An efficient method for introducing block mutations into specific regions of a gene.
The introduction by Kunkel of a method which uses uracil-containing single-stranded DNA as a template for site-directed mutagenesis has greatly simplified the procedure for introducing point mutations into DNA in a phagemid vector. We have extended the use of this method to introduce block mutations into specific regions of DNA, using oligonucleotide primers containing multiple nucleotide mismatches which contain new restriction sites. The oligonucleotides, with up to 15 mismatches, can specifically and stably bind to the predicted target sequence. Because of the high mutation rate and convenient selection by restriction enzyme digestion, this method of introducing site-specific block mutations into DNA is very easy and efficient. We have constructed a series of block mutations in the promoter of the gene for the cytosolic form of P-enolpyruvate carboxykinase (GTP) (EC4.1.1.32) (PEPCK) by this method showing that a block mutation in the cAMP responsive element (CRE-1) completely disrupts protein binding to CRE-1 in vitro and causes a dramatic decrease in the basal transcription from the PEPCK promoter in vivo.
Liu
J S
JS
Case Western Reserve University, School of Medicine.
Roesler
W J
WJ
Hanson
R W
RW
D001483:Base Sequence; D001665:Binding Sites; D000242:Cyclic AMP; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014169:Transformation, Bacterial
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D004247:DNA; D000242:Cyclic AMP; D004262:DNA Restriction Enzymes; D010729:Phosphoenolpyruvate Carboxykinase (GTP)