High-Throughput DNA Extraction and Optimization of PCR Efficiency for Barley SSRs Genotyping

Abstract

Preparation of large quantity and high-quality DNA is a major bottleneck for most genetic studies. Although a number of high-throughput methods are available, but degradation of genomic DNA is still one of the major constraints in many plant species. The present study is designed to evaluate the low-cost, high-throughput genomic DNA extraction method that yields high-quality DNA, which has consistency for SSRs amplification. Five different DNA isolation procedures from three different tissue sources were employed for efficient and easy recovery of high-purity DNA. It was evident that procedures 2 and 3 was more suitable to yield amplifiable DNA from any of the three tissue sources. Analysis of variance revealed that DNA extraction procedures have significant impact on DNA yield and DNA quality while tissue source was not found to impose any significant impact on both DNA yield and DNA quality. Effect of DNA quality on the efficiency of Taq polymerase and SSRs amplification revealed that procedures 2 and 3 was more suitable to yield amplifiable DNA from any of the three tissue sources. It is concluded that during PCR analysis, excessive magnesium amount results in accumulation of nonspecific PCR products, whereas insufficient magnesium results in reduced yield of the desired PCR product. However, template concentration had negligible effects on amplification while annealing temperatures had significant effects on PCR amplification as stringent annealing temperature, especially during the first several cycles, is necessary for proper amplification.