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RExPrimer: an integrated primer designing tool increases PCR effectiveness by avoiding 3' SNP-in-primer and mis-priming from structural variation. 2009

Jittima Piriyapongsa, and Chumpol Ngamphiw, and Anunchai Assawamakin, and Pongsakorn Wangkumhang, and Payiarat Suwannasri, and Uttapong Ruangrit, and Gallissara Agavatpanitch, and Sissades Tongsima
Genome Institute, National Center for Genetic Engineering and Biotechnology, Pathumthani, Thailand. jittima.pir@biotec.or.th

BACKGROUND Polymerase chain reaction (PCR) is very useful in many areas of molecular biology research. It is commonly observed that PCR success is critically dependent on design of an effective primer pair. Current tools for primer design do not adequately address the problem of PCR failure due to mis-priming on target-related sequences and structural variations in the genome. METHODS We have developed an integrated graphical web-based application for primer design, called RExPrimer, which was written in Python language. The software uses Primer3 as the primer designing core algorithm. Locally stored sequence information and genomic variant information were hosted on MySQLv5.0 and were incorporated into RExPrimer. RESULTS RExPrimer provides many functionalities for improved PCR primer design. Several databases, namely annotated human SNP databases, insertion/deletion (indel) polymorphisms database, pseudogene database, and structural genomic variation databases were integrated into RExPrimer, enabling an effective without-leaving-the-website validation of the resulting primers. By incorporating these databases, the primers reported by RExPrimer avoid mis-priming to related sequences (e.g. pseudogene, segmental duplication) as well as possible PCR failure because of structural polymorphisms (SNP, indel, and copy number variation (CNV)). To prevent mismatching caused by unexpected SNPs in the designed primers, in particular the 3' end (SNP-in-Primer), several SNP databases covering the broad range of population-specific SNP information are utilized to report SNPs present in the primer sequences. Population-specific SNP information also helps customize primer design for a specific population. Furthermore, RExPrimer offers a graphical user-friendly interface through the use of scalable vector graphic image that intuitively presents resulting primers along with the corresponding gene structure. In this study, we demonstrated the program effectiveness in successfully generating primers for strong homologous sequences. CONCLUSIONS The improvements for primer design incorporated into RExPrimer were demonstrated to be effective in designing primers for challenging PCR experiments. Integration of SNP and structural variation databases allows for robust primer design for a variety of PCR applications, irrespective of the sequence complexity in the region of interest. This software is freely available at http://www4a.biotec.or.th/rexprimer.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012985 Software Design Specifications and instructions applied to the software. Flowcharts (Computer),Flow Charts (Computer),Flowcharts,Chart, Flow (Computer),Charts, Flow (Computer),Design, Software,Designs, Software,Flow Chart (Computer),Flowchart,Flowchart (Computer),Software Designs
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D017422 Sequence Analysis, DNA A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis. DNA Sequence Analysis,Sequence Determination, DNA,Analysis, DNA Sequence,DNA Sequence Determination,DNA Sequence Determinations,DNA Sequencing,Determination, DNA Sequence,Determinations, DNA Sequence,Sequence Determinations, DNA,Analyses, DNA Sequence,DNA Sequence Analyses,Sequence Analyses, DNA,Sequencing, DNA
D017931 DNA Primers Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide
D019389 Cytochrome P-450 CYP2D6 A cytochrome P450 enzyme that catalyzes the hydroxylation of many drugs and environmental chemicals, such as DEBRISOQUINE; ADRENERGIC RECEPTOR ANTAGONISTS; and TRICYCLIC ANTIDEPRESSANTS. This enzyme is deficient in up to 10 percent of the Caucasian population. CYP2D6,Debrisoquine 4-Hydroxylase,Debrisoquine Hydroxylase,CYP 2D6,Cytochrome P450 2D6,Debrisoquine 4-Monooxygenase,Imipramine 2-Hydroxylase,Sparteine Monooxygenase,2-Hydroxylase, Imipramine,4-Hydroxylase, Debrisoquine,4-Monooxygenase, Debrisoquine,CYP2D6, Cytochrome P-450,Cytochrome P 450 CYP2D6,Debrisoquine 4 Hydroxylase,Debrisoquine 4 Monooxygenase,Hydroxylase, Debrisoquine,Imipramine 2 Hydroxylase,Monooxygenase, Sparteine,P-450 CYP2D6, Cytochrome,P450 2D6, Cytochrome
D020407 Internet A loose confederation of computer communication networks around the world. The networks that make up the Internet are connected through several backbone networks. The Internet grew out of the US Government ARPAnet project and was designed to facilitate information exchange. World Wide Web,Cyber Space,Cyberspace,Web, World Wide,Wide Web, World
D020641 Polymorphism, Single Nucleotide A single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population. SNPs,Single Nucleotide Polymorphism,Nucleotide Polymorphism, Single,Nucleotide Polymorphisms, Single,Polymorphisms, Single Nucleotide,Single Nucleotide Polymorphisms

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