Methods for detecting rare polymorphic variants in genomic DNA sequences

Abstract

Methods for detecting low frequency nuclear mutations in a target sequence from a genomic DNA sequence are disclosed.

What is claimed is:  
     
         1 . A method for detecting low frequency mutations in a target sequence from a DNA sample comprising the steps of: 
 a) enriching a DNA sample for one or more target sequences, wherein the enrichment step comprises sequence-specific hybridization to the target sequences with one or more labeled probes, wherein each labeled probe is complementary to a specific target sequence, resulting in about a 10 3 -fold to about a 10 4 -fold enrichment of target sequences from the DNA sample thereby obtaining a target-enriched sample; and    b) detecting mutations in the target sequence or sequences from the target-enriched sample.    
     
     
         2 . The method of  claim 1 , wherein the enrichment step comprises: 
 a) denaturing double-stranded DNA;    b) contacting the denatured DNA with one or more probes comprising a sequence complementary to one or more target sequences to form a mixture;    c) maintaining the mixture of step b) under conditions such that probe-fragment hybrid molecules are formed; and    d) isolating the probe-fragment hybrids from the mixture,    resulting in a target-enriched sample and a depleted sample.    
     
     
         3 . The method of  claim 2 , wherein the DNA sample fragmented prior to denaturation.  
     
     
         4 . The method of  claim 1 , wherein a probe complementary to a specific target sequence comprises an affinity moiety unique for a specific target sequence.  
     
     
         5 . The method of  claim 1 , wherein a plurality of target sequences is concurrently enriched from a sample resulting in a plurality of target-enriched DNA samples.  
     
     
         6 . The method of  claim 2 , wherein the depleted sample is subjected to a subsequent enrichment step to enrich for one or more target sequences different from the target sequences obtained in the first enrichment step.  
     
     
         7 . The method of  claim 1 , wherein the DNA sample comprises single-stranded DNA molecules.  
     
     
         8 . The method of  claim 1 , wherein the DNA sample comprises double-stranded molecules.  
     
     
         9 . The method of  claim 1 , wherein constant denaturant capillary electrophoresis is used to detect nuclear mutations.  
     
     
         10 . The method of  claim 1 , wherein allele-specific polymerase chain reaction is used to detect nuclear mutations.  
     
     
         11 . The method of  claim 1 , wherein the mutation detected is present at a mutant fraction about or higher than 10 −6 .  
     
     
         12 . A method for detecting low frequency nuclear mutations in a target sequence from a genomic DNA sample comprising the steps of: 
 a) enriching the DNA sample for molecules comprising one or more target sequences, thereby preparing a target-enriched sample comprising mutant and non-mutant sequences, wherein the enrichment step comprises sequence-specific hybridization with one or more labeled probes that hybridize to the target sequences resulting in about a 10 3 -fold to about 10 4 -fold enrichment of molecules comprising target sequences from the DNA sample;    b) subjecting the target-enriched sample to constant denaturant capillary electrophoresis using a wide-bore capillary to separate mutant heteroduplexes from non-mutant homoduplexes;    c) amplifying the heteroduplexes of step b) by high fidelity polymerase chain reaction to obtain amplified polymerase chain reaction products;    d) subjecting the polymerase chain reaction products of step c) to constant denaturant capillary electrophoresis to further enrich the sample for mutants, thereby creating a mutant-enriched sample;    e) subjecting the mutant-enriched sample of step d) to constant denaturant capillary electrophoresis to obtain a mutational spectra; and    f) selecting one or more individual mutant fractions from the mutational spectra for sequence analysis to detect mutations.    
     
     
         13 . The method of  claim 12 , wherein step b) further comprises subjecting mutant heteroduplexes to capillary electrophoresis prior to high fidelity polymerase chain reaction.  
     
     
         14 . The method of  claim 12 , wherein step d) further comprises subjecting the target-enriched sample to one or more additional rounds of constant denaturant capillary electrophoresis prior to obtaining the mutational spectra.  
     
     
         15 . The method of  claim 12 , wherein the mutation detected is present at a mutant fraction about or higher than 10 −6 .  
     
     
         16 . The method of  claim 12 , wherein step a) comprises fragmenting the genomic DNA to obtain double-stranded DNA fragments.  
     
