Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> ISP5230 |
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Authors: | M Piraee L C Vining |
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Institution: | (1) Biology Department, Dalhousie University, Halifax, NS, Canada B3H 4J1, CA |
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Abstract: | Consensus amino acid sequences of FADH2-dependent bacterial halogenases were used to design PCR primers amplifying a halogenase gene fragment from the chloramphenicol
producer Streptomyces venezuelae ISP5230. The sequence-specific degenerate primers (MPF1 and MPR2) were used with a touchdown PCR procedure in the first PCR-assisted
cloning of a halogenase gene fragment. In the region of the 290-bp PCR product containing the reverse primer, the deduced
amino acid sequence exhibited characteristics of a β–α–β fold present in FAD-binding sites of certain monooxygenases. When
used to probe Southern blots of restriction-enzyme-digested DNA, the α-32P]dCTP-labeled PCR product hybridized specifically with DNA fragments from genomic DNA of S. venezuelae ISP5230. Primers MPF1 and MPR2 also allowed amplification by PCR of approximately 290-bp DNA fragments from several other
streptomycetes. The fragments from Streptomyces aureofaciens NRRL2209 and Streptomyces coelicolor A3(2) showed sequence identity with halogenase genes from these species. Thus, the PCR primers are of potential value for
amplification and subsequent isolation of actinomycete halogenase genes. Journal of Industrial Microbiology & Biotechnology (2002) 29, 1–5 doi:10.1038/sj.jim.7000263
Received 25 June 2001/ Accepted in revised form 02 April 2002 |
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Keywords: | : PCR FAD-binding site gene cloning halometabolite |
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