Separation of transforming N-ras mutations in codons 12, 13 and 61 by denaturing gradient gel electrophoresis: investigation based on a set of phagemid constructs.
In the present study we have introduced 19 activating base pair substitutions into N-ras cDNA by use of an in vitro site-directed mutagenesis system. Six mutants were constructed for N-ras codon 12 (exon 1), six for codon 13 (exon 1), and seven for codon 61 (exon 2). Fifteen out of 19 PCR-amplified mutation sequences showed a clear separation from the wild type on denaturing gradient gel electrophoresis runs as homoduplex band, and the rest could be separated after heteroduplex formation with wild-type DNA. These constructs can be used as controls in many screening systems for analyzing activating point mutations of the N-ras gene.
Burvall
K
K
Occupational Medicine, National Institute of Occupational Health, Solna, Sweden.
Ridanpää
M
M
Husgafvel-Pursiainen
K
K
Onfelt
A
A
D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D011905:Genes, ras; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009691:Nucleic Acid Denaturation; D010957:Plasmids; D016133:Polymerase Chain Reaction
D003062:Codon; D004247:DNA