary polynucleotide sequence is at least 10 nucleotides in length.
4 . The method according to claim 1 , wherein the exonuclease is RecJ.
5 . The method according to claim 1 , wherein the exonuclease is Exo VII.
6 . The method according to claim 1 , wherein the nucleic acid sample is unamplified genomic DNA.
7 . The method according to claim 1 , wherein the nucleic acid sample is genomic DNA that has been enriched for a subset of target sequences.
8 . The method according to claim 1 , wherein the full length array probes have a sequence length of between 15 nucleotides and 55 nucleotides.
9 . The method according to claim 1 , wherein the full length array probes have a sequence length of between 50 nucleotides and 71 nucleotides.
10 . The method according to claim 1 , wherein the full length array probes have a sequence length of between 26 nucleotides and 30 nucleotides.
11 . The method according to claim 1 , wherein formamide is added to the step of hybridizing the nucleic acid sample.
12 . The method according to claim 1 , wherein yeast RNA is added to the step of hybridizing the nucleic acid sample.
13 . A method for genotyping a target polymorphism in a ribonucleic acid sample comprising:
a) providing an array comprising a plurality of features
wherein each feature comprises a plurality of probes comprising the same sequence;
wherein the probes in a feature are perfectly complementary to the sequence that is immediately adjacent to a target polymorphism in a target sequence;
wherein a subset of the features comprise probes that are perfectly complementary to a first allele of the target polymorphism but not to a second allele of the target polymorphism;
wherein another subset of the features comprises probes that are perfectly complementary to the second allele of the target polymorphism;
wherein the probe position corresponding to the target polymorphism is modified to include a detectable label; and
wherein the probe further comprises a noncomplementary polynucleotide sequence of at least 7 nucleotides immediately 3′ to the probe position corresponding to the target polymorphism;
b) hybridizing the ribonucleic acid sample to the array to allow formation of probe-target complexes;
c) adding an effective amount of an exoribonuclease enzyme possessing a 5′ to 3′ exonuclease activity, wherein substrate for the exoribonuclease enzyme must be single-stranded nucleic acid;
d) determining the genotype of a polymorphism by detection of the presence of the label either:
i) on the probe complementary to the first allele of a target polymorphism,
ii) on the probe complementary to the second allele of a target polymorphism, or
iii) on both probes complementary to the first and second alleles of a target polymorphism, respectively.
14 . The method according to claim 13 , wherein the label is biotin.
15 . The method according to claim 13 , wherein the noncomplementary polynucleotide sequence is at least 10 nucleotides in length.
16 . The method according to claim 13 , wherein the exoribonuclease is exoribonuclease I or exoribonclease II.
17 . The method according to claim 13 , wherein the nucleic acid sample is whole genome RNA.
18 . The method according to claim 13 , wherein the array probes have a sequence length of between 35 nucleotides and 55 nucleotides.
19 . The method according to claim 13 , wherein formamide is added to the step of hybridizing the nucleic acid sample.
20 . The method according to claim 13 , wherein yeast RNA is added to the step of hybridizing the nucleic acid sample.