Point mutation analysis in a mammalian gene: rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a novel method.
We have developed a very rapid procedure for DNA sequence analysis of induced mutations in a typically large mammalian gene. We are able to determine the nature of chemical carcinogen-induced point mutations in the 25 kb Chinese hamster ovary (CHO) cell dihydrofolate reductase gene within two days starting with 5 to 10 x 10(6) cells. The approach is based on the use of rapidly prepared total RNA from which a 730 bp dhfr cDNA is synthesized by reverse transcriptase and amplified by the polymerase chain reaction (PCR) procedure. Genomic DNA can simultaneously be prepared from the same cells. The amplified double-stranded cDNA is then sequenced directly by the dideoxy method using Taq polymerase in the Thermal Cycler (Perkin Elmer/Cetus). We have previously shown that nonsense codons in the dhfr coding sequence often result in greatly reduced steady-state levels of dhfr mRNA (2). The methods described are suitable for mutants of this type which contain only 1 to 2 copies of mRNA per cell. This approach is readily adaptable to other selectable genes and other cell types provided the necessary primers can be prepared.
Carothers
A M
AM
Columbia University.
Urlaub
G
G
Mucha
J
J
Grunberger
D
D
Chasin
L A
LA
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D019914:Taq Polymerase
D012313:RNA; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase