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:
 generating 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;   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 the collection of capture probes;   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 . A method of reducing the genetic complexity of a population of nucleic acid molecules and analyzing the sequence of nucleic acids in the reduced complexity sample, the method comprising the steps of:
 (a) providing on a solid support target fragments of the population captured by hybridization of a fragmented nucleic acid sample to a plurality of capture probes that are covalently attached to a solid support in known locations;   (b) separating unbound and non-specifically hybridized nucleic acids from the captured target fragments;   (c) amplifying the captured target fragments to obtain a reduced complexity sample; and   (d) analyzing a plurality of the amplified target fragments in the reduced complexity sample to determine at least some sequence of the nucleic acids.   
     
     
         22 . The method according to  claim 20  further comprising the step of ligating an adaptor molecule to at least one end of the target fragments prior to step (a). 
     
     
         23 . The method according to  claim 22  further comprising the step of amplifying the nucleic acid molecules with at least one primer that comprises a sequence that specifically hybridizes to the sequence of the adaptor molecule. 
     
     
         24 . The method according to  claim 23  wherein the probes are selected from the group consisting of a plurality of probes that defines a plurality of exons, introns or regulatory sequences from a plurality of genetic loci, a plurality of probes that defines a complete sequence of at least one single genetic locus, a plurality of probes that defines sites known to contain single nucleotide polymorphisms (SNPs), and a plurality of probes that defines an array designed to capture the complete sequence of at least one complete chromosome. 
     
     
         25 . The method according to  claim 24  wherein the at least one single genetic locus has a size selected from the group consisting of between 100 and 10,000 base pairs or between 4,000 and 500,000 base pairs. 
     
     
         26 . A method for determining nucleic acid sequence information about at least one region of nucleic acid, the method comprising the steps of (i) reducing the genetic complexity of a population of nucleic acid molecules according to a method comprising the steps of: (a) providing on a solid support target fragments of the population captured by specific hybridization to a plurality of capture probes wherein the fragmented target molecules are between 100 and 10,000 base pairs in size, by hybridizing fragmented target molecules to a solid support having a plurality of capture probes covalently attached thereto, (b) separating unbound and non-specifically hybridized nucleic acids from the captured target molecules; (c) amplifying the captured target molecules and eluting the captured target molecules from the solid support; and (ii) determining the nucleic acid sequence of the captured target molecules by sequencing at least a portion of the amplified target molecules. 
     
     
         27 . The method according to  claim 26  wherein the fragmented target molecules are ligated to an adaptor molecule prior to hybridization to the capture probes and wherein the amplification is performed with at least one primer that comprises a sequence that specifically hybridizes to the sequence of the adaptor molecule. 
     
     
         28 . A kit comprising adaptor molecules, and a plurality of capture probes on a solid support, wherein the targets are selected from the group consisting of a plurality of coding regions or regulatory sequences from a plurality of genetic loci and a plurality of sites known to contain SNPs and further comprising at least one additional component selected from the group consisting of DNA polymerase, T4 polynucleotide kinase, T4 DNA ligase, a hybridization buffer, a wash buffer, and an elution buffer. 
     
     
         29 . A method for reducing the complexity of a genomic sample to obtain a sample that is enriched for a collection of target sequences comprising: (i) hybridizing a collection of target-specific primers to a genomic DNA sample comprising adaptor modified fragments, wherein each target-specific primer comprises a 5′ first common sequence that is not complementary to the target sequence and a 3′ region that is complementary to the target sequence; (ii) extending the target-specific primers using the adaptor modified fragments as template to obtain primer extension products that have a second common sequence at the 3′ end of at least some of the extension products; (iii) annealing a splint oligonucleotide that comprises the complement of the first common sequence and the complement of the second common sequence to the extension products, wherein the splint oligonucleotide brings the 5′ and 3′ ends of the extension product together; (iv) ligating the 5′ and 3′ ends of the extension products to form a circular extension product; (v) treat with exonuclease VII to remove uncircularized nucleic acid; and (vi) amplify with PCR. 
     
     
         30 . A method of genotyping a plurality of polymorphisms 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.   
     
     
         31 . The method of  claim 30  wherein said first and second allele-specific primers comprise 1, 2 or 3 phosphorothioate linkages at the 3′ end. 
     
     
         32 . The method of  claim 30  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. 
     
     
         33 . The method of  claim 30  wherein the nucleic acid sample is an amplified genomic DNA sample obtained by amplifying the nucleic acid sample using random primers and DNA polymerase in an isothermal amplification reaction to obtain an amplified nucleic acid sample and further comprising fragmenting the amplified nucleic acid sample prior to step (a). 
     
     
         34 . 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.   
     
     
         35 . The method of  claim 34  wherein said step of cleaving comprises an enzymatic step. 
     
     
         36 . The method of  claim 35  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. 
     
     
         37 . The method of  claim 35  wherein the cleavage region comprises one or more inosines and cleavage comprises incubation with an Endonuclease V. 
     
     
         38 . The method of  claim 34  wherein said step of cleaving comprises photo cleavage of a light sensitive linkage in the capture probe. 
     
     
         39 . 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.   
     
     
         40 . The method of  claim 39  wherein the solid support comprises a plurality of beads. 
     
     
         41 . The method of  claim 40  wherein the beads are coated with anti-digoxigenin, and the capture probes comprise a digoxigenin label. 
     
     
         42 . The method of  claim 39  wherein the capture probes are exonuclease resistant at the 3′ end and the DNA polymerase has 3′ to 5′ exonuclease proof-reading activity. 
     
     
         43 . The method of  claim 42  wherein the DNA polymerase is selected from the group consisting of T4 DNA polymerase,  E coli  Klenow fragment, and T7 DNA polymerase. 
     
     
         44 . The method of  claim 39  wherein the cleavage from the solid support is enzymatic and comprises cleavage with an endonuclease. 
     
     
         45 . The method of  claim 39  wherein the cleavage from the solid support is enzymatic and comprises treatment with uracil DNA glycosylase and heat or acid treatment. 
     
     
         46 . The method of  claim 39 , wherein the cleavage from the solid support is photocleavage and comprises exposure to UV light with a wavelength between 200 and 400 nanometers. 
     
     
         47 . The method of  claim 39  wherein the dU region comprises a plurality of inosine residues and the cleavage step comprises cleavage with Endonuclease V. 
     
     
         48 . The method of  claim 47  wherein the dU region comprises UIUI. 
     
     
         49 . The method of  claim 39  wherein the 3′ end of the capture probes comprises 0, 1, or 3 phosphorothioate linkages.