Verma I M
J Virol. 1978 Jun;26(3):615-29. doi: 10.1128/JVI.26.3.615-629.1978.
Genome-length complementary DNA (cDNA) transcripts were synthesized in vitro by using purified virions of avian myeloblastosis virus. Moloney murine leukemia virus, and clone 124 mouse sarcoma virus. The size of the genomelenth cDNA transcripts was measured on either alkaline sucrose gradients or alkaline agarose gels. The longest cDNA transcripts synthesized by using avian myeloblastosis virus, Moloney murine leukemia virus, and clone 124 mouse sarcoma virus were 7, 9 and 6 kilobases (kb), respectively. The in vitro system used was capable of synthesizing double-stranded DNA, but the plus strands (same polarity as the viral RNA) were only 0.5 to 1.5 kb long. Lone Moloney murine leukemia virus cDNA transcripts were used as templates to synthesize the second plus strand. Essentially two strategies were employed as follows. (i) The 3' ends of the cDNA transcripts were extended by addition of 50 to 100 dAMP residues by terminal deoxynucleotidyl transferase. The (dA)n-tailed cDNA transcripts were used as templates along with an oligomer of dT as primer and Escherichia coli DNA polymerase to synthesize the plus strands. (ii) DNase-digested calf thymus DNA was used to prime the synthesis of plus strands on long cDNA with E. coli DNA polymerase I. In both cases, the synthesis of the plus strands was monitored by increased resistance of the cDNA templates to single-strand-specific S1 nuclease. The double-stranded DNA was fractionated on neutral sucrose gradients. Analysis of the double-stranded DNA synthesized by using oligo(dT) primer showed the plus strands to be about 5 to 6 kb long, whereas the plus strands synthesized by using DNase-digested calf thymus DNA primers were only 0.3 to 0.5 kb long. Double-stranded DNA synthesized by either method has an average size of 6 x 10(6) daltons. Double-stranded DNA was also synthesized by using cDNA transcripts as templates without the addition of any primers. In this case, the plus strands were covalently linked to the template strand and were not representative of the whole parent strand.
利用禽成髓细胞瘤病毒、莫洛尼鼠白血病病毒和124克隆小鼠肉瘤病毒的纯化病毒粒子在体外合成基因组长度的互补DNA（cDNA）转录本。在碱性蔗糖梯度或碱性琼脂糖凝胶上测定基因组长度cDNA转录本的大小。利用禽成髓细胞瘤病毒、莫洛尼鼠白血病病毒和124克隆小鼠肉瘤病毒合成的最长cDNA转录本分别为7、9和6千碱基（kb）。所使用的体外系统能够合成双链DNA，但正链（与病毒RNA极性相同）仅0.5至1.5 kb长。单独的莫洛尼鼠白血病病毒cDNA转录本用作模板来合成第二条正链。基本上采用了以下两种策略。（i）通过末端脱氧核苷酸转移酶添加50至100个dAMP残基来延伸cDNA转录本的3'末端。以（dA）n尾化的cDNA转录本为模板，与dT寡聚物作为引物以及大肠杆菌DNA聚合酶一起合成正链。（ii）用脱氧核糖核酸酶消化的小牛胸腺DNA作为引物，用大肠杆菌DNA聚合酶I在长cDNA上合成正链。在这两种情况下，通过cDNA模板对单链特异性S1核酸酶抗性的增加来监测正链的合成。双链DNA在中性蔗糖梯度上进行分级分离。对使用寡聚（dT）引物合成的双链DNA的分析表明，正链约5至6 kb长，而使用脱氧核糖核酸酶消化的小牛胸腺DNA引物合成的正链仅0.3至0.5 kb长。通过任何一种方法合成的双链DNA的平均大小为6×10⁶道尔顿。也使用cDNA转录本作为模板不添加任何引物来合成双链DNA。在这种情况下，正链与模板链共价连接，并不代表整个亲本链。
