A non-radioactive DNA sequencing method using biotinylated dideoxynucleoside triphosphates and delta Tth DNA polymerase

DNA Res. 1995 Oct 31;2(5):225-7. doi: 10.1093/dnares/2.5.225.

Abstract

We synthesized a set of four biotinylated dideoxynucleoside triphosphates (biotin-9-ddNTPs) and optimized the reaction conditions for non-radioactive cycle sequencing using modified Tth DNA polymerase (delta Tth) and a chemiluminescent detection system. The resulting sequencing ladders showed lower background compared to those with the conventional non-radioactive sequencing method which uses 5'-biotinylated primers, especially when PCR products were analysed. With our method, DNA sequences can be determined at any primer positions without preparing 5'-biotinylated primers for dideoxy chain-termination.

MeSH terms

  • Biotin*
  • DNA Gyrase
  • DNA Topoisomerases, Type II / genetics
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyribonucleotides / chemistry
  • Luminescent Measurements
  • Molecular Structure
  • Sequence Analysis, DNA / methods*
  • Thermus thermophilus / enzymology

Substances

  • Deoxyribonucleotides
  • Biotin
  • DNA-Directed DNA Polymerase
  • DNA Gyrase
  • DNA Topoisomerases, Type II