Cloning a defined region of DNA using a limited action of DNA polymerase: application to dissection of hepatitis B virus surface antigen gene

Gene. 1986;45(1):19-25. doi: 10.1016/0378-1119(86)90127-7.


Using dodecadeoxynucleotides as primers for DNA synthesis and 3'-o-chlorophenyl-phosphorylated dodecadeoxynucleotides as "stoppers" for chain elongation, pre-defined regions of a gene previously cloned in M13 single-stranded (ss) DNA phage were converted into double-stranded (ds) DNA utilizing the action of the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). The resulting ds DNA was freed from the ss region by S1 nuclease treatment. This method can be used to obtain DNA fragments of any size with pre-defined 5' and 3' ends. About 15% of the input ss DNA template molecules are converted into ds DNA fragments. This technique was used to synthesize several DNA fragments from different portions of the hepatitis B virus surface antigen (HBsAg) gene. The products were then ligated into a yeast plasmid vector that carries the E. coli lacZ gene which is located downstream from the yeast acid-phosphatase promotor. Using this system, several fragments of HBsAg were produced in the form of beta-galactosidase fused protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism*
  • Cloning, Molecular / methods*
  • DNA / biosynthesis
  • DNA Polymerase I / metabolism*
  • DNA, Recombinant / metabolism*
  • DNA, Viral / biosynthesis
  • DNA, Viral / genetics*
  • Hepatitis B Surface Antigens / genetics*
  • Hepatitis B virus / genetics*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics


  • Bacterial Proteins
  • DNA, Recombinant
  • DNA, Viral
  • Hepatitis B Surface Antigens
  • Recombinant Fusion Proteins
  • DNA
  • DNA Polymerase I