Optimization of Polymerase Chain Reaction for Inter Simple Sequence Repeat Technique for Four Species of Plants

Abstract:

Polymerase chain reaction optimization for inter simple sequence repeat primers is a key factor to obtain accurate and reproducible results for gene mapping, studying the genetic structure of populations, plant passporting, phylogenetic analysis. Changing temperature conditions, the amount of amplification cycles and concentration of reaction mixture components is allowed to vary the number of bands obtained by this method. This article is result of preliminary research of method selection for molecular analysis. It is aimed to show how to adjust the profile of inter simple sequence repeat fragments by polymerase chain reaction for four model species Stipa lessingiana, Poa intricata, Equisetum fluviatile and Pteridium aquilinum. The working concentrations of magnesium chloride for primer ((СТС)₃GC) and ((АС)₈YG) were 2.5 mM for 0.63 units of Taq DNA polymerase and for primer ((СА)₆GG) it was 4.5 mM for 1.25 units. Sharply defined banding was observed from the minimal amount of DNA 5 ng per reaction, with primer concentration from 10 to 80 pmol and dNTPs concentration 0.2 mM. Optimal hybridization temperatures were 51.9 °C for primers ((АС)₈YG), ((СА)₆GG) and 50.0 °C for ((СТС)₃GC). The best imaging results were obtained when setting up electrophoresis in 1.9% agarose gel

• Periodical: Key Engineering Materials (Volume 683)
• Pages: 511-518
• DOI: https://doi.org/10.4028/www.scientific.net/KEM.683.511
• Online since: February 2016
• Authors: Polina Gudkova, Eugene Bayahmetov
• Keywords: Herbarium Material, ISSR, ISSR Primers, Molecular Markers, PCR, Reproducibility

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