Sequence analysis of a human gene responsible for drug resistance: a rapid method for manual and automated direct sequencing of products generated by the polymerase chain reaction.
We developed a system for rapid, manual and automated sequence analysis by utilizing and modifying methods used in conjunction with the polymerase chain reaction (PCR). We are using these techniques to detect single base mutations in the dihydrofolate reductase (DHFR) gene giving rise to methotrexate (MTX) resistance of tumor cells obtained from patients with malignancies. Amplifying in vitro both genomic DNA and transcripts of the human DHFR we are able to reproducibly generate single-stranded templates. Utilizing [alpha-35S]dATP and both the universal and reverse sequencing primers we obtain sequence information from either strand. The methods described have been successfully used for automated sequencing with the Applied Biosystems Model 370A Sequencer using both modified T7 DNA polymerase and Taq I. DNA polymerase for dideoxy-termination sequencing. The use of this methodology to detect a single base change in a human colon carcinoma cell line, HCT-8, is illustrated.
Dicker
A P
AP
Cornell University Graduate School of Medical Sciences, Memorial Sloan-Kettering Cancer Center.
Volkenandt
M
M
Adamo
A
A
Barreda
C
C
Bertino
J R
JR
D000230:Adenocarcinoma / Q000235:genetics; D001331:Automation; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D004277:DNA, Single-Stranded / Q000138:chemical synthesis; D004259:DNA-Directed DNA Polymerase; D004351:Drug Resistance / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005784:Gene Amplification; D005821:Genetic Techniques; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000302:isolation & purification; D019914:Taq Polymerase; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D019914:Taq Polymerase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D008727:Methotrexate