Sensitive Detection of DNA Lesions by Bulge-Enhanced Highly Specific Coamplification at Lower Denaturation Temperature Polymerase Chain Reaction.

Abstract

Mutagenic modifications of nucleotides or DNA lesions that result from environmental stress have proven to be associated with a variety of diseases, particularly cancer. The method for accurately detecting the lesions is therefore of great importance for biomedical research and toxicity study. We develop a sensitive and low-cost bulge-enhanced coamplification at lower denaturation temperature polymerase chain reaction (COLD-PCR) method for detecting DNA lesions (uracil and 8-oxoguanine) by combining an in vitro base excision repair (BER) pathway and COLD-PCR. The modified bases are converted to bulge via the BER pathway involving converting modified bases to an apurinic/apyrimidinic (AP) site, cleavage at the AP site, and break ligation. The presence of the bulge induces a large change of the hybridization thermodynamics of double-stranded DNA, eventually enhancing the specificity of COLD-PCR. Besides, we used the free energy of hybridization as a reference to optimize the critical denaturation temperature (Tc) of COLD-PCR obtaining more specific amplification than empirical Tc. Taking advantage of the proposed bulge-enhanced COLD-PCR, we are able to identify the presence of DNA lesion-containing strands at low abundance down to 0.01%. This method also exhibits high sensitivity for glycosylase with a detection limit of 10-4 U/mL [3 S/N (signal-to-noise ratio)] that is superior than some recently reported methods. With the design of the repair guide probe, the level of oxidative damage in genomic DNA caused by chemicals and photodynamic therapy (PDT) can be evaluated, heralding more applications in clinical diagnosis and epigenetic study.