Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing (TM) technology
2002 (English)In: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, E-ISSN 1872-857X, Vol. 52, no 2, 71-82 p.Article in journal (Refereed) Published
A new one-step method for fast and efficient preparation of double-stranded DNA template, suitable for use with Pyrosequencing(TM) 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.
Place, publisher, year, edition, pages
2002. Vol. 52, no 2, 71-82 p.
pyrosequencing, DNA sequencing, template preparation, blocking oligonuclemides, single-nucleotide polymorphism, double-stranded DNA, single-nucleotide polymorphisms, polymerase chain-reaction, real-time pyrophosphate
IdentifiersURN: urn:nbn:se:kth:diva-21966ISI: 000178574200001OAI: oai:DiVA.org:kth-21966DiVA: diva2:340664
QC 201005252010-08-102010-08-10Bibliographically approved