Application Guide - QIAxcel Advanced

30 QIAxcel Advanced Application Guide 10/2016 QIAxcel® system — mapping mutant gene loci in Arabidopsis thaliana Nobuyoshi Mochizuki, Kyoto University, Graduate School of Science, Division of Biological Science, Kyoto, Japan In this application note, we describe the assessment of simple sequence length polymorphism (SSLP) and cleaved amplified polymorphisms (CAPS) markers in the mapping of mutant gene loci and the homo/hetero examination of known mutant gene loci using the mutated CAPS or derived CAPS (dCAPS) markers. Both applications utilize the QIAxcel system and the QIAxcel DNA High Resolution Kit, with the OM500 method. The QIAxcel system demonstrated superior separation capability, enabling the resolution of markers differing by only 4 base pairs in size. It is predicted that the QIAxcel system will be a powerful tool for increasing the speed of mapping and genotyping in the future. Introduction There are several methods used in the identification of gene loci responsible for the mutation in Arabidopsis thaliana. Of these, mapping of the F2 populations from crosses between different ecotypes has been a highly effective technique. However, as the entire genomic sequence became available, the increasing numbers of DNA markers made gene cloning much easier and faster using mapping and chromosomal walking. In addition, these DNA markers have often been used to detect polymorphisms of Arabidopsis thaliana. For polymorphism studies, we have routinely performed the following experiment types: • Assessment of SSLP and CAPS markers in the mapping of mutant gene loci • Homo/hetero examination of known mutant gene loci using the mutated CAPS or dCAPS markers • Homo/hetero examination of T-DNA insertion in T-DNA insertion mutations Design of PCR primers for the SSLP and dCAPS markers was extremely challenging, due to the sequence limitation, and the resulting PCR products were small (less than 200 bp). Consequently, the size differences among PCR products or restriction-enzyme–digested fragments are often just a few base pairs. Until recently, polyacrylamide gels or high concentration (2–4%) agarose gels were used to resolve such small size differences between DNA fragments. The introduction of the QIAxcel system has enabled the generation of rapid, reproducible results for these types of analysis.

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