Detection of a single base mutation in the human dihydrofolate reductase gene from a methotrexate-resistant cell line using the polymerase chain reaction.
To test the utility of the polymerase chain reaction in identifying single base mutations in a gene known to give rise to an altered enzyme and drug resistance phenotype, a human colon adenocarcinoma cell line resistant to methotrexate, with a known single base mutation (Srimatkandada et al., J. Biol. Chem. 264:3524, 1989) was examined. Poly A+ RNA was used for cDNA synthesis with reverse transcriptase, deoxynucleoside triphosphates, and 5 microM 3' primer that anneals outside the coding region of the human dihydrofolate reductase. The RNA:DNA hybrid was used as a template for the polymerase chain reaction with the addition of a 5' primer and Thermus aquaticus (Taq)I DNA polymerase. These primers flank the coding region of the human dihydrofolate reductase and define a region of 650 bases. The polymerase chain reaction was carried out for 40 cycles resulting in full length transcripts in microgram amounts clearly visible by ethidium bromide staining on agarose gels. DNA was isolated by standard methods, and double-stranded DNA was sequenced by the chain-termination method using TaqI DNA polymerase. A single point mutation was discovered at position 91 (T----C) resulting in a substitution of serine for phenylalanine at codon 31, as determined previously by classical cDNA cloning and sequencing. Sequence analysis indicated that this base transition resulted in the loss of Eco RI and Xmn I sites and the gain of a HinfI site in the cDNA, which were confirmed by restriction digests.(ABSTRACT TRUNCATED AT 250 WORDS)
Dicker
A P
AP
Cornell University Graduate School of Medical Sciences, New York, NY.
Volkenandt
M
M
Bertino
J R
JR
D000230:Adenocarcinoma; D001483:Base Sequence; D002460:Cell Line; D003062:Codon / Q000235:genetics; D003110:Colonic Neoplasms; D004273:DNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004351:Drug Resistance; D005796:Genes; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D012334:RNA, Neoplasm / Q000235:genetics; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D003062:Codon; D004273:DNA, Neoplasm; D015345:Oligonucleotide Probes; D012334:RNA, Neoplasm; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate