Abstract
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.
Highlights
Recent progress in the chemical synthesis of DNA complementarysequence a t their 3‘ termini
The economyin nucleotides involved only four synthetic fragmentsof 43, 42, chemical synthesis using this approach is substantial 39, and 39 bases long. This represents with a greater than 40% reduction in the amount of a 44%reduction in the amounoft chemical synthesis required chemical synthesis required as compared with thecon- by the conventional approaches [1,6].The IFm gene segment ventional approach
We describe in detail this meth- has been cloned into anE . coli plasmid vector using a threeodologyforthebiochemicalassemblyof long gene part ligation, and the correct DNA sequencoef the synthetic segments from syntheticoligodeoxyribonucleotides. gene segment hasbeen verified subsequent to itscloning
Summary
We have utilized the amino acid coding sequences deduced for a2 [17] to chemically synthesize fouroligodeoxyribonucleotides which, when assembled, comprise the coding sequence for IFa2 amino acid residues 126 through 165. Restrictionendonuclease recognition sites for Eco RI and Pst I were introduced into the syntheticdesign to facilitate cloning of the IFa2 segmentinto a bacterial plasmid vector. Each pair of fragments was designed to have a short region of complementary sequenceat its 3' termini (Fig. 1). When the pairs of fragments are annealed in the presence of deoxyribonucleoside triphosphates, they become a substrate for DNA polymerase I (K1enow)-mediated polymerization to give a full length duplex product The products of thesereactions were electrophoresed in acrylamide t o resolve duplexes from the single-stranded oligonucleotides(Fig. 2).
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