High Resolution Melt Analysis as an SNP Genotyping Tool in Arabidopsis thaliana

Abstract

Single nucleotide polymorphisms (SNPs) are a common type of mutation in most species. An insertion/deletion mutation (in/del) is a type of SNP which denotes the addition or removal of nucleotides, and are genotyped using the same methodology as SNPs. SNPs are typically genotyped by using polymerase chain reaction (PCR) to amplify a specific fragment of the genome and use various sequencing methods to detect the SNPs present. High resolution melt analysis (HRM) is a technique used to precisely measure the annealing temperatures of the product DNA during quantitative PCR. Here, we report HRM analysis to screen for tt4-4, an in/del mutation of the TRANSPARENT TESTA (TT4, At5g 13930) gene in A.thaliana, in which there exists an extra C:G base pair. With HRM we can accurately determine the zygosity of each plant based on the slight differences in annealing temperatures between the amplicons produced by the wildtype and mutant alleles. HRM provides us with an inexpensive, high-throughput method of genotyping SNPs, which is used in a number of applications, including fast medical diagnostics, zygosity testing, and screening for DNA methylation patterns (Wojdacz & Dobrovic 2007, NAR 35(6):e41).

* Indicates faculty mentor.