Nucleic Acids Research, 1991, Vol. 19, No. 11 3055-3060
© 1991
MOLECULAR BIOLOGY |
Targeted gene walking polymerase chain reaction
Department of Pathology, University of Washington Seattle, WA 98195, USA
*To whom correspondence should be addressed
Received January 28, 1991. Revised April 26, 1991. Accepted April 26, 1991.
We describe a modification of a polymerase chain reaction method called ‘targeted gene walking’ that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific ‘walking’ primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.
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