A rapid PCR dependent microtitre plate screening method for DNA sequences altered by site-directed mutagenesis.
A simple and rapid method for identifying DNA sequences altered by site-directed mutagenesis is described. The procedure requires that a restriction endonuclease recognition sequence is either created or removed from the target DNA sequence by the site-directed mutagenesis reaction. In the screening method presented, transformant colonies or M13 plaques are directly subjected to PCR using universal primers that flank the region containing the site-directed changes. The double stranded DNA products generated are then digested, with no purification, by the restriction enzyme whose site is modified, allowing mutant clones to be differentiated from those carrying wild type sequences. The protocol also provides for recovery of the bacterial cultures harbouring the mutated plasmid or M13 for further characterisation. All the procedures are carried out in small volumes in microtitre plates, thereby lowering costs and enabling the investigation of large numbers of clones. The technique allows a considerable amount of effort to be saved compared to conventional screening practices by circumventing time consuming DNA template preparations.
Trower
M K
MK
Molecular Genetics Unit, MRC Centre, Cambridge, England.
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005069:Evaluation Studies as Topic; D005798:Genes, Bacterial; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D010770:Phosphotransferases; D019868:Kanamycin Kinase