Differential amplification of mutant nucleic acids by pcr in a mixture 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) using a pair of PCR primers; and   (c) optionally detecting the products of the PCR;   wherein the distance between the pair of primers in the PCR of step (b) is 3-30 bases;   wherein the mutant nucleic acid differs from the parental nucleic acid in 1 or 2 bases;   wherein the mutant nucleic acid comprises a greater number of adenine (A) and thymine (T) nucleotides than the parental nucleic acid; and   wherein the mutant nucleic acid is 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 mutant nucleic acid is a G/ C →A/ T  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 mutant nucleic acid 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/ C →A/ T  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/ C →A/ T  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/ C →A/ T  mutant nucleic acid of the parental nucleic acid, in which up to 60% of the G/ C  residues have been substituted by A/ T  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 or 2 G/ C →A/ T  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 a viral nucleic acid. 
     
     
         19 . The method as claimed in  claim 18 , wherein the parental nucleic acid is chosen from HIV, poliovirus, and measle virus nucleic acid. 
     
     
         20 . The method as claimed in  claim 19 , wherein the 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) using a pair of PCR primers; and   (c) optionally detecting the products of the PCR;   wherein the distance between the pair of primers in the PCR of step (b) is 3-30 bases;   wherein the mutant nucleic acid differs from the parental nucleic acid in 1 or 2 bases;   wherein the mutant nucleic acid comprises a greater number of guanine (G) and cytosine (C) nucleotides than the parental nucleic acid; and   wherein the mutant nucleic acid is 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 mutant nucleic acid is an A/ T →G/ C  mutant of the parental nucleic acid, which pairs with a fully complementary nucleic acid sequence. 
     
     
         23 . (canceled) 
     
     
         24 . A method for characterizing the origin of parental DNA or for detecting mutations characteristic of human gene disorders, wherein the method comprises enriching a mutant nucleic acid in a mixture of nucleic acids according to the method as claimed in  claim 1  and then further analyzing the products to determine the origin of parental DNA or to detect mutations characteristic of human gene disorders. 
     
     
         25 . A method for detecting a G→A mutant strain of HIV (G→A hypermutants) that is resistant to antiretroviral drug, wherein the method comprises enriching a mutant nucleic acid in a mixture of nucleic acids according to the method as claimed in  claim 1  and then further analyzing the products to detect a G→A mutant strain of HIV that is resistant to antiretroviral drug. 
     
     
         26 . A method for detecting neurovirulent vaccine-derived poliovirus isolates that cause vaccine-associated paralytic poliomyelitis, wherein the method comprises enriching a mutant nucleic acid in a mixture of nucleic acids according to the method as claimed in  claim 1  and then further analyzing the products to detect neurovirulent vaccine-derived poliovirus isolates that cause vaccine-associated paralytic poliomyelitis. 
     
     
         27 . The method as claimed in  claim 21 , wherein the parental nucleic acid is a viral nucleic acid. 
     
     
         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) using a pair of PCR primers; and   (c) optionally detecting the products of the PCR;   wherein the distance between the pair of primers in the PCR of step (b) is 3-30 bases;   wherein the mutant nucleic acid differs from the parental nucleic acid in 1 or 2 bases;   wherein the mutant nucleic acid comprises a greater number of adenine (A) and thymine (T) nucleotides than the parental nucleic acid; and   wherein the mutant nucleic acid is 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) using a pair of PCR primers; and   (c) optionally detecting the products of the PCR;   wherein the distance between the pair of primers in the PCR of step (b) is 3-30 bases;   wherein the mutant nucleic acid differs from the parental nucleic acid in 1 or 2 bases;   wherein the mutant nucleic acid comprises a greater number of guanine (G) and cytosine (C) nucleotides than the parental nucleic acid; and   wherein the mutant nucleic acid is 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.   
     
     
         30 . The method as claimed in  claim 27 , wherein the parental nucleic acid are chosen from HIV, poliovirus, and measle virus nucleic acids. 
     
     
         31 . A method for characterizing the origin of parental DNA or for detecting mutations characteristic of human gene disorders, wherein the method comprises enriching a mutant nucleic acid in a mixture of nucleic acids according to the method as claimed in  claim 21  and then further analyzing the products to determine the origin of parental DNA or to detect mutations characteristic of human gene disorders.