Razin A, Hirose T, Itakura K, Riggs A D
Proc Natl Acad Sci U S A. 1978 Sep;75(9):4268-70. doi: 10.1073/pnas.75.9.4268.
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.
通过体外酶促DNA合成与异源双链体产物的体内复制相结合，噬菌体phiX174的am3裂解缺陷型突变体中的突变碱基已得到校正。携带野生型序列的化学合成寡脱氧核糖核苷酸已被用于以am3 phiX174 DNA为模板引发DNA合成。由am3（+）链和野生型（-）链组成的所得半合成异源双链体，在phiX174 DNA序列的第587位有一个错配碱基对，被用于感染原生质球。通过在野生型宿主大肠杆菌C上进行平行噬菌斑测定来筛选野生型表型，并在琥珀抑制菌株大肠杆菌HF4714上进行测定以确定总后代噬菌体，从而对后代噬菌体进行分析。当使用23个碱基长的合成引物时，发现约三分之一的总后代为野生型。较短的引物产生的野生型百分比更低；它们的引发活性也较差。
