Mutation detection using denaturing gradients

Abstract

Methods for detecting single nucleotide polymorphisms in a sample by applying an energy gradient to a mixture of probe-hybridized target wild type and mutant polynucleotides are provided. A nucleotide probe, having a nucleotide sequence complementary to at least a portion of the wild type or mutant polynucleotide(s), is exposed to a sample containing target wild type polynucleotide(s) and target mutant polynucleotide(s) under hybridizing conditions. Target wild type polynucleotide(s) form homoduplexes with the wild type probe, while target mutated polynucleotide(s) form heteroduplexes with the wild type probe. Target mutant polynucleotide(s) form homoduplexes with the mutant probes, while target wild type polynucleotide(s) form heteroduplexes with the mutant probe. An energy gradient is applied to the duplex-containing sample to induce selective denaturation of the duplexes. The gradient may be a thermal or chemical one and is preferably temporally linear. The hetero- and homo-duplexes deanneal at different times so that the detection of more than one type of polynucleotide(s) is indicative of a single nucleotide polymorphism. Capillary electropheresis, gel electropheresis, high performance liquid chromatography, or microfluidics are typically used for identification of the separated strands.

What is claimed is:  
     
         1 . A method of detecting single nucleotide polymorphisms comprising 
 a. admixing a nucleotide probe with a sample containing target wild type polynucleotide(s) and target mutant polynucleotide(s), said probe having a nucleotide sequence complementary to at least a portion of one strand of either the mutant or wild type polynucleotide(s);    b. inducing hybridization between the probe and target polynucleotide(s), thereby forming duplexes between the probe and target polynucleotide(s), wherein target wild type polynucleotide(s) form homoduplexes with wild type probes and target mutant polynucleotide(s) form heteroduplexes with wild type probes, while target mutant polynucleotide(s) form homoduplexes with mutant probes and target wild type polynucleotide(s) form heteroduplexes with mutant probes;    c. applying an energy gradient to the sample to induce selective denaturation of the duplexes; and    d. detecting the presence of single stranded polynucleotide(s) wherein the detection of more than one type of polynucleotide(s) is indicative of a single nucleotide polymorphism.    
     
     
         2 . A method of  claim 1  in which the gradient is a thermal gradient.  
     
     
         3 . A method of  claim 1  in which the gradient is a chemical gradient.  
     
     
         4 . A method of  claim 1  in which the probe is attached to a solid support.  
     
     
         5 . A method of  claim 1  in which the target polynucleotide(s) are attached to a solid support.  
     
     
         6 . A method of  claim 1  employing capillary electropheresis, gel electropheresis, high performance liquid chromatography, or microfluidics for identification of the separated strands.  
     
     
         7 . A method of detecting single nucleotide polymorphisms comprising 
 a. admixing a nucleotide probe with a sample containing target wild type polynucleotide(s) and target mutant polynucleotide(s), said probe having a nucleotide sequence complementary to at least a portion of one strand of either the mutant or wild type polynucleotide(s);    b. inducing hybridization between the probe and target polynucleotide(s), thereby forming duplexes between the probe and target polynucleotide(s), wherein target wild type polynucleotide(s) form homoduplexes with wild type probes and target mutant polynucleotide(s) form heteroduplexes with wild type probes, while target mutant polynucleotide(s) form homoduplexes with mutant probes and target wild type polynucleotide(s) form heteroduplexes with mutant probes;    c. applying a thermal gradient to the sample to induce selective denaturation of the duplexes; and    d. detecting the presence of single stranded polynucleotide(s) wherein the detection of more than one type of polynucleotide(s) is indicative of a single nucleotide polymorphism.    
     
     
         8 . A method of  claim 7  in which the probe is attached to a solid support.  
     
     
         9 . A method of  claim 7  in which the target polynucleotide(s) are attached to a solid support.  
     
     
         10 . A method of  claim 7  employing capillary electropheresis, gel electropheresis, high performance liquid chromatography, or microfluidics for identification of the separated strands.  
     
