Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 31 Materials and methods Assessment of Col and Ler using the SSLP marker NGA707 The sizes of SSLP marker NGA707 for two ecotypes of Col and Ler are 132 bp and 128 bp, respectively, meaning a difference in size of only 4 bp. Genomic DNA samples from Col, Ler, and a hybrid of Col and Ler cross were extracted using a miniprep system and PCR was performed on a 10 µl scale. PCR products were analyzed using the QIAxcel system together with the QIAxcel DNA High Resolution Kit and the OM500 method. Hetero/homozygote examination of mutants using dCAPS markers When base sequence polymorphisms, derived from the mutations of substitution, insertion, or deletion do not generate or modify a restriction enzyme recognition sequence, the PCR primers can be designed to introduce a new restriction enzyme recognition sequence to enable the easy assessment of polymorphisms (dCAPS method). The gun5-1 mutation is shown as an example (Figure 1 shows the sequence) and has a base substitution (from G to A). PCR products amplified using specially designed, mismatching primers will include the EcoT14 I recognition site in the wild type, but not in the gun5-1 mutant (Figure 1). Results and Discussion Assessment of Col and Ler using the SSLP marker NGA707 Figure 2 shows the analysis of the PCR products using the QIAxcel system in conjunction with the QIAxcel DNA High Resolution Kit. In the hybrid (H) sample, the two DNA bands were easily distinguished and the identification of hybrid was clear with no ambiguity. Presence of additional 2 heteroduplex bands confirmed heterozygosity. Hetero/homozygote examination of mutants using dCAPS markers The predicted sizes of EcoT14 I digested fragments from wild-type and mutant are shown in Figure 3. The EcoT14 I digested samples were analyzed on the QIAxcel system after a 5- to 10-fold dilution with water (Figure 4). The gel view of the QIAxcel BioCalculator Software enables easy assessment of wild-type, heterozygote, and mutant — with no ambiguity. Figure 1. Sequence of the gun5-1 mutant. WT – – – – – –GGATC TAAGGCAT– – – – gun5–1 – – – – – –GGATC TAAG ACAT– – – – Primer2 – – – – – –GGATCCAAG WT product – – – – – –GGATCCAAGGCAT– – – – gun5–1 product – – – – – –GGATCCAAG A CAT– – – – EcoT14 site

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