期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 271, 期 3, 页码 726-730出版社
ACADEMIC PRESS INC
DOI: 10.1006/bbrc.2000.2716
关键词
thermostable DNA polymerase; melanin; reverse transcriptase; polymerase chain reaction; reverse transcription; enzyme inhibition
We found that both RNA and cDNA preparations derived from melanocytes contain a RT-PCR inhibitor that copurified with nucleic acids. Investigation of the candidate inhibitor melanin revealed that it potently blocks PCR at concentrations below 200 ng/ml, whereas 100 mu g/ml melanin was required to inhibit reverse transcription. Melanin and thermostable DNA polymerase preferentially formed a distinct complex with reduced migration velocity as compared to pure polymerase in nondenaturating polyacrylamide gel electrophoresis. The inhibition of the enzyme by melanin could be reversed by diluting solutions of preformed complexes or by adding excess amounts of other proteins such as bovine serum albumin or dry milk. Our findings demonstrate that melanin is a potent inhibitor of thermostable DNA polymerase in vitro and that the inhibitory effect is conferred by a direct and reversible polymerase-melanin interaction. (C) 2000 Academic Press.
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