     
         11 . A method of detecting single nucleotide polymorphisms comprising 
 e. admixing a nucleotide probe with a sample containing target wild type polynucleotide(s) and target mutant polynucleotide(s), said probe having a nucleotide sequence complementary to at least a portion of one strand of either the mutant or wild type polynucleotide(s);    f. inducing hybridization between the probe and target polynucleotide(s), thereby forming duplexes between the probe and target polynucleotide(s), wherein target wild type polynucleotide(s) form homoduplexes with wild type probes and target mutant polynucleotide(s) form heteroduplexes with wild type probes, while target mutant polynucleotide(s) form homoduplexes with mutant probes and target wild type polynucleotide(s) form heteroduplexes with mutant probes;    g. applying a chemical gradient to the sample to induce selective denaturation of the duplexes; and    h. detecting the presence of single stranded polynucleotide(s) wherein the detection of more than one type of polynucleotide(s) is indicative of a single nucleotide polymorphism.    
     
     
         12 . A method of  claim 11  in which the probe is attached to a solid support.  
     
     
         13 . A method of  claim 11  in which the target polynucleotide(s) are attached to a solid support.  
     
     
         14 . A method of  claim 11  employing capillary electropheresis, gel electropheresis, high performance liquid chromatography, or microfluidics for identification of the separated strands.  
     
     
         15 . A method of detecting single nucleotide polymorphisms comprising 
 a. admixing a nucleotide probe with a sample containing target wild type polynucleotide(s) and target mutant polynucleotide(s), said probe having a nucleotide sequence complementary to at least a portion of one strand of the wild type polynucleotide(s);    b. inducing hybridization between the probe and target polynucleotide(s), thereby forming duplexes between the probe and target polynucleotide(s), wherein target wild type polynucleotide(s) form homoduplexes with the wild type probes and target mutant polynucleotide(s) form heteroduplexes with the wild type probes;    c. applying an energy gradient to the sample to induce selective denaturation of the duplexes; and    d. detecting the presence of single stranded polynucleotide(s) wherein the detection of more than one type of polynucleotide(s) is indicative of a single nucleotide polymorphism.    
     
     
         16 . A method of detecting single nucleotide polymorphisms comprising 
 a. admixing a nucleotide probe with a sample containing target wild type polynucleotide(s) and target mutant polynucleotide(s), said probe having a nucleotide sequence complementary to at least a portion of one strand of the mutant polynucleotide(s);    b. inducing hybridization between the probe and target polynucleotide(s), thereby forming duplexes between the probe and target polynucleotide(s), wherein target mutant polynucleotide(s) form homoduplexes with the mutant probes and target wild type polynucleotide(s) form heteroduplexes with the mutant probes;    c. applying an energy gradient to the sample to induce selective denaturation of the duplexes; and    d. detecting the presence of single stranded polynucleotide(s) wherein the detection of more than one type of polynucleotide(s) is indicative of a single nucleotide polymorphism.    
     
     
         17 . A method of detecting single nucleotide polymorphisms comprising 
 a. admixing a nucleotide probe with a sample containing target wild type polynucleotide(s) and target mutant polynucleotide(s), said probe having a nucleotide sequence complementary to at least a portion of one strand of either the mutant or wild type polynucleotide(s);    b. inducing hybridization between the probe and target polynucleotide(s), thereby forming duplexes between the probe and target polynucleotide(s), wherein target wild type polynucleotide(s) form homoduplexes with wild type probes and target mutant polynucleotide(s) form heteroduplexes with wild type probes, while target mutant polynucleotide(s) form homoduplexes with mutant probes and target wild type polynucleotide(s) form heteroduplexes with mutant probes;    c. applying an energy gradient to the sample to induce selective denaturation of the duplexes; and    d. detecting the presence of single stranded polynucleotide(s) wherein the detection of only one type of polynucleotide(s) may be indicative of a single nucleotide polymorphism;    e. comparing the elution time of said single strand to that of a homozygous wild type sample.