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PieceMaker: selection of DNA fragments for selector-guided multiplex amplification
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
2005 (English)In: Nucleic Acids Research, ISSN 0305-1048, Vol. 33, no 8, e72- p.Article in journal (Refereed) Published
Abstract [en]

We describe PieceMaker, a software tool for the design of applications of selector probes-oligonucleotide probes that direct circularization of target nucleic acid molecules. Such probes can be combined in parallel to circularize a selection of fragments from restriction digested total genomic DNA. These fragments can then be amplified in a single PCR using a common primer pair, yielding substrates for subsequent analyses, such as parallel genotyping or sequencing. However, designing multiplex selector assays is a laborious task. The PieceMaker program alleviates this problem by selecting restriction enzymes to generate suitable fragments for selection, and generating the output data required to design the selector probes.

Place, publisher, year, edition, pages
2005. Vol. 33, no 8, e72- p.
Keyword [en]
Computational Biology, DNA Restriction Enzymes/metabolism, DNA; Circular/chemistry, Genomics, Oligonucleotide Probes/*chemistry, Polymerase Chain Reaction, Research Support; Non-U.S. Gov't, Software
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-75748DOI: doi:10.1093/nar/gni071PubMedID: 15860769OAI: oai:DiVA.org:uu-75748DiVA: diva2:103659
Available from: 2006-02-16 Created: 2006-02-16 Last updated: 2009-05-07Bibliographically approved
In thesis
1. Selector Technology: For Multiplex DNA Analysis
Open this publication in new window or tab >>Selector Technology: For Multiplex DNA Analysis
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A majority of methods for identifying sequences in the human genome involve target sequence amplification through PCR. This work presents novel methods for amplifying circularized DNA and presents solutions for some major limitations of PCR.

We have developed a novel method to amplify circularized DNA molecules based on a serial rolling-circle replication reaction, called circle to circle amplification (C2CA). Amplified DNA circles can be detected in array-based analyses or in real-time using molecular beacons. The amplification mechanism allows higher precision in quantification than in exponential amplification methods like PCR, and more products can be generated than in PCR.

A major limitation of PCR is that amplification artifacts arise when large numbers of specific primer pairs are simultaneously added to a reaction. We have developed a solution to this problem that enables multiplex PCR amplification of specific target sequences without producing amplification artifacts. The procedure is based on oligonucleotide constructs, called selectors. The selectors identify defined target nucleic acid sequences, and they act as ligation templates to direct circularization of these targets. The selectors contain a general primer-pair motif that allows the circularized targets to be amplified in multiplex using a universal PCR primer pair. We also developed a computer program, PieceMaker, that finds an optimal design of selector probes for a given selector application. We demonstrate the method by performing a 96-plex PCR of specific DNA sequences with high success-rate and reproducibility.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 44 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 65
Keyword
Genetic engineering, DNA technology, Genteknik
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-5921 (URN)91-554-6330-4 (ISBN)
Public defence
2005-10-07, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2005-09-14 Created: 2005-09-14 Last updated: 2009-10-14Bibliographically approved

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Stenberg, JohanDahl, FredrikLandegren, UlfNilsson, Mats

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