Abstract:
The purpose of this study is to optimize PCR system to achieve efficient amplification for AT-rich DNA fragments.Four additives and four KOD-based high-fidelity DNA polymerases were studied for their amplification effects of four AT-rich fungal mitochondrial DNA fragments (AT contents: 72%~80%) with variable lengths of 1.0~5.4 kb. When applied alone, only magnesium ion facilitated the amplification, and the other three additives (bovine serum albumin(BSA), dimethyl sulfoxide(DMSO) and formamide) failed to produce the expected amplicon, which were contrary to their well-known facilitative effects on GC-rich DNA amplification. The presence of BSA or DMSO showed no impact on the amplification effect of magnesium ion, whereas the presence of formamide expressed inhibition effect on the facilitation function of magnesium ion. The four KOD-based polymerases (KOD-Plus-, KOD-Plus-Neo, KOD FX and KOD FX Neo) generated expected amplicons when magnesium ion was present in the PCR system, but KOD-Plus- and KOD-Plus-Neo failed to generate expected amplicon when magnesium ion was absent. This study verified that magnesium ion was critical for the successful amplification of AT-rich DNA fragments, and the optimal concentration was 1.75 mmol/L.The results provide a basis for the amplification of AT-rich DNA fragments in other eukaryotes.
