Differential amplification of mutant nucleic acids by PCR in a mixure of nucleic acids

Abstract

A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: (a) providing a nucleic acid mixture comprising a parental nucleic acid and a mutant nucleic acid of the parental nucleic acid; and (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR); wherein the mutant nucleic acid is a G→A mutant of the parental nucleic acid, which pairs with a fully complementary nucleic acid sequence to form an AT-rich nucleic acid variant of the parental nucleic acid; and wherein the AT-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T p ) that allows amplification of the parental nucleic acid to thereby enrich the mutant nucleic acid in the nucleic acid mixture.

1 . A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: 
 (a) providing a nucleic acid mixture comprising a parental nucleic acid and a mutant nucleic acid of the parental nucleic acid;    (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR); and    (c) optionally detecting the products of the PCR;    wherein the mutant nucleic acid is an AT-rich nucleic acid variant of the parental nucleic acid; and    wherein the AT-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T p ) that allows amplification of the parental nucleic acid, to thereby enrich the mutant nucleic acid in the nucleic acid mixture.    
     
     
         2 . The method as claimed in  claim 1 , wherein the AT-rich nucleic acid variant is a G→A mutant of the parental nucleic acid, which pairs with a fully complementary nucleic acid sequence.  
     
     
         3 . The method as claimed in  claim 1 , wherein the AT-rich nucleic acid variant is a small deletion mutant of the parental nucleic acid.  
     
     
         4 . The method as claimed in  claim 3 , wherein the deletion comprises 1 or 2 bp.  
     
     
         5 . The method as claimed in  claim 1 , wherein the nucleic acid mixture contains the parental nucleic acid and one G→A mutant of the parental nucleic acid, which is selectively amplified compared to amplification of the parental nucleic acid.  
     
     
         6 . The method as claimed in  claim 1 , wherein the nucleic acid mixture contains the parental nucleic acid and more than one G→A mutant nucleic acid of the parental nucleic acid, wherein each mutant nucleic acid is selectively amplified compared to amplification of the parental nucleic acid.  
     
     
         7 . The method as claimed in  claim 1 , wherein the nucleic acid mixture contains the parental nucleic acid and one G→A mutant nucleic acid of the parental nucleic acid, in which up to 60% of the G residues have been substituted by A in the parental nucleic acid, and wherein mutant nucleic acids in the mixture are selectively amplified compared to amplification of the parental nucleic acid.  
     
     
         8 . The method as claimed in  claim 1 , wherein products of the PCR are detected by gel electrophoresis in agarose or acrylamide gel, capillary electrophoresis, or chromatography.  
     
     
         9 . The method as claimed in  claim 1 , wherein the products of the PCR are detected by gel filtration or ion-exchange chromatography.  
     
     
         10 . The method as claimed in  claim 6 , wherein products of the PCR are identified by relative location in the gel.  
     
     
         11 . The method as claimed in  claim 1 , wherein the mutant nucleic acid is a hypermutated variant of the parental nucleic acid in the nucleic acid mixture and the denaturing temperature is about 1° C. lower than T p .  
     
     
         12 . The method as claimed in  claim 2 , wherein the mutant nucleic acid contains 1 to 18 G→A mutations compared to the parental nucleic acid.  
     
     
         13 . The method as claimed in  claim 1 , wherein step b) of amplification of nucleic acids by PCR is carried out with modified bases.  
     
     
         14 . The method as claimed in  claim 13 , wherein the modified bases are dUTP, 4-methyl dCTP, 5-bromo dCTP, or 5-iodo dCTP, or mixtures thereof.  
     
     
         15 . The method as claimed in  claim 1 , wherein step b) of amplification of nucleic acids by PCR is carried out with non-standard PCR buffer comprising tetraethyl-ammonium chloride, methanol, or polyethylene glycol.  
     
     
         16 . The method as claimed in  claim 1 , wherein the parental nucleic acid comprises 40 to 500 bases.  
     
     
         17 . The method as claimed in  claim 1 , wherein the parental nucleic acid comprises 40 to 80 bases.  
     
     
         18 . The method as claimed in  claim 1 , wherein the parental nucleic acid is comprised of HIV-1 or HIV-2 nucleic acids.  
     
     
         19 . The method as claimed in  claim 1 , wherein the parental nucleic acid is a viral nucleic acid (HIV, poliovirus, measle virus).  
     
     
         20 . The method as claimed in  claim 1 , wherein parental nucleic acid is a poliovirus nucleic acid.  
     
     
         21 . A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: 
 (a) providing a nucleic acid mixture comprising a parental nucleic acid and a mutant nucleic acid of the parental nucleic acid;    (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR); and    (c) optionally detecting the products of the PCR;    wherein the mutant nucleic acid is a GC-rich nucleic acid variant of the parental nucleic acid; and    wherein the GC-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T p ) that allows amplification of the parental nucleic acid, to thereby enrich the mutant nucleic acid in the nucleic acid mixture;    and wherein PCR is carried out in a reaction medium containing deoxyinosine triphosphate (dITP), or in a reaction medium containing deoxy 2,6-diaminopurine triphosphate (dDTP), or in a reaction medium containing dITP and dDTP.    
     
     
         22 . The method as claimed in  claim 21 , wherein the GC-rich nucleic acid variant is a A→G mutant of the parental nucleic acid, which pairs with a fully complementary nucleic acid sequence.  
     
     
         23 . The method as claimed in  claim 15 , wherein the GC-rich nucleic acid variant is a T→C mutant of the parental nucleic acid, which pairs with a fully complementary nucleic acid sequence.  
     
     
         24 . Use of the method as claimed in  claim 1  for characterizing the origin of parental DNA or for detecting mutations characteristic of human gene disorders.  
     
     
         25 . Use of the method as claimed in  claim 5  for detecting a G ->A mutant strain of HIV (G ->A hypermutants) that is resistant to antiretroviral drug.  
     
     
         26 . Use of the method as claimed in  claim 10  for detecting neurovirulent vaccine-derived poliovirus isolates that cause vaccine-associated paralytic poliomyelitis.  
     
     
         27 . The method as claimed in  claim 15 , wherein the parental and mutant nucleic acids are from measle virus.  
     
     
         28 . A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: 
 (a) providing a nucleic acid mixture comprising a mutant nucleic acid of a parental nucleic acid;    (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR); and    (c) optionally detecting the products of the PCR;    wherein the mutant nucleic acid is an AT-rich nucleic acid variant of the parental nucleic acid; and    wherein the AT-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T p ) that allows amplification of the parental nucleic acid, to thereby enrich the mutant nucleic acid in the nucleic acid mixture.    
     
     
         29 . A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: 
 (a) providing a nucleic acid mixture comprising a mutant nucleic acid of a parental nucleic acid;    (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR); and    (c) optionally detecting the products of the PCR;    wherein the mutant nucleic acid is a GC-rich nucleic acid variant of the parental nucleic acid; and    wherein the GC-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (T p ) that allows amplification of the parental nucleic acid, to thereby enrich the mutant nucleic acid in the nucleic acid mixture; and    wherein PCR is carried out in a reaction medium containing deoxyinosine triphosphate (dITP), or in a reaction medium containing deoxy 2,6-diaminopurine triphosphate (dDTP), or in a reaction medium containing dITP and dDTP.