Isolation of Adaptive Genes from Shorea curtisii Dyer ex King via PCR

Ang, Swee Kim (2005) Isolation of Adaptive Genes from Shorea curtisii Dyer ex King via PCR. [Final Year Project Report] (Unpublished)

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Abstract

Two pairs of primers for heat shock protein 90 (HSP90) and abscisic acid induced guard cell protein (ABGI) which are adaptive genes responsible for heat stress and drought stress, respectively were designed and synthesized based on the sequence of HSP90 in Catharanthus roseus and ABGI in Vicia faba. These primers were used in amplification of HSP90 and ABGI from the genomic DNA of Shorea curtisii via polymerase chain reaction (PCR). Optimization of PCR produced single distinct DNA fragment with approximately 750bp HSP90 and 830bp ABGI. Verification of PCR inserts from recombinant plasmids was conducted via restriction digestion method using EcoR1 and PCR method using the same primer sets, and also T7/SP6 Promoter primers for further confirmation purpose. Restriction digestion successfully detected HSP90 PCR insert but failed to detect ABGI PCR insert with desired size. PCR fragments with size about 750bp and 830bp, which matched the size of HSP90 PCR insert and ABGI PCR insert respectively, were re-amplified from respective recombinant plasmids using the same primer sets. By using T7/SP6 Promoter Primers in HSP90 PCR insert and ABGI PCR insert verification, the amplified DNA fragment was about 920bp and 1000bp respectively. The recombinant plasmids were sent for DNA sequencing of PCR products in HSP90 and ABGI amplification. The DNA sequencing was in progress and sequence analysis was not able to be performed.

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