Method for preparing single-stranded DNA templates for Pyrosequencing using vector ligation and universal biotinylated primers

Anal Biochem. 2006 Sep 15;356(2):194-201. doi: 10.1016/j.ab.2006.04.043. Epub 2006 May 12.


In Pyrosequencing, the addition of nucleotides to a primer-template hybrid is monitored by enzymatic conversion of chemical energy into detectable light. The technique yields both qualitative and quantitative sequence information because the chemical energy is released by a stoichiometric split off of pyrophosphates from incorporated deoxynucleotide triphosphates and a defined nucleotide dispensation order is given. Because Pyrosequencing works best if single-stranded DNA templates are used, template generation usually requires PCR with a target-specific biotinylated primer and a subsequent purification involving interaction of the biotin label with immobilized streptavidin. To circumvent the need for numerous and expensive template-specific biotinylated primers, we developed a method that uses the ligation of amplified DNA fragments into a plasmid vector, thereby facilitating subsequent PCR using a universal vector-specific biotinylated primer. This approach allows easy and straightforward isolation of single-stranded templates of any PCR product. As a proof of principle, we used the method for genotyping two single-nucleotide polymorphisms in the human genes CARD15 and A2M and for characterization of four multisite variations in the human DEFB104 gene.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotinylation
  • DNA Primers / genetics*
  • DNA, Single-Stranded / genetics*
  • Gene Frequency / genetics
  • Genotype
  • Humans
  • Nod2 Signaling Adaptor Protein / genetics
  • Polymerase Chain Reaction / methods
  • Polymorphism, Single Nucleotide / genetics
  • Sequence Analysis, DNA / methods*
  • Templates, Genetic


  • DNA Primers
  • DNA, Single-Stranded
  • NOD2 protein, human
  • Nod2 Signaling Adaptor Protein