Enzymatic multiplication of a chemically synthesized DNA fragment.
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 complete 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.
D000818:Animals; D002417:Cattle; D004247:DNA / Q000096:biosynthesis; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology; D008722:Methods; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000096:biosynthesis; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology
D004269:DNA, Bacterial; D009841:Oligonucleotides; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D004254:DNA Nucleotidyltransferases