Method for preparing single-stranded DNA templates for Pyrosequencing using vector ligation and universal biotinylated primers.
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.
Groth
Marco
M
Genome Analysis, Leibniz Institute for Age Research-Fritz Lipmann Institute, 07745 Jena, Germany. mgroth@fli-leibniz.de
Huse
Klaus
K
Reichwald
Kathrin
K
Taudien
Stefan
S
Hampe
Jochen
J
Rosenstiel
Philip
P
Birkenmeier
Gerd
G
Schreiber
Stefan
S
Platzer
Matthias
M
D019921:Biotinylation; D017931:DNA Primers / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D005787:Gene Frequency / Q000235:genetics; D005838:Genotype; D006801:Humans; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic
D017931:DNA Primers; D004277:DNA, Single-Stranded; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein