Enzymatic multiplication of a chemically synthesized DNA fragment

Abstract

A synthetic DNA fragment of 19 residues was enlarged by the enzymatic addition of deoxyadenylate residues to its 3'-end with calf thymus terminal deoxynucleotidyl transferase. The 3'-terminus of this elongated DNA strand was blocked with 2',3'-dideoxyadenylate to prevent hydrolysis by the 3'-exonuclease function of E. coli DNA polymerase I. This elongated and 3'-blocked fragment was annealed to an oligomeric primer and used as a template for the synthesis of a complementary copy of the synthetic 19-mer. The product of such a repair synthesis was separated by gel filtration and analyzed by nearest neighbor techniques.All template strands were copied with complgte repair in over 90% of the chains. Facile recovery of the elongated template by virtue of its size permitted repetition of the copy process, thus allowing accumulation of the desired strand.

Determination of the 3′ terminal nucleotide of DNA fragments

“…Terminal deoxynucleotidyl transferase from calf thymus was purified as described (5), except that the final hydroxylapatite chromatography was omitted. The enzyme from Sephadex G-100 chromatography was further purified by iso-electric focusing (6). No detectable exo-or endonucleolytic activity was found under our conditions of use.…”

“…hibitors of DNA synthesis, blocking chain extension after their addition due to the absence of a 3'-hydroxyl linkage group. Likewise, DNA and DNA oligomers, after addition of a dideoxyribonucleotide, are resistant to-the pyrophosphorolysis and 3'exonuclease functions of DNA polymerase (6,15). In contrast to the addition of ribonucleotides to DNA oligomers with terminal transferase (3), only one molecule of a dideoxyribonucleotide can be added.…”

Construction of a double-stranded deoxyribonucleotide sequence of 45 base pairs designed to code for S-peptide2-14of bovine ribonuclease A

“…50 Units of T4 ligase were added and the mixture incubated at 00. At 24,48, and 72 hr. the reaction was heated, reannealed, and 50 units of ligase added each time.…”

PCR and Scientific Invention: The Trial of DuPont vs. Cetus

“…In the Chemical Research Department of Hoffmann-La Roche in New Jersey the problem of enzymatic duplication of synthetic DNA was also being approached (Olsen et al, 1975). Responding to the same template reassociation problem as the researchers in Khorana's lab, the Roche group proposed another cumbersome solution without commenting and probably without considering the far simpler alternative of just diluting the complementary template strands sufficiently so that they would not interfere with each other's priming and extension.…”