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Detection of a single base mutation in the human dihydrofolate reductase gene from a methotrexate-resistant cell line
A P Dicker1, M Volkenandt, J R Bertino
1Cornell University Graduate School of Medical Sciences, New York, NY.
Abstract:
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)
Insights
Polymerase chain reaction (PCR) effectively identifies single base mutations in genes, as demonstrated by detecting a specific mutation in the human dihydrofolate reductase gene linked to drug resistance. This method facilitates rapid genetic analysis for altered enzyme phenotypes.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Drug resistance in cancer cells can arise from specific genetic mutations.
- Identifying these single base mutations is crucial for understanding disease mechanisms and developing targeted therapies.
- The human dihydrofolate reductase (DHFR) gene is a key target in cancer treatment, and mutations can confer resistance to drugs like methotrexate.
Purpose of the Study:
- To evaluate the utility of the polymerase chain reaction (PCR) for identifying single base mutations.
- To confirm a known mutation in the human DHFR gene associated with methotrexate resistance in a colon adenocarcinoma cell line.
- To assess PCR's efficiency in detecting genetic alterations leading to altered enzyme function and drug resistance phenotypes.
Main Methods:
- RNA extraction and complementary DNA (cDNA) synthesis using reverse transcriptase.
- Polymerase chain reaction (PCR) amplification of the human DHFR coding region using TaqI DNA polymerase.
- DNA sequencing via the chain-termination method and restriction digestion analysis.
Main Results:
- PCR successfully amplified the target 650-base region of the human DHFR gene, producing microgram amounts of full-length transcripts.
- DNA sequencing identified a single point mutation (T to C at position 91), resulting in a serine-phenylalanine substitution at codon 31.
- Restriction digest analysis confirmed the mutation by showing the loss of Eco RI and Xmn I sites and the gain of a HinfI site.
Conclusions:
- The polymerase chain reaction is a highly effective tool for pinpointing single base mutations in genes.
- This technique can accurately identify genetic alterations responsible for altered enzyme function and drug resistance.
- PCR-based mutation detection offers a rapid and reliable alternative to classical cDNA cloning and sequencing methods.