Analysis of nicked DNA by matched ion polynucleotide chromatography

Abstract

The present invention provides a method for calibrating a MIPC column wherein the calibration relates to the determination of the organic solvent component in the mobile phase required to elute dsDNA fragments of different base pair lengths at specific retention times. Since a MIPC column affords highly reproducible separations, once calibrated, the base pair length of unknown dsDNA fragments can be determined by comparing their retention times to those obtained on a standard calibration chromatogram. The standard calibration chromatogram is obtained by chromatographing a standard dsDNA ladder containing fragments of known base pair length. In addition, a method is provided to determine the presence of nicks in dsDNA using MIPC under fully denaturing conditions, e.g., 80° C. In one embodiment, this method is applied to the detection of mutations in dsDNA.

What is claimed is:  
     
         1 . A method for calibrating a Matched Ion Polynucleotide Chromatography column comprising: 
 (a) applying to the column a sample containing a mixture of double stranded DNA fragments of known base pair length;    (b) eluting said fragments;    (c) detecting the eluted fragments; and    (d) identifying the solvent concentration in the mobile phase at which each DNA fragment in the mixture elutes whereby a relationship is derived between the organic solvent concentration in the mobile phase required for eluting DNA fragments of different base pair length from the column as a function of base pair length.    
     
     
         2 . A method of  claim 1 , wherein said mixture comprises a DNA ladder.  
     
     
         3 . A method of  claim 2 , wherein said mixture has been pre-treated with a ligase.  
     
     
         4 . A method of  claim 1 , wherein said mixture comprises a restriction enzyme digest.  
     
     
         5 . A method for determining the presence of a nick in a known fragment of double stranded DNA comprising: 
 (a) applying said fragment to a Matched Ion Polynucleotide Chromatography column;    (b) eluting said fragment under denaturing conditions;    (c) detecting the single stranded DNA species eluted in step (b); and    (d) quantifying the single stranded DNA species from step (c) wherein at least three single stranded DNA species are detected if said fragment has a nick.    
     
     
         6 . A method of  claim 5 , wherein said denaturing conditions comprise an elevated level, sufficient for completely denaturing said fragment, of at least one of temperature, urea concentration, dimethylformamide concentration, organic solvent concentration, counterion concentration, and pH.  
     
     
         7 . A method for determining the presence of nicked DNA in a sample of double stranded DNA fragments comprising: 
 (a) applying a first aliquot of said sample to a Matched Ion Polynucleotide Chromatography column;    (b) eluting the fragments in said first aliquot under non-denaturing conditions;    (c) detecting the DNA species eluted in step (b);    (d) determining the number of DNA species detected in step (c);    (e) applying a second aliquot of said sample to a Matched Ion Polynucleotide Chromatography column;    (f) eluting the fragments in said second aliquot under denaturing conditions;    (g) detecting the DNA species eluted in step (f);    (h) determining the number of DNA species detected in step (g); and    (i) comparing the number in step (d) to the number in step (h) to determine whether or not the number in step (h) exceeds twice the number in step (d) whereby the presence of nicked DNA is indicated if the number in step (h) exceeds twice the number in step (d).    
     
     
         8 . A method of  claim 7 , wherein said denaturing conditions comprise an elevated level, sufficient for completely denaturing the fragments in said sample, of at least one of temperature, urea concentration, dimethylformamide concentration, organic solvent concentration, counterion concentration, and pH.  
     
     
         9 . A method for analyzing a sample of double stranded DNA to determine the presence of a mutation therein comprising: 
 (a) contacting said sample with a mutation site binding reagent under conditions which allow said reagent to nick a strand of said double stranded DNA at or near the site of a mutation;    (b) applying a first aliquot of the product of step (a) to a Matched Ion Polynucleotide Chromatography column;    (c) eluting the fragments in said first aliquot under non-denaturing conditions;    (d) detecting the DNA species eluted in step (c);    (e) determining the number of DNA species detected in step (d);    (f) applying a second aliquot of the product of step (a) to a Matched Ion Polynucleotide Chromatography column;    (g) eluting the fragments in said second aliquot under denaturing conditions;    (h) detecting the DNA species eluted in step (g);    (i) determining the number of DNA species detected in step (h); and    (j) comparing the number in step (e) to the number in step (i) to determine whether or not the number in step (i) exceeds twice the number in step (e) whereby the presence of nicked DNA is indicated if the number in step (i) exceeds twice the number in step (e).    
     
     
         10 . A method of  claim 9 , wherein said sample of double stranded DNA is the product of a hybridization of a DNA sample suspected of containing a mutation with corresponding wild type DNA.  
     
     
         11 . A method of  claim 9  wherein said mutation site binding reagent is an enzyme.  
     
     
         12 . A method of  claim 9  wherein said enzyme is selected from the group consisting of S1 nuclease, mung bean endoucleose, CEL 1, mismatch repair enzymes, MutY protein, MutS protein, MutH protein, MutL protein, cleavase, exonuclease III, and HINF1.  
     
     
         13 . A method of  claim 9  wherein said mutation site binding reagent is a non-proteinaceous chemical reagent.  
     
     
         14 . A method of  claim 13  wherein said chemical reagent is an organometallic DNA intercalator.  
     
     
         15 . A method of  claim 14  wherein said intercalator contains rhodium or ruthenium.  
     
     
         16 . A method of  claim 15  wherein said intercalator is selected from the group consisting of bis(2,2′-bipyridyl)chrysenequinone diimine rhodium(III), bis(2,2′-bipyridyl)chrysenequinone diimine rhodium(III), (2,2′-bipyridyl)-bis(phenanthrenequinone) diimine rhodium(III), (bis(phenanthroline)dipyridophenazine ruthenium(II), and bis(phenanthroline)dipyridophenazine ruthenium(III).  
     
     
         17 . A chromatographic method for analyzing a sample of double stranded DNA to determine the presence of a mutation in said sample, the method comprising: 
 (a) separating a first aliquot of said sample using Matched Ion Polynucleotide Chromatography to produce a first chromatogram comprising peaks or other shapes which represent separated components of the sample;    (b) contacting another aliquot of said sample with a mutation site binding reagent under conditions which allow said reagent to nick a strand of DNA at or near the site of a base pair mismatch;    (c) separating the product of step (b) by the chromatographic method of step (a) to produce a second chromatogram; and    (d) comparing the chromatogram of step (c) to the chromatogram of step (a), wherein a change in the retention time or the number of peaks or other shapes in the chromatogram of step (c) indicates the presence of a mutation in said sample.    
     
     
         18 . A method of  claim 17  wherein the separation of step (a) is performed under non-denaturing conditions.  
     
     
         19 . A method of  claim 17  wherein the separation of step (a) is performed under denaturing conditions.  
     
     
         20 . A method of  claim 17 , wherein said sample of double stranded DNA is the product of a hybridization of a DNA sample suspected of containing a mutation with corresponding wild type DNA.  
     
     
         21 . A method of  claim 17 , wherein said mutation binding reagent is an enzyme.  
     
     
         22 . A method of  claim 21 , wherein said enzyme is selected from the group consisting of mismatch repair enzymes, S1 nuclease, mung bean endonuclease, CEL 1, MutY protein, MutS protein, MutH protein, MutL protein, cleavase, exonuclease III, and HINF1.  
     
     
         23 . A method of  claim 17  wherein said mutation site binding reagent is a non-proteinaceous chemical reagent.  
     
     
         24 . A method of  claim 23  wherein said chemical reagent is an organometallic DNA intercalator.  
     
     
         25 . A method of  claim 24  wherein said intercalator contains rhodium or ruthenium.  
     
     
         26 . A method of  claim 24  wherein said intercalator is selected from the group consisting of bis(2,2′-bipyridyl)chrysenequinone diimine rhodium(III), bis(2,2′-bipyridyl)chrysenequinone diimine rhodium(III), (2,2′-bipyridyl)-bis(phenanthrenequinone) diimine rhodium(III), (bis(phenanthroline)dipyridophenazine ruthenium(II), and bis(phenanthroline)dipyridophenazine ruthenium(III).