Book of Abstracts: Albany 2003
June 17-21 2003
Unexpected Digestion of DNA in One-Base Mismatch PNA/DNA Duplex by Nuclease S1 and Its Application to SNPs Detection
PNA and single-strand-specific nuclease were used here for SNPs detection. After treatment by single-strand-specific nuclease such as S1 and mung bean, the DNA strand in PNA/DNA duplex, which involves a one-base mismatch, was completely digested. However, for full-matched PNA/DNA duplex, PNA protected the DNA strand quite efficiently so that most of the DNA strand remained undigested. Thus, if the PNA is completely complementary to DNA, the PNA/DNA duplex remains after the treatment by single-strand-specific nuclease; if the PNA is non-complementary to DNA (even one-mismatch), the DNA is digested to mono-nucleotides, and no PNA/DNA duplex exists. This difference was also visualized by adding 3,3'-diethylthiadicarbocyanine, which changes its color from blue to purple upon binding to DNA/PNA duplexes. These findings were successfully applied to the detection of single nucleotide polymorphisms (SNPs). When the target site in the sample DNA is completely complementary with the PNA probe, a notable amount of DNA/PNA duplex remains and thus the solution exhibits purple color. In the presence of even one mismatch between PNA and DNA, however, the DNA is completely digested and the dye shows its intrinsic blue color. Furthermore, the SNPs in the apolipoprotein E gene of human DNA have been successfully genotyped using this technique. The mechanism for the digestion of one-base mismatch PNA/DNA duplex was also discussed, and two possible processes were proposed. This method could become a fast, inexpensive, and facile genetic screening detection by visual inspection, with no need for expensive and complicated equipment.
Research Center for Advanced Science and Technology