In silico simulation of
Terminal Restriction Fragment Length Polymorphism (T-RFLP)
This script is an implementacion of in silico PCR-RFLP simulation tool.
In order to undertand how this tool works, we will simulate the following experiment:
The selected primers will amplify a 328 bp fragment from 16S ribosomal RNA.
In E. coli genome there are 7 copies of the target sequence, so 7 amplicons will be obtained. .
One primer (in red) is labelled, and the other one is not.
In this simulation tool, both bands may be labelled.
Endonuclease restriction of amplicons and detection on fragments
One or two endonucleases may be used in the experiment.
In this experiment only one endonuclease is used:
BspLI cuts at GGN'NCC and yields blunt ends.
BspLI cleaves within one of the amplified bands, yielding two fragments of 136 and 191bp.
All 328 bands and the 191 bp band are labelled.
In the results page we will get a table like the one below:
Position means the exact nucleotide within genome wheare the amplicon starts
Length of amplicon is the length of the PCR productCleavage positions within amplicon show the exact position where restriction takes place. In this case, endonuclease BspLI cleaves the third amplicon in the list at positions 136 and 137 (overlapping cleavage sites are present). Althought fragments of sizes 136, 137, 191 and 192 will be yielded, the script will only computed bands of lengths 136 and 191 for simulation of the band patterns below.
Fragment length is the length of each fragment. When the amplicon is not restricted the total length of the amplicon is shown
Red fragment length: those are the labelled fragments (the ones containing the labelled primer)
Simulation of electrophoretic band pattern
Although detection of bands will often be performed by capillary electrophoresis, we will use simulated band patterns in a regular gel.
Only labelled fragments are shown.
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