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Nucleic Acids Research, Vol 25, Issue 6 1303-1304, Copyright © 1997 by Oxford University Press

ARTICLES

Construction of a deletion library using a mixture of 5'-truncated primers for inverse PCR (IPCR)

H Pues, B Holz and E Weinhold
Max-lnstitut fur Molekulare Physiologie, Abteilung Physikalische Biochemie, Rheinlanddamm 201, D-44139 Dortmund, Germany.

A quick in vitro mutagenesis method for the construction of nested deletion libraries was developed. Many deletions can be obtained in a single inverse PCR (IPCR) by replacing one of the two primers with a mixture of 5'-truncated oligodeoxynucleotides. Since chemical DNA synthesis proceeds from the 3'to the 5'end, such a mixture of 5'- truncated oligodeoxynucleotides can easily be obtained in a single automated DNA synthesis under reduced coupling efficiency. This deletion mutagenesis method yields many different deletions in a defined short DNA segment and is, therefore, best suited for a deletion analysis at base pair level. Applications might include functional analysis of regulatory DNA segments and protein engineering work that requires libraries for the expression of N-terminal, C-terminal or internal truncated proteins as well as fusion proteins having different splice sites.
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