Method for detecting mutated alleles

Abstract

A method for detecting mutated alleles in an excess of wild type alleles in a sample by isolating sample DNA; amplifying a target DNA sequence; and separating mutated DNA sequences from wild type DNA sequences by virtue of the preferential binding of the wild type sequences to carrier-bound complementary oligonucleotides. The amplification and separation steps may be iterated through one or more additional cycles to enhance sensitivity.

That which is claimed is:  
     
         1 . A method for detecting the presence of tumor cells in an examination sample from a patient suspected of having cancer, wherein the method comprises detecting mutated alleles in an excess of wild type alleles by the steps of: 
 isolating DNA from the examination sample;    amplifying from the isolated DNA a DNA sequence region that contains a target DNA sequence suspected of containing one or more mutations;    converting the amplified DNA sequence region to single stranded DNA;    separating mutated single stranded DNA sequences from unmutated single stranded DNA sequences by a separation step that employs preferential binding of the unmutated single stranded DNA sequences to one or more oligonucleotides; wherein 
 the oligonucleotides comprise DNA sequences of 12 to 25 bases;  
 the oligonucleotide DNA sequences are complementary to DNA sequences in the unmutated target DNA sequence and together include all of the unmutated target DNA sequence; and  
 the oligonucleotides are covalently bound to carrier materials.  
   
     
     
         2 . The method according to  claim 1 , wherein the examination sample comprises a material selected from the group consisting of stool, sputum, bronchial lavage, urine, tissue biopsy material, saliva, and smear material.  
     
     
         3 . The method according to  claim 1 , wherein the examination sample comprises stool from a patient suspected of having colorectal carcinoma.  
     
     
         4 . The method according to  claim 1 , wherein the examination sample comprises a sputum sample or a bronchial lavage sample from a patient suspected of having bronchial carcinoma.  
     
     
         5 . The method according to  claim 1 , wherein the examination sample comprises urine from a patient suspected of having bladder carcinoma.  
     
     
         6 . The method according to  claim 1 , wherein the examination sample comprises a tissue biopsy sample.  
     
     
         7 . A method for detecting the presence of tumor cells in an examination sample from a patient suspected of having cancer, wherein the method comprises detecting mutated alleles in an excess of wild type alleles by the steps of: 
 isolating from an examination sample a DNA sample comprising unmutated wild typed alleles and suspected mutated alleles, wherein the nucleotide sequence is known for the unmutated wild typed alleles;    amplifying the DNA sample by PCR;    introducing the amplified DNA sample into at least one chromatographic column wherein the at least one chromatographic column comprises at least one carrier-bound oligonucleotide sequence that either alone in a single column or in multiple columns is complementary to the entire region of interest of the unmutated wild type sequence suspected of including at least one point mutation, and wherein the wild type alleles bind to the carrier bound oligonucleotide DNA sequences and the mutated alleles do not bind under hybridizing conditions maintained at the melting temperature of the wild-type specific oligonucleotide and its complementary sequence or if the mutated alleles do bind they are eluted from the column before the wild type alleles when a buffer is added; and detecting the mutation in the mutated alleles eluted from the at least one chromatographic column.    
     
     
         8 . The method according to  claim 7 , wherein the method is used to separate K-ras or p53 mutated alleles from an excess of unmutated K-ras or p53 wild type alleles, respectively.  
     
     
         9 . The method according to  claim 7 , wherein the mutated alleles comprise at least one point mutation.  
     
     
         10 . The method according to  claim 7 , wherein the multiple columns are arranged in parallel or in series for the separation process.  
     
     
         11 . The method according to  claim 7 , wherein the at least one carrier bound oligonucleotide sequence comprises sense and/or anti-sense oligonucleotides that are complementary to the wild type alleles.  
     
     
         12 . The method according to  claim 7 , wherein the carrier bound oligonucleotide sequence comprises from 16 to 20 base pairs.  
     
     
         13 . The method according to  claim 7 , wherein the carrier bound oligonucleotide is bound to a carrier material that is selected from the group consisting of glasses, gel materials, and polymer materials.  
     
     
         14 . The method according to  claim 7 , wherein the at least one carrier-bound oligonucleotide comprises a plurality of oligonucleotides having different sequences, and mutated single stranded DNA sequences are separated from unmutated single stranded DNA sequences by preferential binding of the unmutated single stranded DNA sequences to the different sequence oligonucleotides in series.  
     
     
         15 . The method according to  claim 7 , wherein the at least one carrier-bound oligonucleotide comprises a plurality of oligonucleotides having different sequences in multiple, and mutated single stranded DNA sequences are separated from unmutated single stranded DNA sequences by preferential binding of the unmutated single stranded DNA sequences to the different sequence oligonucleotides configured in a parallel flow arrangement.