Abstract
Most methods developed for detecting known single nucleotide polymorphisms (SNP) and deletion–insertion polymorphisms (DIP) are dependent on sequence conservation around the SNP/DIP and are therefore not suitable for application to heterogeneous organisms. Here we describe a novel, versatile and simple PCR-RFLP procedure baptised ‘derived Polymorphic Amplified Cleaved Sequence’ (dPACS) for genotyping individual samples. The notable advantage of the method is that it employs a pair of primers that cover the entire fragment to be amplified except for one or few diagnostic bases around the SNP/DIP being investigated. As such, it provides greater opportunities to introduce mismatches in one or both of the 35–55 bp primers for creating a restriction site that unambiguously differentiates wild from mutant sequences following PCR-RFLP and horizontal MetaPhorTM gel electrophoresis. Selection of effective restriction enzymes and primers is aided by the newly developed dPACS 1.0 software. The highly transferable dPACS procedure is exemplified here with the positive detection (in up to 24 grass and broadleaf species tested) of wild type proline106 of 5-enolpyruvylshikimate-3-phosphate synthase and its serine, threonine and alanine variants that confer resistance to glyphosate, and serine264 and isoleucine2041 which are key target-site determinants for weed sensitivities to some photosystem II and acetyl-CoA carboxylase inhibiting herbicides, respectively.
Highlights
Single nucleotide polymorphisms (SNPs) are the most common type of variation in the genome [1]
We propose a variant of the PCR-RFLP approach named the ‘derived Polymorphic Amplified Cleaved Sequence’ which provides greater possibilities for genotyping single nucleotide polymorphisms (SNP) and deletion–insertion polymorphisms (DIP) compared to the Cleaved Amplified Polymorphic Sequence (CAPS) and derived Cleaved Amplified Polymorphic Sequence (dCAPS) methodologies
A single nucleotide polymorphism was detected among the 48 individuals, namely the targeted adenine to guanine transition at the first base of codon 264, resulting in the serine to glycine mutation in the D1 protein of photosystem II
Summary
Single nucleotide polymorphisms (SNPs) are the most common type of variation in the genome [1]. There are as many as 10 million SNPs or around one in every 1000 nucleotides [2] When they occur in exons and cause a change in protein structure and function, they may have profound phenotypic effects on the organism [3]. Several such SNPs are documented including an adenine to thymine change in the β-globin gene, resulting in sickle-cell anaemia [4]. While not as frequent as SNPs, deletion–insertion polymorphisms (DIPs or indels) are widely spread in the genome [14]. Indels which are not a multiple of three nucleotides give rise to frameshift mutations, thereby coding for an entirely different set of amino acids or resulting in a premature stop codon [17]
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