     
         17 . The method of  claim 16 , wherein a double-stranded DNA sample is enriched for one or more target sequences, said enrichment comprising the steps of: 
 a) denaturing the double-stranded DNA;    b) contacting the denatured DNA with a plurality of probes comprising a sequence complementary to one or more target sequences to form a mixture;    c) maintaining the mixture of step b) under conditions such that probe-fragment hybrid molecules are formed; and    d) isolating the probe-fragment hybrids from the mixture,    resulting in a target-enriched sample and a depleted sample.    
     
     
         18 . The method of  claim 17 , wherein the probe comprises an affinity moiety unique for a specific target sequence.  
     
     
         19 . The method of  claim 18 , wherein the isolation of the probe-fragment hybrid is accomplished by contacting the probe-fragment hybrid with a binding partner molecule affixed to a solid support matrix, wherein the binding partner molecule binds to the affinity moiety of the probe.  
     
     
         20 . The method of  claim 19 , wherein mutant heteroduplexes are subjected to capillary electrophoresis prior to hifiPCR.  
     
     
         21 . The method of  claim 19 , wherein the enrichment further comprises one or more additional rounds of constant denaturant capillary electrophoresis prior to obtaining the mutational spectra.  
     
     
         22 . A method of mutational analysis to detect nuclear gene mutations at mutant fractions at or above 10 −6  in a target sequence comprising subjecting a DNA sample comprising one or more target sequences to constant denaturant capillary electrophoresis and high fidelity polymerase chain reaction to obtain a mutational spectrum to detect nuclear gene mutations, wherein, prior to constant denaturant capillary electrophoresis and high fidelity polymerase chain reaction, the DNA sample is enriched for a target sequence and wherein the enrichment comprises two steps wherein the first step comprises a sequence-specific hybridization coupled with a biotin-streptavidin capture system to enrich for DNA molecules comprising the target sequences, and wherein the second step comprises a mutant enrichment using constant denaturant capillary electrophoresis using a wide bore capillary.  
     
     
         23 . The method of  claim 22 , wherein the double-stranded DNA fragments are enriched for fragments comprising one or more target sequence, said enrichment comprising the steps of: 
 a) denaturing the double-stranded DNA fragments;    b) contacting the denatured fragments with a probe comprising a sequence complementary to a known target sequence;    c) maintaining the probe and DNA fragments under conditions such that a probe-fragment hybrid molecule is formed;    d) isolating the probe-fragment hybrid; and    e) regenerating a double-stranded fragment,    thereby generating an enriched pool of DNA fragments.    
     
     
         24 . A method for preparing a plurality of target-enriched DNA samples, wherein each enriched DNA sample comprises one or more particular target sequences, said method comprising the steps of: 
 a) contacting a DNA sample with a plurality of probes that can hybridize to a plurality of target sequences under conditions suitable for hybridization, wherein each probe that hybridizes to a specific target sequence comprises an affinity moiety wherein the affinity moiety is unique for each target sequence, thereby forming a hybridized sample mixture containing a plurality of affinity-labeled target sequences;    b) contacting the hybridized sample mixture of step a) with a plurality of binding partners to the affinity moieties in step a), wherein each binding partner is attached to a particular solid support matrix and wherein each binding partner binds to a specific affinity moiety; and    c) separating the particular solid support matrices,    thereby separating the particular target sequences, resulting in separate target-enriched DNA samples.    
     
     
         25 . The method of  claim 24 , wherein the DNA samples are enriched concurrently.  
     
     
         26 . The method of  claim 24 , wherein at least one paramagnetic solid support and at least one non-magnetic solid support is used.  
     
     
         27 . The method of  claim 24 , wherein at least one fluorescently-labeled solid support is used.  
     
     
         28 . The method of  claim 24 , wherein more than one fluorescently-labeled solid support comprising microsphere beads are separated based on fluorescent differences.  
     
     
         29 . The method of  claim 24 , wherein the DNA sample is fragmented prior to hybridization.  
     
     
         30 . The method of  claim 24 , wherein mutant enrichment is performed on target-enriched DNA samples.  
     
     
         31 . The method of claim  30 , wherein mutant enrichment is performed by constant denaturant capillary electrophoresis.  
     
     
         32 . The method of claim  30 , wherein mutant enrichment is performed by allele-specific polymerase chain reaction.