Cloning a defined region of DNA using a limited action of DNA polymerase: application to dissection of hepatitis B virus surface antigen gene.
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.
D001426:Bacterial Proteins / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000096:biosynthesis; D004256:DNA Polymerase I / Q000378:metabolism*; D004274:DNA, Recombinant / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D011993:Recombinant Fusion Proteins; D004247:DNA; D004256:DNA Polymerase I