Chemical synthesis enzymatic elongation and enzymatic joining of mutated primers for ΦX174 replication

摘要

The deoxyoligonucleotide TCTA*AAACAT⁺⁾ and ATTAAC*CTCCT have been synthesized chemically. Both are complementary to adjacent regions of the N-terminal part of gene G of Ø×174 plus strand DNA except for the two positions marked by asteriks. In spite of the mismatches arising from these positions, the “mutated” primers can be elongated in the presence of DNA polymerase I and Ø×174 wildtype DNA as template. The elongation products TCTAAAACATG and ATTAACCTCCTAAG and the elongated primer AGAAATAAAAG described earlier, fit without gaps to the N-terminal part of gene G and, after phosphorylation of the respective 5′ ends, can be linked by the means of polynucleotide ligase.