An alternate method for synthesis of double-stranded DNA segments.
Recent progress in the chemical synthesis of DNA has now made it possible to rapidly synthesize single-stranded DNAs over 40 bases in length. We have taken advantage of these longer DNAs in assembling and cloning a 132-base pair gene segment coding for amino acids 126 through the stop codon of human leukocyte interferon alpha 2. The method used involves DNA polymerase I-mediated repair synthesis of synthetic oligonucleotide substrates having short stretches of complementary sequence at their 3' termini. In the presence of DNA polymerase I and the four deoxyribonucleoside triphosphates, those primer-templates are converted to full length double-stranded DNAs. The economy in chemical synthesis using this approach is substantial with a greater than 40% reduction in the amount of chemical synthesis required as compared with the conventional approach. We describe in detail this methodology for the biochemical assembly of long gene segments from synthetic oligodeoxyribonucleotides.
D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000138:chemical synthesis*; D004256:DNA Polymerase I; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics; D007372:Interferons / Q000235:genetics*; D008722:Methods; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids
D009838:Oligodeoxyribonucleotides; D004247:DNA; D007372:Interferons; D004256:DNA Polymerase I; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific