DNA Isolation and Optimization of ISSR-PCR Reaction System in Oryza sativa L.

Abstract

Inter simple sequence repeats (ISSRs) have been utilized widely for molecular markers in analyzing the genetic diversity and phylogenetic and regions in the genome flanked by microsatellite sequences. PCR amplification of these regions using a single primer yields multiple amplification products that can be used as a dominant multilocus marker system for the study of genetic variation in various organisms. For this study provides, DNA isolation, adjusting in six factors (Buffer, MgCl2, dNTPs, ISSR primers, Template DNA and Taq polymerase ) at six levels, and optimization of PCR temperature for the ISSR reaction was 60-45 °C, primers screening on indica rice ( Oryza sativa ). In this research, simple method of DNA isolation by using seedling. The objective of the present investigation was to assess the optimizations and quantification. Has been shown that stalk enhanced the maximum value of genomic. The results show that 100 ISSR primers were examined as well as, 56 ISSR primers was productively amplified. Optimum components for PCR reactions were 5.0 μl of 5X PCR Buffer, 1.5 μl of 25mM MgCl2, 1 μl of 10 mM dNTP, 1 μl of 10 Μm ISSR primers, 2 μl Template DNA, and 0.1 μl of 5 units/ml Taq polymerase. Based on this study, has brought out some information on the relationship between these ISSR primers will be applied further for molecular profiling as well as response evaluation in rice varieties