Cationic copolymers that enhance wild-type-specific suppression in BNA-clamp PCR and preferentially increase the T m of fully matched complementary DNA and BNA strands

Biol Methods Protoc. 2022 Mar 30;7(1):bpac009. doi: 10.1093/biomethods/bpac009. eCollection 2022.


Mutation detection is of major interest in molecular diagnostics, especially in the field of oncology. However, detection can be challenging as mutant alleles often coexist with excess copies of wild-type alleles. Bridged nucleic acid (BNA)-clamp PCR circumvents this challenge by preferentially suppressing the amplification of wild-type alleles and enriching rare mutant alleles. In this study, we screened cationic copolymers containing nonionic and anionic repeat units for their ability to (i) increase the Tm of double-stranded DNA, (ii) avoid PCR inhibition, and (iii) enhance the suppression of wild-type amplification in BNA-clamp PCR to detect the KRAS G13D mutation. The selected copolymers that met these criteria consisted of four types of amines and anionic and/or nonionic units. In BNA-clamp PCR, these copolymers increased the threshold cycle (C t) of the wild-type allele only and enabled mutation detection from templates with a 0.01% mutant-to-wild-type ratio. Melting curve analysis with 11-mer DNA-DNA or BNA-DNA complementary strands showed that these copolymers preferentially increased the Tm of perfectly matched strands over strands containing 1-bp mismatches. These results suggested that these copolymers preferentially stabilize perfectly matched DNA and BNA strands and thereby enhance rare mutant detection in BNA-clamp PCR.

Keywords: DNA melting temperature; PCR clamping; bridged nucleic acid; cationic copolymers; mutant detection; rare allele enrichment.