Open AccessEfficient correction of a mutation by use of chemically synthesized DNA.
Author(s) -
Aharon Razin,
Tatsuro Hirose,
Keiichi Itakura,
Arthur D. Riggs
Publication year - 1978
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.75.9.4268
Subject(s) - biology , microbiology and biotechnology , escherichia coli , heteroduplex , spheroplast , dna , mutant , dna replication , wild type , dna synthesis , base pair , primer (cosmetics) , thymidine , chemistry , genetics , gene , organic chemistry
The mutated base in the am3 lysis-defective mutant of the bacteriophage phiX174 has been corrected by a combined in vitro enzymatic DNA synthesis and in vivo replication of the heteroduplex product. Chemically synthesized oligodeoxyribonucleotides carrying the wild-type sequence have been used to prime DNA synthesis with am3 phiX174 DNA serving as a template. The resultant semisynthetic heteroduplex composed of an am3(+) strand and a wild-type (-) strand, with one mismatched base pair at position 587 on the phiX174 DNA sequence, was used to infect spheroplasts. The progeny phage were analyzed by a parallel plaque assay on wild-type host, Escherichia coli C, to screen for wild-type phenotype, and on E. coli HF4714, an amber suppressor strain, to determine the total progeny phage. When a 23-base-long synthetic primer was used, about one-third of total progeny were found to be wild type. Shorter primers yielded lower percentages of wild type; they also had poorer priming activity.