Retrieving and Aligning Homologous Protein Sequences Using Blast and ClustalX
- Download ClustalX to your desktop.
- Open the Notepad file containing the Cytochrome C Oxidase III (COX3) sequence you saved in the previous exercise (ORF Finder).
- Copy and Paste the COX3 nucleotide sequence into ORF Finder, select the "Invertebrate Mitochondrial" genetic code, and click "ORF Find."
- Click on the long green open reading frame (+1). Imagine that this was the sequence you just received by email from the sequencing company... your immediate impulse would be to Blast it against the protein database to see if your reading frame was the protein you were looking for. ORF Finder has made that easy:
- Notice that the program is already set to Blastp (the amino acid sequence version of Blast) and that the Database is set to nr (non-redundant). Click BLAST.
- Go to Taxonomy Reports and save six or seven sequences into a single file that you might name Cox3_peps. I selected a couple of mollusks, a couple of echinoderms, a couple of mammals, and one polychaete.
- Go to your desktop and double-click ClustalX
- If you select FILE --> Load Sequences you should be able to navigate to your desktop and open the Cox3_peps file. Do a complete alignment.
- Identify the conserved regions... Our goal is to output a nice alignment and to highlight the portions coded for by the primers. How would you proceed?
