Bioinformatics Advance Access originally published online on June 28, 2007
Bioinformatics 2007 23(17):2331-2333; doi:10.1093/bioinformatics/btm343
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QPRIMER: a quick web-based application for designing conserved PCR primers from multigenome alignments
Center for Computational Biology, Molecular Biology Institute, Institute for Genomics and Proteomics, Department of Chemistry and Biochemistry, University of California, Los Angeles, 90095-1570, USA
*To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT 1 INTRODUCTION2 FEATURES AND WEB...ACKNOWLEDGEMENTSREFERENCES |
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Summary: We have developed a quick web-based application for designing conserved genomic PCR and RT-PCR primers from multigenome alignments targeting specific exons or introns. We used Pygr (The Python Graph Database Framework for Bioinformatics) to query intervals from multigenome alignments, which gives us less than a millisecond access to any intervals of any genome within multigenome alignments. PRIMER3 was used to extract optimal primers from a gene of interest. QPRIMER creates an electronic genomic PCR image from a set of conserved primers as well as summary pages for primer alignments and products. QPRIMER supports human, mouse, rat, chicken, dog, zebrafish and fruit fly.
Availability: http://www.bioinformatics.ucla.edu/QPRIMER/
Contact: leec{at}mbi.ucla.edu
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1 INTRODUCTION |
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TOPABSTRACT1 INTRODUCTION2 FEATURES AND WEB...ACKNOWLEDGEMENTSREFERENCES |
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Polymerase chain reaction (PCR) is the most important experimental technique for comparative studies as well as gene cloning in order to amplify specific genes of interest from various organisms. There is a need for fast and flexible tool to design proper conserved primer candidates from multiple alignments. EC_oligos (Liu et al., 2004) and DualPrime (Andersson et al., 2005) can design conserved primers between two sequences. Software that can design conserved primers from multiple alignments also have been developed. Primaclade (Gadberry et al., 2005) can handle many types of multiple alignments and Primer Premier 5 (PREMIER Biosoft International) is only available commercially.
Availability of multigenome alignments (Blanchette et al., 2004) at UCSC Genome Browser (Karolchik et al., 2003) provides an important new platform for designing conserved genomic PCR as well as RT-PCR primers of various organism. But, web-based applications utilizing multigenome alignments have not been reported, due in part to the challenges of fast database query from more than a billion alignment intervals. Pygr (A Python Graph Database Framework for Bioinformatics, http://bioinformatics.ucla.edu/pygr/) recently announced new features—NCList (Alekseyenko and Lee, 2007), which is designed for querying intervals of any genomic location of any genomes from multigenome alignments within 1 ms.
QPRIMER is a novel, automatic web-based application for designing conserved genomic PCR and RT-PCR primers from multigenome alignments on the web using Pygr and PRIMER3 (Rozen and Skaletsky, 2000). User-friendly and intuitive graphical interface enable one to browse any genomic location and design conserved PCR primers on the fly. Furthermore, it usually takes <5 s to get conserved genomic PCR primers.
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2 FEATURES AND WEB INTERFACE FOR DESIGNING CONSERVED PRIMERS |
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TOPABSTRACT1 INTRODUCTION2 FEATURES AND WEB...ACKNOWLEDGEMENTSREFERENCES |
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Most software requires sequences or their multiple alignments as an input. Users need to do many time-consuming steps in order to get optimal primer sets. An important new feature of QPRIMER is its combination of genome browser and PRIMER3 on the web. As shown in Figure 1A and C, the user can browse a specific gene of interest using UCSC-like genome browser based on its genomic location. The user can select any region in the gene structure as a target for amplification, simply by clicking on it. Convenient zooming (700 bp) provides up to 1 bp to 1 pixel mapping for single nucleotide resolution.
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Once the user has selected start and end positions for the primer target area (Fig. 1A), a PRIMER3 option window will be displayed at the bottom of page (Fig. 1B). The maximum product length is 1 kb by default for typical genomic PCR primers, but this can be increased in order to design RT-PCR primers covering a target exon. Total calculation time will be also increased. It should be noted that QPRIMER selects primers from only exonic regions. Number of PRIMER3 output is another important parameter because we need many combinations of forward and reverse primers within conserved blocks. QPRIMER chooses conserved primers by comparing synteny blocks retrieved by Pygr from multigenome alignment with optimal primer sets derived from PRIMER3. Total calculation time depends on total length of search area (product size). It will take <5 s for usual genomic PCR primer design, product size <1 kb.
Genomic alignments of each primer pairs, organism information, product size and sum of mismatches as well as target area will be displayed as shown in Figure 1C (detailed information will be available at QPRIMER web site). Success of conserved primer design is solely dependent on whether the specific gene sequence is conserved over the target organisms or not. If the given gene is diverse or newly created, one may not get primer candidates. QPRIMER will give a short warning message noticed failure to retrieve conserved blocks from multigenome alignment within a second—Pygr performance—in this case.
If user clicks one of the primer sets in Figure 1C, electronic genomic PCR image will be displayed in a new window as shown in Figure 1D. Thickness and darkness of PCR bands represent total number of mismatches in that organism, better alignments will be thick and dark. Primer sequences, PRIMER3 summary, multiple alignments and products from various organisms will be displayed followed by genomic PCR image as shown in Figure 1E. QPRIMER does not check multiple hits for all genomes because it will require huge amount of calculation and not suitable as a web-based application. But, QPRIMER provides linkouts to UCSC In-Silico PCR (http://genome.ucsc.edu/cgi-bin/hgPcr). One can click the linkouts in order to check whether multiple hits exist or not. There would be no multiple hits for most cases because QPRIMER selects primers only within exonic region, not within Repeats.
In summary, one can easily design conserved PCR primers on the web via a simple, fast web interface.
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ACKNOWLEDGEMENTS |
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TOPABSTRACT1 INTRODUCTION2 FEATURES AND WEB...ACKNOWLEDGEMENTSREFERENCES |
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We wish to thank Dr Meenakshi Roy and Min Jae Lee for valuable comments on this work and testing this application. This work is supported by DOE grant DE-FC02-02ER63421 and NIH grant U54 RR021813.
Conflict of Interest: none declared.
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FOOTNOTES |
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Associate Editor: Keith Crandall
Received on April 11, 2007; revised on April 11, 2007; accepted on June 23, 2007
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REFERENCES |
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TOPABSTRACT1 INTRODUCTION2 FEATURES AND WEB...ACKNOWLEDGEMENTSREFERENCES |
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Alekseyenko AV, Lee CJ. Nested Containment List (NCList): a new algorithm for accelerating interval query of genome alignment and interval databases. Bioinformatics (2007) 23:1386–1393.
Andersson A, et al. Dual-genome primer design for construction of DNA microarrays. Bioinformatics (2005) 21:325–332.
Blanchette M, et al. Aligning multiple genomic sequences with the threaded blockset aligner. Genome Res (2004) 14:708–715.
Gadberry MD, et al. Primaclade – a flexible tool to find conserved PCR primers across multiple species. Bioinformatics (2005) 21:1263–1264.
Karolchik D, et al. The UCSC Genome Browser Database. Nucleic Acids Res (2003) 31:51–54.
Liu S, et al. EC_oligos: automated and whole-genome primer design for exons within one or between two genomes. Bioinformatics (2004) 20:3668–3669.
Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol (2000) 132:365–386.[Medline]
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