Polymerase Chain Reaction

2.0 Polymerase Chain Reaction (PCR)

2.1 Introduction: Plants produce secondary metabolites and polysaccharides interfere with genomic isolation procedures and it down-stream reactions (Amani, et al., 2011). Secondary metabolites are like alkaloids, flavanoids, phenolic compounds, polysaccharides, quinnes and terpenes. The polymerase chain reaction (PCR) analysis is mainly used to measure high quality DNA to make sure successful amplification with reproducible results. PCR is also a sensitive technique and not time consuming which enhance in DNA extraction step to isolate DNA from tissue sample for PCR analysis. The technique generally depends on a thermostable DNA-dependent DNA polymerase (Taq DNA polymerase). Thus, this enzyme was isolated from thermophilic bacteria which endure high temperature at 95ºC. This is a stable amplification process and it may denature DNA. PCR reaction involves 3 critical steps in the thermal cycle and it may produce billions of copies of genes. These 3 significant steps are denaturation, annealing and polymerization. Denaturation heated up to 95ºC for 30 seconds to denature the genomic DNA template into single strands. Then in annealing step, the sample is cooled down till 55ºC by allowing the primers to anneal to the complementary sequences in genomic DNA. After that, the sampled is raised again to 72ºC (optimal temperature) in the polymerization step (elongation step) for DNA synthese by Tag DNA-polymerase. This cycle is repeated up to 35 cycles with the same methods. Result of the PCR analysis is identified between the sequence in the 2 primers and it can be determined on the agarose gel.

2.2 MATERIALS AND METHODS: The DNA sample is extracted from transformed and untransformed explants in the previous week. The tissue samples were collected into a sterile microcentrifuge tubes were labelled with group name and the type of the tested sample. The tissue

samples were mixed with the PCR mixture of 7.5µl of 10X Polymerase...