Re-usable DNA template for the polymerase chain reaction.
DNA covalently bound to an uncharged nylon membrane was used for consecutive amplifications of several different genes by PCR. Successful PCR amplifications were obtained for membrane-bound genomic and plasmid DNA. Membrane-bound genomic DNA templates were re-used at least 15 times for PCR with specific amplification of the desired gene each time. PCR amplifications of specific sequences of p53, p16, CYP1A1, CYP2D6, GSTM1 and GSTM3 were performed independently on the same strips of uncharged nylon membrane containing genomic DNA. PCR products were subjected to restriction fragment length polymorphism analysis, single-strand conformational polymorphism analysis and/or dideoxy sequencing to confirm PCR-amplified gene sequences. We found that PCR fragments obtained by amplification from bound genomic DNA as template were identical in sequence to those of PCR products obtained from free genomic DNA in solution. PCR was performed using as little as 5 ng genomic or 4 fg plasmid DNA bound to membrane. These results suggest that DNA covalently bound to membrane can be re-used for sample-specific PCR amplifications, providing a potentially unlimited source of DNA for PCR.
Sheikh
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Department of Pathology and Laboratory Medicine, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA.
Lazarus
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D002352:Carrier Proteins / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics; D004247:DNA / Q000032:analysis; D005091:Exons; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008567:Membranes, Artificial; D009062:Mouth Neoplasms / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational; D013698:Templates, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D002352:Carrier Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D008567:Membranes, Artificial; D016159:Tumor Suppressor Protein p53; D004247:DNA; D019363:Cytochrome P-450 CYP1A1; D019389:Cytochrome P-450 CYP2D6; D005982:Glutathione Transferase