Nucleic Acids Research, 1989, Vol. 17, No. 11 4015-4023
© 1989
MOLECULAR BIOLOGY |
A method for introducing random single point deletions in specific DNA target sequences using oligonucleotides
Department of Biochemistry and the Biotechnology Laboratory, University of British Columbia Vancouver, BC V6T 1W5, Canada
*To whom correspondence should be addressed
Received April 26, 1989. Accepted May 10, 1989.
We describe a method for the generation of random point deletions in any target DNA sequer using synthetic mixed oligonucleotides. A mixed pool of oligonucleotides, which contain sinj nucleotide deletions randomly distributed throughout the full length, was generated by a modificati of the synthesis cycle of an automated DNA synthesiser that allowed the inefficient incorporati of nucleotide monomers during each cycle of synthesis. A family of oligonucleotides was used prime in vitro synthesis of the complementary strand of a cloned DNA fragment in an M13 vec which had previously been passaged through a dut–, ung– Escherichia coli host. Strong selecti for progeny from the newly synthesised strand is provided by transforming the heteroduplex in a dut+, ung+ host. This procedure introduced point deletions at 10–25% efficiency. It has be used to introduce point deletions into operator sequences which bind the yeast regulatory prote encoded by MATal and MAT
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+Present address: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK