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

A method of differential DNA denaturation PCR (3DI-PCR), wherein the method consists essentially of:

• (a) providing a nucleic acid mixture comprising a parental DNA having up to about 500 bp and a GC allele of the parental DNA having up to about 500 bp;
• (b) carrying out PCR in the nucleic acid mixture in a reaction medium containing deoxyinosine (dITP), deoxy 2,6-diaminopurine triphosphate (dDTP), or dITP and dDTP, wherein Guanine bases (G) are converted into Inosine bases (I) and Adenosine bases (A) are converted into 2,6-diaminopurine bases (D);
• and
• (c) subjecting nucleic acids of (b) to PCR using primers having about 20-25 bp at a denaturation temperature 1-10° C. lower than the lowest denaturation temperature (Tp) that allows amplification of the parental nucleic acid, to thereby selectively amplify a region of about 20-80 bp of the GC allele of the parent DNA in the nucleic acid mixture.

A method of inverse differential DNA denaturation PCR (3DI-PCR), wherein the method consists essentially of:

• (a) providing a nucleic acid mixture comprising a parental DNA and a mutant DNA of the parental DNA containing 2-5 GC alleleic sites;
• (b) carrying out PCR in the nucleic acid mixture in a reaction medium containing deoxyinosine (dITP), deoxy 2,6-diaminopurine triphosphate (dDTP), or dITP and dDTP, wherein Guanine bases (G) are converted into Inosine bases (I) and Adenosine bases (A) are converted into 2,6-diaminopurine bases (D);
• and
• (c) subjecting nucleic acids of (b) to PCR using primers having about 20-25 bp at a denaturation temperature 1-10° C. lower than the lowest denaturation temperature (Tp) that allows amplification of the parental nucleic acid, to thereby produce a differentially amplified PCR fragment of 60-80 bp of the GC allele of the parent nucleic acid in the nucleic acid mixture.