In silico PCR amplification


The aim of in silico PCR is to provide an easy way to obtain the theoretical PCR results we may expect from DNA, by using up to date sequenced bacterial genomes (including, optionally, plasmids when available).

TEMPLATE DNA

Template DNA will correspond to the sequenced bacterial DNA chosen in the form, and when available, plasmids may be included in the experiment. The genera will be chosen at PCR main page, and the species or strain will be chosen in the corresponding form.

When the bacterial species has more than one chromosome (p.e Agrobacterium tumefaciens, circular chromosome), both will be used. In the experiment bacterial chromosomic DNA will be considered as circular unless is it linear (p.e. Agrobacterium tumefaciens, linear chromosome). Plasmids will be always considered circular.

PRIMERS

During in silico experiments one or two primers may be used, which must be at least 10 nucleotides long (A+T+C+G must be at least 10). Degenerated nucleotides may be included in the sequence (letter codes must follow NC-UIBMB recommendations).

Primers must be introduced in the form in 5'-3' direction, and the theoretical amplification will consider all possible combinations which allow amplification (primer 1 to primer 1, primer 2 to primer 2, primer 1 to primer 2 and primer 2 to primer 1).

In all cases it will be considered primers have been design correctly, so they will not form dimmers, hairpin or any other aberrant results.

Maximum number of mismatches allowed (recognition errors between primers and DNA template) is two when only two primers are used (mismatches are limited to one when primers are inputed in fasta format). As a consequence, the theoretical experiment will not be totally stringent. Even this way, in silico PCR amplification may be considered as very restrictive (Sommer Tautz got amplification with 17 nucleotides long primers under non-stringent conditions with nine mismatches). We will try to increase the number of mismatches in the future (up to 4 mismatches for 20 mer primers is our goal).

The length of amplicons may be up to 10000 bp, and it can be customized. We have considered 3000 bp as a default value. The selected length includes primers.

RESULTS

The results page will show the starting position of the amplicon in the chromosome or plasmid and the length of each amplicon. Amplicons obtained in each chromosome or plasmid will be shown separately in tables.

Amplicons will also be shown in a picture which includes a 100 bp ladder. On the right of the picture the sorted length of all amplicons will be shown.

When clicking the starting position of amplicons in the tables, the following information will be provided:

  • Source of DNA, and starting and ending positions of amplicons
  • DNA sequence of amplicons
  • List of ORF to which the amplicons belong to.

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