Derived Polymorphic Amplified Cleaved Sequence (dPACS) Assay.

Abstract

The derived polymorphic amplified cleaved sequence (dPACS) assay is a simple polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP)-based procedure for detecting known single-nucleotide polymorphisms (SNPs) and deletion-insertion polymorphisms (DIPs). It is relatively straightforward to carry out using basic and commonly available molecular biology kits. The method differs from other PCR-RFLP assays in that it employs 35-55bp primer pairs that encompass the entire targeted DNA region except for a few diagnostic nucleotides being examined. In so doing, it allows for the introduction of nucleotide mismatches in one or both primers for differentiating wild from mutant sequences following polymerase chain reaction, restriction digestion and MetaPhor gel electrophoresis. Primer design and the selection of discriminating enzymes are achieved with the help of the dPACS 1.0 program. The method is exemplified here with the positive detection of serine 264-psbA, a key determinant for the effective binding of some photosystem II inhibitors to their target. A serine-to-glycine mutation at codon 264 of psbA causes resistance to serine-binding photosystem II herbicides in several grasses and broad-leaf weeds, including Amaranthus retroflexus, which is employed in this study.