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Nucleic Acids Research 2006 34(6):e49; doi:10.1093/nar/gkl103
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Published online 31 March 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

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Tri-nucleotide threading for parallel amplification of minute amounts of genomic DNA

Erik Pettersson, Mats Lindskog, Joakim Lundeberg and Afshin Ahmadian*

Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center SE-106 91 Stockholm, Sweden

*To whom correspondence should be addressed. Tel: +46 8 5537 8333; Fax: +46 8 5537 8481; Email: afshin.ahmadian{at}biotech.kth.se

Received January 23, 2006. Revised February 13, 2006. Accepted March 8, 2006.

Efforts to correlate genetic variations with phenotypic differences are intensifying due to the availability of high-density maps of single nucleotide polymorphisms (SNPs) and the development of high throughput scoring methods. These recent advances have led to an increased interest for improved multiplex preparations of genetic material to facilitate such whole genome analyses. Here we propose a strategy for the parallel amplification of polymorphic loci based on a reduced set of nucleotides. The technique denoted Tri-nucleotide Threading (TnT), allows SNPs to be amplified via controlled linear amplification followed by complete removal of the target material and subsequent amplification with a pair of universal primers. A dedicated software tool was developed for this purpose and variable positions in genes associated with different forms of cancer were analyzed using sub-nanogram amounts of starting material. The amplified fragments were then successfully scored using a microarray-based PrASE technique. The results of this study, in which 75 SNPs were analyzed, show that the TnT technique circumvents potential problems associated with multiplex amplification of SNPs from minute amounts of material. The technique is specific, sensitive and can be readily adapted to equipment and genotyping techniques used in other research laboratories without requiring changes to the preferred typing method.


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