Optimization of PCR amplification of Arabidopsis microsatellite loci

SPAMPINATO, C.; CHIRINOS ARIAS, M.C.

Rosario

Simposio; Genómica Funcional de Plantas; 2017

Microsatellites or simple sequence repeats are tandem repeat motifs of 1 to 6 bp. These short repetitive elements are found in all genomes analyzed to date in both coding and noncoding regions. They are highly polymorphic, abundant, codominantly inherited and distributed throughout the genome. Microsatellite instability (MSI) is the molecular fingerprint of defective mismatch repair (MMR) system. MMR system improves the fidelity of DNA replication by correcting single base-base mismatches and unpaired nucleotides that arise through replication errors. The pathway involves mismatch recognition by MutS proteins (MSH2, MSH6 and MSH7) followed by recruitment of MutL proteins (MLH1 and PMS1). Several endogenous microsatellite loci were previously scored to demonstrate MSI in Arabidopsis plants deficient in MSH2 (1). Consequently, PCR-based MSI analysis is a useful screening tool for evaluating MMR activity. Here, four dinucleotide microsatellite markers with different sequence and repeat length distributed on different chromosomes were studied.Arabidopsis genomic DNA was PCR amplified with all primer sets (Table 1). PCR products were electrophoretically resolved on 6% denaturing polyacrylamide-urea gels and detected by silver staining. Artifactual bands are frequently observed in denaturing gel electrophoresis probably due to secondary DNA structures (2,3). In order to eliminate extra non-specific bands, PCR optimization was performed. Single specific PCR products were obtained using touchdown PCR, combining annealing and extension into one step and reducing PCR cycle numbers. This modified protocol will be used to reveal MSI in wild-type and MMR-deficient parental lines and their progenies after genotoxic stress.