Construction of a deletion library using a mixture of 5'-truncated primers for inverse PCR (IPCR).
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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Max-lnstitut für Molekulare Physiologie, Abteilung Physikalische Biochemie, Rheinlanddamm 201, D-44139 Dortmund, Germany.
Holz
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Weinhold
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D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis / Q000235:genetics; D015723:Gene Library; D014644:Genetic Variation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D013824:Thermus / Q000201:enzymology / Q000235:genetics
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases