Detection of cryII gene from Bacillus thuringiensis using Polymerase Chain Reaction (PCR)

Abstract

Bacillus thuringiensis is one species of bacteria that has been applied as a microbiological control agent for pests and a vector of plant disease. The availability of Cry proteins in B. thuringiensis can be acted as a specific insect exterminator that only toxic to certain insects. The cryII gene is an example of a type of cry gene that encodes a CryII Protein. The CryII protein is toxic to Lepidoptera insects which can attack Helicoverpa armigera species which is a corn borer. Polymerase Chain Reaction (PCR) is a general method that can be used to amplify the gene. This research purposed to design a good primer candidate for cryII gene amplification from B. thuringiensis. In silico analysis for designing cryII primer was carried out using some software, such as BLAST for searching cryII gene sequence, Bioedit for sequences alignment, and DINAmelt for analyzing dimer structure of primers. Ten primer candidates were successfully obtained based on the result of the primer 3 software. A pair of primer was selected to amplify the cryII gene, with forward primer 5’-GGTAGTGGACCACAGCAGAC-3’and reverse primer 5’-TCTTCTGGCGCCAAATGGAT-3’. This primer has fulfilled good primer characteristics because it does not cause dimer structure and the resulting amplicons do not form secondary structures. Amplification of the cryI gene by PCR method using selected primer resulting in a PCR product with a length of approximately 800 bp.

Algorithm Model Determination of DNA Primer Design For The Success PCR Process

“…DNA, Deoxyribonucleic Acid is the blueprint of life for almost all other organisms [20] and contains the information cells need to synthesize proteins and replicate themselves. Polymerase Chain Reaction (PCR) is an enzymatic method to multiply exponentially a certain DNA nucleotide sequence In-Vitro [14,21]. The success in carrying out DNA Primer Design in Polymerase Chain Reaction (PCR) is strongly influenced by the characteristics of the primers used [1,12].…”