AdvanCETM FS96 Used for TILLING in Barley Allowed Cost Efficient and More Sensitive Mutant Detection

TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetic approach which involves a random mutagenesis and screening for the point mutations in the gene of interest as a part of functional gene analysis. Usually the first step is performed by the Ethyl methanesulfonate (EMS) mutagenesis treatment, whereas the second step needs a more sophisticated DNA-screening system.  The AdvanCE^TM FS96 system in combination with the AdvanCE^TM TILLING kit can be used as an attractive alternative compared to other more popular systems such as the LI-COR^® gel-based detection system. The cost- effectiveness of the first platform comes mainly from the usage of unlabeled primers. Moreover, the primer labels could be very often connected to the decreased performance of the PCR reaction in the case of LI-COR (Stein 2011, personal communication). The detection threshold in case of the AdvanCE^TM FS96 system is also a great advantage that allows scaling-down the volume of PCR and digestion reactions; besides, the digestion was much more efficient when contained less PCR product. The analysis was performed on the selected 2-dimensional 96 well plates containing pooled DNA from the barley cv. “Barke” TILLING population (Gottwald et al. 2009, BMC Res Notes 2:258). The above mentioned advantages of the AdvanCE^TM FS96 system allowed for the detection of new mutations that could not be detected with LI-COR screens before. The SNPs were confirmed by Sanger sequencing using ABI 3730 XL technology.  Pros and cons of both systems will be discussed.