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

Authors:
Marco Groth, Klaus Huse, Kathrin Reichwald, Stefan Taudien, Jochen Hampe, Philip Rosenstiel, Gerd Birkenmeier, Stefan Schreiber, Matthias Platzer
Year of publication:
2006
Volume:
356
Issue:
2
Issn:
0003-2697
Journal title abbreviated:
Anal. Biochem.
Journal title long:
Analytical biochemistry
Impact factor:
2.305
Abstract:
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.