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Friday, September 20, 2013

Sequence Alignment and Primer Designing Using Bioedit


BioEdit is a biological sequence alignment editor written for Windows 95/98/NT/2000/XP/7. An intuitive multiple document interface with convenient features makes alignment and manipulation of sequences relatively easy on your desktop computer. Several sequence manipulation and analysis options and links to external analysis programs facilitate a working environment which allows you to view and manipulate sequences with simple point-and-click operations.




BioEdit's features include:
  • Several modes of hand alignment
  • Automated ClustalW alignment
  • Automated Blast searches (local and WWW)
  • Plasmid drawing and annotation
  • Accessory application configuration
  • Restriction mapping 
  • RNA comparative analysis tools
  • Graphical matrix data viewing tools
  • Shaded alignment figures
  • Translation-based nucleic acid alignment
  • ABI trace viewing, editing and printing

Let's see how to do a sequence alignment using bioedit software....

First thing you have to do is to get the sequences of your interest, it can be whole genome sequence or particular gene.This can be retrieved from NCBI (National Center for Biotechnology Information) .

For Example let's search for Haemagluttin gene (HA) of H1N1

You can save the selected gene sequence in FASTA format, which can be used in Bioedit software.


You can import multiple sequence files into one window
File > New Alignment. File> Import> Sequence alignment file> choose text file that save the FASTA sequences.

Once all the sequence have been selected you can run Clustal W from the Accessory application menu

choose all sequence> Accessory Application> ClastalW Multiple alignment> Run ClastalW> OK> Alignment> Find Conserved Regions> Start




The result will show how many conserved regions found and details of each region. Then choose one region as the template

 


Then use the chosen one region as template for real-time PCR primer and probe design. Before the region is used as template, checking specificity of this region by alignment of this region is required by using “Nucleotide BLAST”

 

Conserved regions are important because these regions will have least mutations, so these are ideal region for designing primers and probe for qPCR.

Primers can be Picked up from the conserved region using one of the many softtwares, to mention few

  • Primer 3
  • Primer Premier
  • NCBI
  • Oligo Designer, etc
References and Links:

Download BioEdit
BioEdit Help Files