Cloning of Thermostable DNA Polymerase Gene from a Thermophilic Brevibacillus sp. Isolated from Sikidang Crater, Dieng Plateu, Central Java  

Oleh:

Witasari, Lucia Dhiantika ; Prijambada, Irfan D. ; Widada, Jaka ; Wibawa, D. Andang Arif

Jenis: Article from Journal - ilmiah nasional - terakreditasi DIKTI
Dalam koleksi: Indonesian Journal of Biotechnology vol. 15 no. 2 (2010), page 72-78.
Topik: Thermostable DNA Pol I; Brevibacillus sp.; PCR; Cloning
Fulltext: 13-44-1-PB_thom.pdf (193.93KB)

Isi artikel

Thermostable DNA polymerase has an important role for amplifying small amount of DNA through polymerase chain reaction (PCR). Thermophillic bacteria Brevibacillus sp. was isolated from Sikidang Crater, Dieng Plateu, Central Java. Previous study showed that crude protein of the isolate could be used in PCR. Unfortunately, like most native thermos-table enzymes, the thermos-table DNA polymerase of the isolate is synthesized in a very low level and therefore is cumbersome to purify. The purpose of this research is to clone thermos-table DNA polymerase gene of the isolate. The DNA polymerase gene was amplified by means of PCR using specific primers. The amplified fragment was then isolated, purified, and ligated into the pGEM-T cloning vector. The recombinant plasmid was then transformed to competent E. coli JM109 cells using heat shock method. The cloned thermos-table DNA polymerase gene from the thermophilic isolate was then characterized for its nucleotide base sequence. The result showed that the DNA Pol I gene was successfully be amplified from the isolate DNA genom, resulting in ± 2,7 kb DNA fragment in length. Sequence analysis of segment of targeted gene showed high similarity to that of thermos-table DNA polymerase genes from other Bacillus.

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