Use of a phage vector for rapid synthesis and cloning of single-stranded cDNA.
We have developed a technique for synthesis of single stranded complementary DNA (ss cDNA) using specifically designed phage ssDNA as vector primer. This vector (pPBS27) was constructed by introducing a poly(dT) tail adjacent to the XbaI site of pTZ18R, which can exist either as a plasmid in Escherichia coli or as a ssDNA phage. The pPBS27 phage vector is linearized with XbaI using a restriction-site-directed fragment and used to anneal a mixture of poly(A) + RNA for cDNA synthesis by reverse transcriptase. The RNA is then hydrolysed with NaOH and a poly(dG) tail added to the 3' end of the vector-cDNA with terminal transferase. The linear hybrid ssDNA is then closed by annealing with a 15-mer site-directed fragment oligodeoxynucleotide molecule and ligated with T4 DNA ligase. Almost 10(5) E. coli transformants per microgram of vector primer can be obtained in two days.
D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D010957:Plasmids
D004277:DNA, Single-Stranded; D004247:DNA; D004262:DNA Restriction Enzymes