In silico simulation of DNA fingerprinting

This tool will simulate the following general procedure:
  • Several labelled DNA fragments of a target sequence are obtained. It will be the probe. A typical target sequence used in this kind of experiment is ribosomal DNA. Labelling methods may be radioactive (32P) or not (biotin, digoxigenin).
  • Genomic DNA from a microorganism is extracted. 
  • DNA is endonuclease digested with one or two enzymes.
  • Fragments will be separated by electrophoresis and transferred to suitable media. Some of these fragments will contain part or a complete target sequence .
  • Labelled DNA (the probe) will be hybridized to electrophoretically separated fragments.
  • Hybridized bands will be detected
Different species or strains may yield different band patterns, and this data may be used for identification and epidemiology.

Selection of genome
When simulating this technique, in the first step we will be required to select a prokaryotic genera. After this selection, it is required to select a genome sequence, and other parameters for simulation of the experiment..

Design of probes: target sequence
Target sequence is defined in this tool as the DNA sequence we have obtained several copies from. The DNA copies will be labelled, and they will be later used for hybridization againts a endonuclease digested DNA fragments from the sample. These labelled copies are the probes.

This tool allows two procedures to define the target sequence:
  • By PCR: the target sequence will be PCR amplified, and during amplification or after it, the copies of the target sequences will be labelled. Different strategies may be used to label the PCR fragments: usa of at least one labelled primer, addition of labelled nucleotides during polymerization or labelling amplicons after amplification. Labelling of DNA may be by radioactive or non-radioactive methods.
Obtaining a labelled probe by PCR

  • By name: this alternative makes it easier to find the gene we want to use in the experiment. After introducing the name of the gene (e.g. 16S RNA), a list of matching ORF or RNA names will be returned.

    The list of genes also indicates the starting and ending position of the sequence within the genome. If the checkbox on the left is selected, the sequence defined between start and end positions will be considered as a target sequence (so that probes will hybridize to it later in the experiment). The starting and ending positions may be customised.

Complete restriction digest of genome and electrophoresis of fragments
In this step, DNA from the bacterial genome is extracted and restricted with an endonuclease. The form allows selecting one endonuclease from a list of all commercially available endonucleases.

After digestion, the sequence with capacity to hybridize with the probe (the target sequence) will be included in a unique fragment or in several ones. In the image below the target sequence (blue) is separated into 3 fragments of different sizes.

Restriction and electrophoresis of DNA

Hybridization with the probe and detection of bands
Electophoretically separated DNA fragments are hybridized with previously developped probe (red). In this example, only 3 fragments will hybridize with the probe. Detection of bands will depend on labelling technique.

Hibridazation with labelled probe

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