Dataset Information

MFEprimer-2.0: a fast thermodynamics-based program for checking PCR primer specificity.

ABSTRACT: Evaluating the specificity of polymerase chain reaction (PCR) primers is an essential step in PCR primer design. The MFEprimer-2.0 server allows users to check primer specificity against genomic DNA and messenger RNA/complementary DNA sequence databases quickly and easily. MFEprimer-2.0 uses a k-mer index algorithm to accelerate the search process for primer binding sites and uses thermodynamics to evaluate binding stability between each primer and its DNA template. Several important characteristics, such as the sequence, melting temperature and size of each amplicon, either specific or non-specific, are reported on the results page. Based on these characteristics and the user-friendly output, users can readily draw conclusions about the specificity of PCR primers. Analyses for degenerate primers and multiple PCR primers are also supported in MFEprimer-2.0. In addition, the databases supported by MFEprimer-2.0 are comprehensive, and custom databases can also be supported on request. The MFEprimer-2.0 server does not require a login and is freely available at http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0. More over, the MFEprimer-2.0 command-line version and local server version are open source and can be downloaded at https://github.com/quwubin/MFEprimer/wiki/Manual/.

SUBMITTER: Qu W

PROVIDER: S-EPMC3394324 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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MFEprimer-2.0: a fast thermodynamics-based program for checking PCR primer specificity.

Qu Wubin W Zhou Yang Y Zhang Yanchun Y Lu Yiming Y Wang Xiaolei X Zhao Dongsheng D Yang Yi Y Zhang Chenggang C

Nucleic acids research 20120611 Web Server issue

Evaluating the specificity of polymerase chain reaction (PCR) primers is an essential step in PCR primer design. The MFEprimer-2.0 server allows users to check primer specificity against genomic DNA and messenger RNA/complementary DNA sequence databases quickly and easily. MFEprimer-2.0 uses a k-mer index algorithm to accelerate the search process for primer binding sites and uses thermodynamics to evaluate binding stability between each primer and its DNA template. Several important characteris ...[more]

PMID: 22689644

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Project description:BackgroundIn some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this target gene approach are called "intron-flanking primers" or because they are located in exonic sequences which are usually conserved between related species, "conserved primers". They are useful for large-scale single nucleotide polymorphism (SNP) discovery and marker development, especially in species, such as wheat, for which a large number of ESTs are available but for which genome sequences and intron/exon boundaries are not available. To date, no suitable high-throughput tool is available for this purpose.ResultsWe have developed, the ConservedPrimers 2.0 pipeline, for designing intron-flanking primers for large-scale SNP discovery and marker development, and demonstrated its utility in wheat. This tool uses non-redundant wheat EST sequences, such as wheat contigs and singleton ESTs, and related genomic sequences, such as those of rice, as inputs. It aligns the ESTs to the genomic sequences to identify unique colinear exon blocks and predicts intron lengths. Intron-flanking primers are then designed based on the intron/exon information using the Primer3 core program or BatchPrimer3. Finally, a tab-delimited file containing intron-flanking primer pair sequences and their primer properties is generated for primer ordering and their PCR applications. Using this tool, 1,922 bin-mapped wheat ESTs (31.8% of the 6,045 in total) were found to have unique colinear exon blocks suitable for primer design and 1,821 primer pairs were designed from these single- or low-copy genes for PCR amplification and SNP discovery. With these primers and subsequently designed genome-specific primers, a total of 1,527 loci were found to contain one or more genome-specific SNPs.ConclusionThe ConservedPrimers 2.0 pipeline for designing intron-flanking primers was developed and its utility demonstrated. The tool can be used for SNP discovery, genetic variation assays and marker development for any target genome that has abundant ESTs and a related reference genome that has been fully sequenced. The ConservedPrimers 2.0 pipeline has been implemented as a command-line tool as well as a web application. Both versions are freely available at http://wheat.pw.usda.gov/demos/ConservedPrimers/.

Dataset Information

MFEprimer-2.0: a fast thermodynamics-based program for checking PCR primer specificity.

Publications

MFEprimer-2.0: a fast thermodynamics-based program for checking PCR primer specificity.

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