This site uses Primer3 to generate PCR primer candidates for all genomic regions specified in your input file. For each input region, Primer3 is run twice with different criteria to increase the number of viable primers found. Each run yields a maximum of 20 primers. To ensure primer specificity, this tool tests primer candidates using a version of Jim Kent's in silico PCR. In addition, when making primers for hg19, genomic positions containing common SNPs (minor allele frequency > 5%) are excluded from primer sequences in order to reduce the chance of PCR failures.
Your input text file should include the following tab-delimited columns:
The entire region from start position to stop position will be included in the primer product, up to a product size of 10,000bp. Regardless of whether you use this parameter, the default minimum product size is 200bp. Any extra columns included in the input file will be ignored and re-printed in output. Using a header line will make the output more readable, but it's not required. (The use of multiple header lines is not allowed.)
Here's what an input file might look like:
Identifier Chromosome Position 101 chr3 54,881,527 102 chr9 24,000,020 103 chrX 5,043,122
In this example, columns 2 and 3 are the chromosome and start position columns, and the optional stop position column has been omitted.
The "Identifier" column would not be used in primer design, but it would be re-printed in the output files.
The BestPrimers output file contains detailed information on the two best primer pairs found for each input region as determined by Primer3. Primers failing the isPCR test will not be included in this file, with one exception: Primers for sex chromosomes are allowed to match exactly one region on the opposite sex chromosome, and are marked in the XY_Oppo_Match field. (This field is irrelevant for genomes without sex chromosomes.) Both primers in the BestPrimers file will come from Run 1 of Primer3 unless that run yields fewer than two primer pairs, in which case primers will be taken from Run 2. For some reference genomes, particularly ones that are highly repetitive or not well characterized, it's possible that most primers will fail in-silico PCR. In that case, you may need to use the AllPrimers output file to select primers despite their failing isPCR.
The AllPrimers file lists all primers returned by Runs 1 and 2 of Primer3 for all input regions, including primers that fail the isPCR test. Primers in this file are ordered by Primer ID, which follows the format chr_startPos_stopPos_RunNum_PrimerNum. The run number corresponds to this tool's Run 1 or Run 2 of Primer3, and Primer Number is a number from 1-20. (With default settings, Primer3 returns up to 20 primers, and it ranks them from 1-20.)
The FailedRegions file lists all of the input regions for which no primers were printed in the BestPrimers file. Either Primer3 failed to find any primers, or all of Primer3's primers failed the isPCR test.
The Regions50 and Regions1000 files contain, for your convenience, the fasta sequences used by this tool for the 50- and 1000-base neighborhoods around the input file's target regions. See below for more information about the reference builds used by this tool.
This program runs Primer3 version 2.3.6 twice with different settings. Primer3 has hundreds of optional settings; any settings not listed in this tool's Run 1 and Run 2 settings files retain their default values. For details, see the Primer3 manual. If you would like, you can also upload your own Primer3 settings files, in which case you should keep these points in mind:
Do not include any type of sensitive or private information in your input file. The best practice is to include only the list of target loci in your input file.
This website was created by Jeff Mandell. The back-end script that does the work of designing primers using Primer3 and gfPcr was designed and implemented by Jeff Mandell, with advice from Michael Walker and Stephan Sanders.
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