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

Abstract

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.