Enzymatic methods for genotyping on arrays

Abstract

The invention relates to methods for enzymatic, genotyping of polymorphisms on solid supports. In some aspects the methods include ligation of allele or base specific interrogation probes to an array probe. The array probe is labeled by ligation of the interrogation probe. Ligation is dependent on the identity of the base immediately adjacent to the end of the array probe. In other aspects array bound probes are labeled by template dependent extension.

What is claimed is: 
     
         1 . A method for genotyping a plurality of target polymorphisms in a nucleic acid sample comprising:
 obtaining an array comprising a plurality of features wherein each feature comprises a plurality of array probes of the same sequence, wherein the array probes in a feature are perfectly complementary to the sequence that is immediately 5′ of a target polymorphism in a target sequence and wherein the probe terminates at a base that is perfectly complementary to a first allele of said target polymorphism but not to a second allele of said target polymorphism, wherein said array also comprises a second feature that comprises probes that are perfectly complementary to the second allele;   hybridizing the nucleic acid sample to the array, in the presence of non-specific competitor selected from the group consisting of yeast RNA and formamide, to allow array probes and target sequences to form complexes and washing to remove nucleic acid that is not hybridized specifically to a feature of the array;   adding a collection of interrogation probes to the array to allow hybridization of the interrogation probes to target sequences on the array;   adding a ligase to allow interrogation probes to be ligated to the array probes in a template dependent manner;   detecting a hybridization pattern and determining from the hybridization pattern the presence or absence of labeled interrogation probe at features of the array and thereby determining the genotype of a plurality of target polymorphisms.   
     
     
         2 . The method of  claim 1  wherein the plurality of polymorphisms comprises single nucleotide polymorphisms, single base deletions and single base insertions. 
     
     
         3 . (canceled) 
     
     
         4 . A method for determining the genotype of a plurality of polymorphisms comprising: obtaining an array of support bound array probes wherein the array comprises at least 1,000,000 different probe sequences and each probe sequence is at a known or determinable location in the array, wherein the array comprises at least one array probe that is specific for each polymorphisms in said plurality of polymorphisms;
 hybridizing a target sample to the array to form complexes between the array probes and complementary target sequences in the target sample;   optionally removing unhybridized target sequences;   adding a plurality of interrogation probes and a ligase to allow ligation of interrogation probes to array probes;   removing unligated interrogation probes and determining the location of ligated interrogation probes to determine the genotype of at least some of said plurality of polymorphisms.   
     
     
         5 . The method of  claim 4  wherein the interrogation probes are a mixture of N9 labeled with a single label. 
     
     
         6 . The method of  claim 4  wherein the interrogation probes are a mixture of N9 differentially labeled depending on the identity of the 3′ base. 
     
     
         7 . The method of  claim 4  wherein the array comprises at least a first array probe and a second array probe for each polymorphism to be genotyped and wherein said first array probe and said second array probe are identical in sequence except at the base present at the 5′ end of the array probe. 
     
     
         8 . The method of  claim 4  wherein for the array probes are complementary to the target at a region that does not include the polymorphism but is immediately adjacent to the polymorphism wherein the interrogation probe hybridizes to a region of the target that includes the polymorphism. 
     
     
         9 . A method for genotyping a plurality of SNPs in a nucleic acid sample, said method comprising:
 (a) obtaining an array comprising a plurality of array probes wherein for each SNP to be genotyped said array comprises an array probe that is perfectly complementary to the region immediately 5′ of the SNP up to but not including the complement of the SNP;   (b) hybridizing the nucleic acid sample to allow formation of probe-target complexes;   (c) incubating the array with a mixture of interrogation probes, wherein each interrogation probe comprises a 3′ fixed base, a plurality of variable bases and a 5′ label that is indicative of the identity of the 3′ fixed base and wherein the mixture comprises interrogation probes with at least two different 3′ fixed bases and at least two different 5′ labels, wherein interrogation probes hybridize to target in probe-target complexes so that the 3′ end of the interrogation probe is immediately adjacent to the 5′ end of the array probe;   (d) ligating the 5′ end of the array probe and the 3′ end of the interrogation probe, thereby labeling the array probe with the 5′ label; and   (e) determining the identity of the SNP by identifying the 5′ label.   
     
     
         10 . The method of  claim 9  wherein formamide is added to the step of hybridizing the nucleic acid sample. 
     
     
         11 . The method of  claim 9  wherein yeast RNA is added to the step of hybridizing the nucleic acid sample. 
     
     
         12 . The method of  claim 9  wherein said array probes are between 35 and 55 bases. 
     
     
         13 . The method of  claim 9  wherein said array probes are between 50 and 71 bases. 
     
     
         14 . The method of  claim 9  wherein said array probes are 26 to 30 bases. 
     
     
         15 . The method of  claim 9  wherein the nucleic acid sample is unamplified genomic DNA. 
     
     
         16 - 19 . (canceled)