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Design of multiplex PCR primers using heuristic algorithm for sequential deletion applications

Paper ID Volume ID Publish Year Pages File Format Full-Text
15321 1403 2009 8 PDF Available
Title
Design of multiplex PCR primers using heuristic algorithm for sequential deletion applications
Abstract

The sequential deletion method is commonly applied to locate the functional domain of a protein. Unfortunately, manually designing primers for multiplex polymerase chain reaction (PCR) is a labor-intensive task. In order to speed up the experimental procedure and to improve the efficiency of producing PCR products, this paper proposes a multiplex PCR primers (MPCRPs) designer to design multiple forward primers with a single 3′-UTR reverse primer for extracting various N-terminal truncated mutants to quickly locate the functional domain of a cDNA sequence. Several factors, including melting temperature, primer length, GC content, internal self-complement, cross-dimerization, terminal limitation, and specificity, are used as the criteria for designing primers. This study obtains a near-optimal solution of primer sets that can be placed in as few test tubes as possible for one multiplex PCR experiment.ResultsHomo sapiens ribosomal protein L5, Homo sapiens xylosyltransferase I, and Bacteriophage T4 gene product 11 were used as test examples to verify efficacy of the proposed algorithm. In addition, the designed primers of Homo sapiens ribosomal protein L5 cDNA were applied in multiplex PCR experiments. A total of 48 forward primers and one reverse primer were designed and used to duplicate N-terminal truncated mutants of different lengths from the protein. The primers were classified into eight tube groups (i.e., test tubes) held within the same temperature range (53–57 °C), and the validity of the PCR products were verified using polyacrylamide gel electrophoresis (PAGE) with the functional domain correctly located. A software implementation of the proposed algorithm useful in assisting the researcher to design primers for multiplex PCR experiments was developed and available upon request.

Keywords
Multiplex polymerase chain reaction (PCR); Polyacrylamide gel electrophoresis (PAGE); Sequential deletion; N-terminal truncated mutants
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Publisher
Database: Elsevier - ScienceDirect
Journal: Computational Biology and Chemistry - Volume 33, Issue 2, April 2009, Pages 181–188
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us