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Electronic PCR calling parameters

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Sequence input

You may either enter sequences (in FASTA format) or enter GenBank/EMBL/DDBJ Accession numbers into the text box.


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STS database: all markers/non-repetitive markers

The search is performed in the UniSTS database. If the non-repetitive option is selected, markers that satisfy one of the following criteria are excluded from search list:
  • marker maps to >1 chromosome
  • or hits >1 LocusLink
  • or hits >2 places on the contig(s)
  • or hits >1 draft genomic sequences


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Word hashing: size and contiguity

The database is searched using hashing of wordsize consecutive nucleotides on the 3' end of each primer. Wordsize is usually a trade off for search speed and sensitivity (see alignment quality): the longer the words are, the faster search is, the shorter the words are, the less is a probability to miss a hit.

Using discontiguios words improves sencitivity by allowing mismatches within word boundary, but also significantly increases search time.



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Primer alignment quality

Alignment quality is controlled by number of mismatches and number of gaps allowed in each primer sequence. The higher these numbers are, the more potential STSs will be found, but more false hits are possible. Reasonable values are "1 mismatch" and "1 gap".

Neither mismatches nor gaps are allowed within wordsize nucleotides from the 3' end of the primer, except mismatches in case of discontiguous words.



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STS product size deviation

Setting this value to non-zero allows the detection of hits with a size that is out of the previously reported (expected) PCR product size range.



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STS input

Depending on tab selected (try Example button after you select the dataset):
Text input
Four-column format, exactly one STS per line, columns are: label for sts, left primer, right primer, and product size (can be two dash-separated numbers for range of sizes).
Table input
Just fill in fields of form according to column names
UniSTS input
Type in Genbank accession numbers or integer UniSTS IDs
Note that if you do not set the product size, the default range of 100-350 nucleotides is used.


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