Team:Goettingen/Project/Bioinformatical Tool

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Revision as of 12:16, 12 August 2012

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Link List of Bioinformatical Tools

Here, an useful link list of computational and bioinformatical tools is provided. All of these programs are regularly used by the students to do the cloning process.

A plasmid Editor ApE - Use of ApE
Blasting of Sequences - BLAST: Basic Local Alignment Search Tool of NCBI
T_m calculator - Calculator for Primer Melting Temperature
Double Digest Finder - Double Digest Finder of Thermo Scientific
Enzyme Finder - Enzyme Finder of NEB
NEBcutter V2.0 - NEBcutter V2.0
ORF Finder - ORF Finder of NCBI
RE Base - The Restriction Enzyme Database RE Base of NEB

A plasmid Editor ApE

The plasmid Editor ApE by M. Wayne Davis is a free ware program conceived for both Windows (XP, Vista and 7) and Mac (OS X v10.5 and above). It can be downloaded from http://biologylabs.utah.edu/jorgensen/wayned/ape/], [06/29/2012]. The program offers lots of applications required for cloning processes, e.g. construction of plasmid maps, primer design, sequence alignments, management of sequences, ORF finder, Tm calculator, translation of nucleotide sequences and a lot more. ApE is compatible for the handling of common sequencing formatted .seq and .ab1 files. For further information visit the ApE homepage. Moreover, the hoster´s of the program take care about an ApE Wiki, [08/01/2012] where you can find help and advice if questions in the use of ApE pop up.

Primer Design with ApE

Primers depend mainly on the chosen criteria. Yet, the specifity and the tendency to form hair-pins drastically reduces straight forward PCR amplification of genes. ApE also offers a primer design feature.

Highlight the sequence to which the primer should bind. Recognize that given standard minimum and maximum length is 20 bp and 25 bp, respectively. Nevertheless, these options can be altered.

Select "Tools -> Find Primers". A new window Find primers will emerge offering lots of different manipulatable attributes.

Click "OK" to see the possible primers.

Primer Binding with ApE

[[File:ApE Primer01.jpg|thumb|250px|'''Fig. 1: ApE window for binding of primers''']] In this example, ApE is used to find the binding site on Lambda DNA for primer with a specific sequence.

Open ApE and paste the desired sequence, for which the primer is designated, in the sequence field (or open according .ape file, if made before.

Go to Edit>Find (or use Ctrl+F).

Paste the primer sequence and check "also find rev-com of string" (the primer may be identical to (a part of) the opposite DNA strand.) and click the "Find Next" button, as shown in Fig. 1.

The in blue highlighted sequence appears to be the desired primer sequence annealing to the template sequence. Check also if primer is indeed the reverse complement. Below the icon bar, one can find information about the length of the primer 30 nucleotides and the binding site.
For an illustrative description of each steps, please follow http://www.bioinformatics.nl/molbi/SCLResources/Bioinformatics.htm#Finding_primer_binding_sites_using_ApE; [06/29/2012] .

Find restriction sites and fragment lenghts with ApE

Start ApE and open desired sequence.

Go to the Enzyme Selector (or use Ctrl+E) and click on the wanted restriction enzymes; the amount of restriction sites will be indicated in brackets.

One can now continue with different options:

searching different restriction enzymes with particular properties applying the various buttons in the "Select panel",

or highlighting the restiction site within the given sequence by pressing "Highlight" button,

or simulating a restriction in silico by klicking on the "Digest" button.

A new window will appear showing the digestion results. Additional information about the different bands can be received by hovering the mouse arrow over the bands, map or text. A convenient way to see the respective sequence behind a digested fragment is by simply klicking on the fragment within the digest window. For a description with the use of pictures for each step, please follow http://www.bioinformatics.nl/molbi/SCLResources/ApE_and_lambda.htm; [06/29/2012].

Blasting Sequences

For blasting two Sequences:

Open two sequences on ApE.
Select "Tools -> Align Two Sequences".

For blasting multiple Sequences:

Open multiple sequences on ApE.
Select "Tools -> Align Sequences".

It is important to note that the opening chronology of the ApE files will matter in the order of the alignment sequences. Those will show up in according sequence from top to bottom. Mismatches will be highlighted in red color, whereas matches will use the respective nucleotide linked with a dash. By double-clicking on any base pair within the sequence alignment, the sequence corresponding to this location will appear in the sequence ApE window.

Finding the ORF

Under "ORFs -> Find Next (or Ctrl+>) / Find Previous (or Ctrl+<)" open reading frames, i. e sequences beginning with a start codon ATG and end with one of the stop codons, become visible.
These ORFs can be translated using "ORFs -> Selection Translate" for direct amino acid sequence in the current displayed selection or by creating a new window "ORFs (or Ctrl+T)-> Translate" listing the aa sequence and enabling to link the amino acids with the nucleotides, respectively. The translated sequence can be chosen in 1- or 3-letter code.

General Remarks

While copying features from one file to another, it is obligatory to have the file containing those features still open.
Addition of extra enzymes is possible only via the "Enzyme Editor (or Ctrl+E) -> Enzymes -> New Enzyme". To save these changes in the program folders ApE, use "Enzyme Editor (or Ctrl+E) -> File -> Save Current Enzymes as Default". For permanent saving exceeding new installing of the program, copy your Default file within the ApE program pathway. After installing paste this file in the corresponding folder.

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