(Published in Journal of Institute of Science and Technology, Vol. 15, 2007-08, pp. 90-102.)
Random Amplified Polymorphic DNA-polymerase chain reaction analysis of Bacillus thuringiensis isolates from the Khumbu region of Everest Base Camp
G. S. Sahukhall, U. T. Shrestha1, B. Lekhak2, A. Singh2, V. P. Agrawal1
1. Research Laboratory for Biotechnology and Biochemistry (RLABB);
Maitidevi Plaza, Maitidevi, Kathmandu, Nepal
2. Central Department of Microbiology, Tribhuvan University;
Kirtipur, Kathmandu, Nepal.
G. S. Sahukhall, U. T. Shrestha1, B. Lekhak2, A. Singh2, V. P. Agrawal1
1. Research Laboratory for Biotechnology and Biochemistry (RLABB);
Maitidevi Plaza, Maitidevi, Kathmandu, Nepal
2. Central Department of Microbiology, Tribhuvan University;
Kirtipur, Kathmandu, Nepal.
ABSTRACT
Genetic similarity and diversity among the Bacillus thuringiensis isolates were examined using Random Amplified Polymorphic DNA-polymerase chain reaction (RAPD-PCR). Relationship between the species was determined by comparing their unique RAPD fingerprint information. A total of 86 endotoxin producing B. thuringiensis strains isolated from soil samples of Khumbu region were tested against a series of 100 decamer RAPD primers (codes 201-300). Nine primers: 208, 254, 256, 268, 275, 276, 284, 292 and 299 with GC%: 50-80, were found to amplify genomic DNA fragments with reproducible polymorphisms. Primer 284 with GC% 70 that produced more polymorphic bands was used to obtain RAPD profiles. The reaction mixture was optimized in 25ul system with PCR buffer containing 10mM Tris HCl, 50 mM KCl, 3 mM MgCl2 with pH 8.3.; 200μm dNTPs each, 1U Taq polymerase, 40 pmol decamer primers, 20 ng template DNA and 1% DMSO. The thermal programme was set as: initial denaturation temperature at 94oC for 5 mins followed by 35 cycles with denaturation at 94oC for 1 min, annealing at 36oC for 1 min and extension at 72oC for 2 mins with final extension temperature at 72oC for 10 mins. Higher polymorphic fragments were produced in the range between 700-900 base pairs. The range of 400-700 and 1200-1600 bp were also highly polymorphic. The Discriminatory capacity (D) of the RAPD-PCR was 0.9901 and hence the cold tolerant B. thuringiensis isolates from high altitude regions were very rich in genomic polymorphism.
Genetic similarity and diversity among the Bacillus thuringiensis isolates were examined using Random Amplified Polymorphic DNA-polymerase chain reaction (RAPD-PCR). Relationship between the species was determined by comparing their unique RAPD fingerprint information. A total of 86 endotoxin producing B. thuringiensis strains isolated from soil samples of Khumbu region were tested against a series of 100 decamer RAPD primers (codes 201-300). Nine primers: 208, 254, 256, 268, 275, 276, 284, 292 and 299 with GC%: 50-80, were found to amplify genomic DNA fragments with reproducible polymorphisms. Primer 284 with GC% 70 that produced more polymorphic bands was used to obtain RAPD profiles. The reaction mixture was optimized in 25ul system with PCR buffer containing 10mM Tris HCl, 50 mM KCl, 3 mM MgCl2 with pH 8.3.; 200μm dNTPs each, 1U Taq polymerase, 40 pmol decamer primers, 20 ng template DNA and 1% DMSO. The thermal programme was set as: initial denaturation temperature at 94oC for 5 mins followed by 35 cycles with denaturation at 94oC for 1 min, annealing at 36oC for 1 min and extension at 72oC for 2 mins with final extension temperature at 72oC for 10 mins. Higher polymorphic fragments were produced in the range between 700-900 base pairs. The range of 400-700 and 1200-1600 bp were also highly polymorphic. The Discriminatory capacity (D) of the RAPD-PCR was 0.9901 and hence the cold tolerant B. thuringiensis isolates from high altitude regions were very rich in genomic polymorphism.
1 comments:
Congrat........
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