Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing technology.
A new one-step method for fast and efficient preparation of double-stranded DNA template, suitable for use with Pyrosequencing technology, has been developed. In the new method, two different types of oligonucleotides were used to prevent reannealing of remaining PCR primers to the template: oligonucleotides complementary to the PCR primers and 3'-end modified oligonucleotides with the same sequence as the PCR primers. Advantages with the new strategy are: (i) faster and simpler template preparation procedure (one-step); (ii) no need for exonuclease I treatment; and (iii) less problem with unspecific priming from loop structures and dimers. By careful oligonucleotide design, and/or by addition of single-stranded DNA-binding protein, problems with unspecific sequence signals due to mispriming can be reduced. The new method was used for analysis of genotype variations within the renin-angiotensin-aldosterone system.
Department of Biotechnology, SCFAB, Royal Institute of Technology, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.
Tommy
T
Nordström
Anders
A
Alderborn
Pål
P
Nyrén
D001483:Base Sequence; D001709:Biotechnology; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D004277:DNA, Single-Stranded; D019281:Dimerization; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012084:Renin-Angiotensin System; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004247:DNA