PCR-assisted large insertion/deletion mutagenesis.
A mutagenesis protocol is presented that allows the exchange or simultaneous insertion/deletion (INDEL) of large fragments of DNA. The technique uses long single-stranded DNA molecules synthesized by asymmetric PCR. Insertion of large DNA fragments (> 300 bp) was accomplished using these single-stranded DNAs without the need for restriction sites or for subcloning. We have used the method to exchange large DNA fragments in the study of DNA regulatory regions and protein domain function.
Tessier
D C
DC
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec.
Thomas
D Y
DY
D002403:Cathepsins / Q000235:genetics; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D006801:Humans; D016254:Mutagenesis, Insertional / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D010206:Papain / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014169:Transformation, Bacterial
D017931:DNA Primers; D004277:DNA, Single-Stranded; D002403:Cathepsins; D010206:Papain; C028217:cathepsin S