Team:UTP-Software/SoftwareTool

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== Project Details ==
== Project Details ==
==== S<sup>2</sup>MT in detail ====
==== S<sup>2</sup>MT in detail ====
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Our software was implemented using MATLAB because it gives the user plenty of tools that he may use after, to continue working with the desired DNA sequence.
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The algorithm developed does three main things:
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<li> First, the user introduces the DNA sequence or link to the BioBrick in the part registry. And the program scans the desired sequence to look for the restriction sites within the assembly standards that are used in the iGEM competition. </li>
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<li> Then the user is prompt to specify in case of incompatibility with the standards, which RFC he would like to fix. Once selected, the program designs the primers required for the Site directed mutagenesis procedure that will allow the sequence to be compatible with the standard.</li>
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<li> And last, if the program successfully created the required primer, it will print the sequence, giving the user the start position where that primer should be applied. </li>
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In some cases, it’s impossible to fix some sequences, because based on the protocol, the output primer may not comply with the steps that are listed in the “Protocol Followed to design the primers” section. Meaning that another standard should be selected.  </ul></div>
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==== Protocol Followed to design the primers:<sup>[1]</sup> ====
==== Protocol Followed to design the primers:<sup>[1]</sup> ====
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Revision as of 11:53, 26 September 2012

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Project Details

S2MT in detail

    Our software was implemented using MATLAB because it gives the user plenty of tools that he may use after, to continue working with the desired DNA sequence.

    The algorithm developed does three main things:

  • First, the user introduces the DNA sequence or link to the BioBrick in the part registry. And the program scans the desired sequence to look for the restriction sites within the assembly standards that are used in the iGEM competition.
  • Then the user is prompt to specify in case of incompatibility with the standards, which RFC he would like to fix. Once selected, the program designs the primers required for the Site directed mutagenesis procedure that will allow the sequence to be compatible with the standard.
  • And last, if the program successfully created the required primer, it will print the sequence, giving the user the start position where that primer should be applied.
    • In some cases, it’s impossible to fix some sequences, because based on the protocol, the output primer may not comply with the steps that are listed in the “Protocol Followed to design the primers” section. Meaning that another standard should be selected.

Protocol Followed to design the primers:[1]

  • Both the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid.
  • Primers should be between 25 and 45 bases in length, and the melting temperature (Tm) of the primers should be greater than or equal to 78°C. The following formula is commonly used for estimating the Tm of primers:
    • Tm = 81.5 + 0.41(%GC) − 675 / N  - where N is the primer length in base pairs.
  • The desired mutation (deletion or insertion) should be in the middle of the primer with ~10–15 bases of correct sequence on both sides.
  • The primers optimally should have a minimum GC content of 40% and should terminate in one or more C or G bases.
  • Primers need not be 5´ phosphorylated but must be purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Failure to purify the primers results in a significant decrease in mutation efficiency.
  • It is important to keep primer concentration in excess. Stratagene suggests varying the amount of template while keeping the concentration of the primer constantly in excess.

References

[1]. Stratagene. QuikChange™ Site-Directed Mutagenesis Kit. Catalog #200518. Revision #108005h. Available in: [http://web.physics.ucsb.edu/~deborah/pro/pro_pdf/Stratagene%20QuikChange.pdf].

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