Provided here is a tool for sequence annotation directed PCR primer design targeting C. elegans promoter regions for the construction of promoter::GFP constructs using fusion PCR (PCR stitching). The design process is automated--only the gene name is required as input. Basic and advanced design parameters may be adjusted, and an optional e-PCR test is available to check primer specificity against the whole C. elegans genome.

• Gene : Enter the gene name for the promoter of interest, as found in Wormbase (under "Gene Name,Cosmid"). If the three-letter locus name is only known, check WormBase for the cosmid gene name.

• Number or primer sets : Number of external primer sets to be designed. Primers are listed by descending Primer Quality (Q value).

• PCR product size range : Primers will be designed to amplify a product (external, first round PCR) near the specified size range. Maximum size is 5 kb. By default, product size is reduced to avoid amplifying part of an upstream gene on the same strand. See Advanced Design Parameters below.

• Max. Q Value : This is a PRIMER3 penalty score that indicates deviation from the specified optimal design parameters. Lower penalty score indicates a better primer pair. Deviations from the optimum primer size and Tm have a large influence on the penalty score. To read more about PRIMER3 and penalty score, follow the link below.

• Allow primers in upstream neighbors : PCR product size may be reduced if a genes is found upstream, coding on the same strand (see Example output). Allowing primers in upstream neighbors forces a PCR product of the specified size, regardless of the presence of an upstream gene coding on the same strand.

• Number of nested sets : The construction of a promoter::GFP fusion using PCR stitching requires the design of primers for two rounds of PCR.
  
  

  Round 1: Design a pair of external primers to amplify a putative promoter region (the external amplicon)

  
  

  Round 2: Design a nested 5' primer for PCR fusion of the amplified GFP and external amplicon.

  
  
  To create a series of promoter::GFP fusions with progressively smaller regions within the external amplicon (omitting regions at the 5' end of the external amplicon), multiple nested primers can be designed. Each fusion PCR would then involve amplifying the putative promoter region using the external primers, followed by a different nested primer in the second round PCR. Primer separation size can be specified (see below).

• Primer separation : Specifiy the number of bases between the external primer and nested primer, or between sets of nested primers. In the example above all forward A primers are separated by approximately 150 bp.

• Default is 'ON'; this is slower but this test is recommended to avoid unexpected PCR prodcuts. External primers are checked against the whole C. elegans genome and nested primers are checked against the amplicon produced by the external primers. By default, e-PCR will search for products within 2000 bp of the expected product size. The stringency of the e-PCR check can be adjusted by changing the word size and allowed mismatches.

• Output from PCR fusion primer design : Gene information (location, coordinates, size, orientation), the location of the nearest upstream neighbor, and any adjustment to the requested external amplicon size (eg. to avoid an upstream neighbor) are indicated in the gene information section. A map shows the gene of interest in the context of surrounding genes. The primer design results table shows the primer sequences and coordinates, primer pair quality, primer Tm, and the expected PCR product size. If the e-PCR check is requested, the PCR product size(s) displayed will be the results from the e-PCR check. A primer map shows the location of the primers.

• Click on the image below to see the output generated from the request below.

• Primer3 documentation: WormPrimer uses Primer3 software (Rozen and Skaletsky, 1996, 1997, 1998) to design and pick primers. For a detailed description of Primer3 click here.

• e-PCR: Read more about electronic PCR (Schuler, 1997).