Multiplexing methods for indentifying nucleic acids using denaturing liquid chromatography

Abstract

A method is provided for the separation of nucleic acid samples. The method includes: providing a test mixture of a plurality of nucleic acid samples, wherein each sample is labeled with a spectrally detectable tag; applying the test mixture to a reversed phase solid support; eluting the mixture under partially denaturing conditions; and detecting spectrally resolved signals produced by the nucleic acid samples labeled with the detectable tags.

What is claimed is:  
     
         1 . A method for separating nucleic acid samples in a test mixture, the method comprising: 
 providing a test mixture comprising a plurality of nucleic acid samples, wherein each sample is labeled with a spectrally detectable tag;    applying the test mixture to a reversed phase support;    eluting the test mixture under partially denaturing conditions to separate at least one of the nucleic acid samples from the test mixture; and    detecting spectrally resolved signals produced by the nucleic acid samples labeled with the detectable tags.    
     
     
         2 . The method of  claim 1  wherein the nucleic acid samples comprise PCR heteroduplex and homoduplex products.  
     
     
         3 . The method of  claim 1  wherein applying a mixture to a reversed phase support comprises applying the mixture to a high performance liquid chromatography column comprising a reversed phase support.  
     
     
         4 . The method of  claim 1  wherein each detectable tag comprises a fluorescent dye.  
     
     
         5 . The method of  claim 4  wherein providing a test mixture comprises: 
 combining a different fluorescent dye with each nucleic acid sample to form a labeled nucleic acid sample; and  
 combining the labeled nucleic acid samples to form a test mixture.  
 
     
     
         6 . The method of  claim 5  wherein combining a different fluorescent dye with each nucleic acid sample comprises combining a different fluorescent dye with each nucleic acid sample during polymerase chain reaction amplification of the nucleic acid sample.  
     
     
         7 . The method of  claim 1  wherein the nucleic acid sample comprises DNA or RNA.  
     
     
         8 . The method of  claim 1  wherein eluting the test mixture under partially denaturing conditions comprises eluting with a mobile phase comprising an ion-pairing agent and an organic solvent.  
     
     
         9 . The method of  claim 1  wherein the partially denaturing conditions comprise a temperature of about 50° C. to about 80° C.  
     
     
         10 . The method of  claim 1  wherein detecting spectrally resolved signals comprises use of a spectrophotofluorometric HPLC detector.  
     
     
         11 . The method of  claim 1  wherein the tag is a spectrally detectable tag selected from the group of an electromagnetic tag and an electrochemical tag.  
     
     
         12 . The method of  claim 11  wherein the tag is a spectrally detectable tag selected from the group of a spectrophotometric tag and a spectrofluorometric tag.  
     
     
         13 . The method of  claim 1  wherein detecting spectrally resolved signals produced by the nucleic acid samples labeled with the detectable tags comprises passing the separately labeled nucleic acid samples through a detection zone of a detector substantially simultaneously, each sample generating a specific signal which is spectrally resolved from the specific signals of the other nucleic acid samples.  
     
     
         14 . The method of  claim 13  wherein the detector excites the detectable tags at one wavelength and detects emissions at multiple wavelengths.  
     
     
         15 . The method of  claim 13  wherein the detector excites the detectable tags using zero-order excitation.  
     
     
         16 . A method for detecting genotypic variations, the method comprising: 
 providing a pre-mixture comprising one unlabeled nucleic acid sample and two or more reference genotypes comprising labeled nucleic acid, wherein each labeled nucleic acid is labeled with a different detectable tag and the unlabeled nucleic acid is present in an excess amount relative to the total amount of labeled nucleic acid;    denaturing and reannealing the pre-mixture to form a test mixture comprising labeled/unlabeled nucleic acid duplexes;    applying the test mixture to a reversed phase support;    eluting the test mixture under partially denaturing conditions to separate at least one of the labeled/unlabeled nucleic acid duplexes from the test mixture;    detecting spectrally resolved signals produced by the labeled nucleic acid to define an elution profile for each tag; and    deducing from the elution profiles the composition of the unlabeled nucleic acid sample.    
     
     
         17 . The method of  claim 16  wherein the tag is a spectrally detectable tag selected from the group of an electromagnetic tag and an electrochemical tag.  
     
     
         18 . The method of  claim 17  wherein the tag is a spectrally detectable tag selected from the group of a spectrophotometric tag and a spectrofluorometric tag.  
     
     
         19 . The method of  claim 16  wherein the labeled and unlabeled nucleic acid comprise PCR products.  
     
     
         20 . The method of  claim 16  wherein applying the test mixture to a reversed phase support comprises applying the mixture to a high performance liquid chromatography column comprising a reversed phase support.  
     
     
         21 . The method of  claim 16  wherein each spectrally detectable tag comprises a fluorescent dye.  
     
     
         22 . The method of  claim 16  wherein detecting spectrally resolved signals produced by the labeled nucleic acid comprises passing the separately labeled nucleic acid samples through a detection zone of a detector substantially simultaneously, each sample generating a specific signal which is spectrally resolved from the specific signals of the other nucleic acid samples.  
     
     
         23 . The method of  claim 22  wherein the detector excites the detectable tags at one wavelength and detects emissions at multiple wavelengths.  
     
     
         24 . The method of  claim 22  wherein the detector excites the detectable tags using zero-order excitation.  
     
     
         25 . A method for determining haplotypes, the method comprising: 
 providing a pre-mixture comprising one unlabeled nucleic acid sample and four or more reference haplotypes comprising labeled nucleic acid, wherein each labeled nucleic acid sample includes two or more alleles, each reference haplotype is labeled with a different detectable tag, and the unlabeled nucleic acid is present in an excess amount relative to the total amount of labeled nucleic acid;    denaturing and reannealing the pre-mixture to form a test mixture comprising labeled/unlabeled nucleic acid duplexes;    applying the test mixture to a reversed phase support;    eluting the test mixture under partially denaturing conditions to separate at least one of the labeled/unlabeled nucleic acid duplexes from the test mixture;    detecting spectrally resolved signals produced by the labeled nucleic acid to define an elution profile for each tag; and    deducing from the elution profiles the composition of the unlabeled nucleic acid sample.    
     
     
         26 . The method of  claim 25  wherein the tag is a spectrally detectable tag selected from the group of an electromagnetic tag and an electrochemical tag.  
     
     
         27 . The method of  claim 26  wherein the tag is a spectrally detectable tag selected from the group of a spectrophotometric tag and a spectrofluorometric tag.  
     
     
         28 . The method of  claim 27  wherein each spectrally detectable tag comprises a fluorescent dye.  
     
     
         29 . The method of  claim 25  wherein detecting spectrally resolved signals produced by the labeled nucleic acid comprises passing the separately labeled nucleic acid samples through a detection zone of a detector substantially simultaneously, each sample generating a specific signal which is spectrally resolved from the specific signals of the other nucleic acid samples.  
     
     
         30 . The method of  claim 29  wherein the detector excites the detectable tags using zero-order excitation.