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Anal Biochem. 2006 Sep 15;356(2):194-201. Epub 2006 May 12.

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

Author information

1
Genome Analysis, Leibniz Institute for Age Research-Fritz Lipmann Institute, 07745 Jena, Germany. mgroth@fli-leibniz.de

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.

PMID:
16737678
DOI:
10.1016/j.ab.2006.04.043
[Indexed for MEDLINE]

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