Nucleic Acids Research, 1988, Vol. 16, No. 15 7351-7367
© 1988
Articles |
A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions
Department of Human Genetics, Cetus Corporation, Emeryville, CA 94608, USA 1Department of Biochemistry, U.C. Berkeley Berkeley, CA 94720, USA
Received May 19, 1988. Accepted July 8, 1988.
Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specffic interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.
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