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Nucleic Acids Research 2005 33(8):e71; doi:10.1093/nar/gni070
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Published online 28 April 2005

© The Author 2005. 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{at}oupjournals.org

Methods Online

Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments

Fredrik Dahl, Mats Gullberg, Johan Stenberg, Ulf Landegren and Mats Nilsson*

Department of Genetics and Pathology, Rudbeck Laboratory Se-75185 Uppsala, Sweden

*To whom correspondence should be addressed. Tel: +46 018 471 4816; Email: Mats.nilsson{at}genpat.uu.se

Received February 11, 2005. Revised April 1, 2005. Accepted April 1, 2005.

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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