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Open Access Methodology article

Confronting two-pair primer design for enzyme-free SNP genotyping based on a genetic algorithm

Cheng-Hong Yang12, Yu-Huei Cheng1, Li-Yeh Chuang3* and Hsueh-Wei Chang4567*

Author Affiliations

1 Department of Electronic Engineering, National Kaohsiung University of Applied Sciences, Kaohsiung, Taiwan

2 Department of Network Systems, Toko University, Chiayi, Taiwan

3 Department of Chemical Engineering & Institute of Biotechnology and Chemical Engineering, I-Shou University, Kaohsiung, Taiwan

4 Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan

5 Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan

6 Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

7 Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

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BMC Bioinformatics 2010, 11:509  doi:10.1186/1471-2105-11-509

Published: 13 October 2010



Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) method produces allele-specific DNA bands of different lengths by adding four designed primers and it achieves the single nucleotide polymorphism (SNP) genotyping by electrophoresis without further steps. It is a time- and cost-effective SNP genotyping method that has the advantage of simplicity. However, computation of feasible CTPP primers is still challenging.


In this study, we propose a GA (genetic algorithm)-based method to design a feasible CTPP primer set to perform a reliable PCR experiment. The SLC6A4 gene was tested with 288 SNPs for dry dock experiments which indicated that the proposed algorithm provides CTPP primers satisfied most primer constraints. One SNP rs12449783 in the SLC6A4 gene was taken as an example for the genotyping experiments using electrophoresis which validated the GA-based design method as providing reliable CTPP primer sets for SNP genotyping.


The GA-based CTPP primer design method provides all forms of estimation for the common primer constraints of PCR-CTPP. The GA-CTPP program is implemented in JAVA and a user-friendly input interface is freely available at webcite.