Methods for Genotyping

Abstract

Novel methods and kits are disclosed for reducing the complexity of a nucleic acid sample to interrogate a collection of target sequences, for example, to discriminating between alleles at polymorphic positions in a genome. Complexity reduction can be accomplished by extension of a capture probes followed by amplification of the extended capture probe using common primers. The capture probes may be locus specific and allele-specific. The amplified sample may be hybridized to an array designed to interrogate the desired fragments for the presence or absence of a polymorphism. In some aspects 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.

1 . A method of amplifying a collection of target sequences from a nucleic acid sample the method comprising:
 fragmenting the nucleic acid sample;   ligating an adaptor comprising a second common sequence to the fragments, wherein the adaptor is ligated to the fragments so that the strand that is ligated to the 5′ end of the fragment strands comprises the second common sequence and the strand that is ligated to the 3′ end of the fragments lacks the complement of the second common sequence and is blocked from extension at the 3′ end;   hybridizing the adaptor-ligated fragments to a collection of capture probes comprising a plurality of different species of primers wherein each species comprises a first common sequence that is common to each capture probe in the collection of capture probes and a 3′ variable region that is specific for a target sequence in the collection of target sequences;   extending the capture probes to obtain extension products; and   amplifying the extended capture probes with first and second common sequence primers to obtain an amplified collection of target sequences.   
     
     
         2 . The method of  claim 1  wherein an amino group is used to block extension at the 3′ end of the adaptor strand that is ligated to the 3′ end of the fragments. 
     
     
         3 . A method of analyzing a nucleic acid sample comprising:
 amplifying a collection of target sequences from the nucleic acid sample according to the method of  claim 1 ;   hybridizing the amplified collection of target sequences to an array; and   analyzing the hybridization pattern to detect the presence or absence of target sequences from the collection of target sequences.   
     
     
         4 . A method of genotyping one or more polymorphic locations in a sample comprising:
 preparing an amplified collection of target sequences from the sample according to the method of  claim 1 ;   hybridizing the amplified collection of target sequences to an array designed to interrogate at least one polymorphic location in the collection of target sequences; and   analyzing the hybridization pattern to determine the identity of the allele or alleles present at one or more polymorphic location in the collection of target sequences.   
     
     
         5 . A method for analyzing sequence variations in a population of individuals comprising;
 obtaining a nucleic acid sample from each individual;   amplifying a collection of target sequences from each nucleic acid sample according to the method of  claim 1 ;   hybridizing each amplified collection of target sequences to an array designed to interrogate sequence variation in the collection of target sequences to generate a hybridization pattern for each sample; and   analyzing the hybridization patterns to determine the presence or absence of sequence variation in the population of individuals.   
     
     
         6 . The method of  claim 1  wherein the nucleic acid sample is fragmented by digestion with one or more restriction enzymes. 
     
     
         7 . The method of  claim 1  wherein prior to amplification the extension products are enriched in the sample to be amplified. 
     
     
         8 . The method of  claim 1  wherein labeled nucleotides are incorporated into the extension products and the extension products are enriched by affinity chromatography. 
     
     
         9 . The method of  claim 8  wherein the labeled nucleotides are labeled with biotin and avidin, streptavidin or an anti-biotin antibody is used to isolate extension products. 
     
     
         10 . The method of  claim 1  wherein prior to amplification the extended capture probes are made double stranded and single stranded nucleic acid in the sample is digested. 
     
     
         11 . The method of  claim 10  wherein the single stranded nucleic acid in the sample is digested with a nuclease. 
     
     
         12 . The method of  claim 11  wherein the nuclease is Exonuclease I. 
     
     
         13 . The method of  claim 1  wherein prior to amplification the extended capture probes are circularized and uncircularized nucleic acid in the sample is digested. 
     
     
         14 . The method of  claim 13  wherein extended capture probes are circularized by a method comprising:
 hybridizing an oligonucleotide splint to the extended capture probes, wherein the splint is complementary to the first and second common sequences, thereby juxtaposing the 5′ and 3′ ends of extended capture probes; and 
 ligating the ends of the extended capture probes to form circular extended capture probes. 
 
     
     
         15 . The method of  claim 13  wherein the uncircularized nucleic acid remaining in the sample is digested with a nuclease. 
     
     
         16 . The method of  claim 15  wherein the nuclease is Exonuclease III. 
     
     
         17 . The method of  claim 1  wherein there are 100 to 1,500 different target sequences in the collection of target sequences. 
     
     
         18 . The method of  claim 1  wherein there are 1,000 to 10,000 different target sequences in the collection of target sequences. 
     
     
         19 . The method of  claim 1  wherein there are 10,000 to 1,000,000 different target sequences in the collection of target sequences. 
     
     
         20 .- 49 . (canceled) 
     
     
         50 . The method of  claim 1  wherein the nucleic acid sample is in solution.