UserGuide:ePCR

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ePCR

The ePCR search page serves two functions: (1). Find coordinates in the genome that can theoretically produce a PCR product given a pair of primer sequences, and (2). Find the smallest encompassing genomic object (explained below) and intersecting genes given a genomic position. Function (A) Search Type I:

I_ePCR1.jpg

Step 1: The user provides a list of assays, each row with 4 columns: assay_name, sequence1, sequence2, predicted_product_length. The columns should be separated by one or more blank space. You may not know the product length a priori. Just give a generic number, say 2000, and set the tolerance liberally in Step3 and/or adjust after some trials.

Step 2: After pasting the list in the Text box, select "This is a <>List of primer pairs".

Step 3: Enter in base-pairs how much the product size can be tolerated to differ from that specified in column 4, in "Allow product length difference of: bp". The user may tune this parameter according to trial results.

Step 4: Choose the number (0-4) in nucleotides of primer sequence mismatch that can be tolerated with "Allow oligo mismatches of: bp".

Step 5: Choose the format of the results; HTML is recommended for most users.

Step 6: Click on "Run e-PCR". Search Type II:

I_ePCR2.jpg

Step 1: The user provides a list of assay names (in single column format), that already exist in WormBase and can be searched as "Primer Pair".

Step 2: Select "This is a <>List of assay names".

Step 3-6: Same as in Search Type I.

Function (B) Search Type III:

I_ePCR3.jpg

Step 1: The User provides a 2 column list of Names and Positions (a pair of chromosomal positions).

Step 2: Click on "This is a <>List of positions".

Step 3-6: Same as in Search Type I. Result Page

The result is a list of 7 columns.

I_ePCR4.jpg

Column 1: Assay, assay name.

Column 2: Chromosomal Pos, the chromosome, beginning and ending nucleotide coordinates for the predicted 'PCR' product. For each assay, there may be more than one product.

Column 3: Genbank Pos, the GenBank accession number for the smallest enclosing genomic sequence object and the corresponding nucleotide coordinates for the PCR product. The GenBank accession may not precisely correspond to the insert of a physical clone in that object. Click on the hyper link for details at NCBI.

Column 4: Cosmid/Link Pos, the smallest enclosing cosmid or sequence link object and the corresponding nucleotide coordinates for the PCR product in that object. Again, these may not precisely correspond to the insert of a physical clone.

Column 5: Genes Covered, (predicted) gene that intersects with the PCR product. There can be more than one gene per PCR product.

Column 6: Exons Covered, those exons in the gene that intersect with the PCR product.

Column 7: Exons in Gene, the total number of exons in the gene that intersects with the PCR product.

More information on the e-PCR procedure can be found in: Schuler GD. Sequence mapping by electronic PCR. Genome Res 1997 May;7(5):541-50.