Efficient incorporation of positively charged 2', 3'-dideoxynucleoside-5'-triphosphates by DNA polymerases and their application in 'direct-load' DNA sequencing

Nucleic Acids Res. 2003 Aug 15;31(16):4769-78. doi: 10.1093/nar/gkg669.

Abstract

A series of charge-modified, dye-labeled 2', 3'-dideoxynucleoside-5'-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dye-labeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction purification is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modified nucleotides have also been shown to be more efficiently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5'-triphosphates.

MeSH terms

  • Base Sequence
  • Chromatography, High Pressure Liquid / methods
  • Coloring Agents / chemistry
  • DNA / chemistry
  • DNA / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Dideoxynucleosides / chemistry*
  • Lysine / analogs & derivatives*
  • Lysine / chemistry
  • Molecular Sequence Data
  • Molecular Structure
  • Nucleotides / chemistry
  • Nucleotides / genetics
  • Nucleotides / metabolism*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / methods*

Substances

  • Coloring Agents
  • Dideoxynucleosides
  • Nucleotides
  • trimethyllysine
  • DNA
  • DNA-Directed DNA Polymerase
  • Lysine