Scenario
3: Comparison of protein sequence against database
How to Blast the DG47 DNA sequence against the lef
gene
Two stages: (1) Obtain the DNA sequence of lef
from Entrez and (2) compare DG47 to that sequence using pairwise Blast.
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| Go to http://www.ncbi.nlm.nih.gov/ | This is the National Center for Biological Information (NCBI) site, home of Blast and much else. Why not bookmark it? |
| Click on the Entrez on the blue bar near the top | Entrez is a tool to download individual sequences held in GenBank |
| Click in the Search box (upper left) and select nucleotide | GenBank has many types of sequence. For example, it has the protein sequence for the lef gene, which you don't want. |
| In the for box (next to the Search box) and type in lef lethal factor, and click the GO button. | You might also type in M29081, the GenBank accession number, which you could have obtained from the BlastP search you did earlier. You could even have gotten to the sequence directly by clicking on the link embedded in the BlastP search results. |
| Scan down until you see ...(lef) gene, complete cds. It will be accession number M29081. | Don't be waylaid by the many other sequences -- partial sequences and bigger sequences that happen to contain lef. Complete cds means the accession contains the complete coding sequence. |
| Save the file and extract the DNA sequence to make a new file in FastA format. | Do this by hand in Word (or similar) or modify the program you wrote in response to Problem Set 2P, problem 6. |
| Return to the NCBI home page | |
| Click on the Blast on the blue bar near the top | |
| Scroll down to Pairwise BLAST and click in the BLAST 2 Sequences | This version of Blast allows you to choose the flavor of blast and the sequences that will be compared. You could also do this on your own computer, since you've downloaded Blast. |
| Paste the DG47 DNA sequence into the first Search box. If you don't have the sequence, get it now by clicking here | Copy and paste the entire file, including the FastA header (beginning >), since the header is used by Blast as a label. |
| Paste the lef DNA sequence into the second Search box. | You got the lef sequence five steps back. Again, copy the FastA header as well. |
| Make sure that blastn is showing in the Program box, otherwise click on the box and select blastn. | BlastN compares two nucleotide sequences. |
| Click the Format button. | This will open up a new window, which will refresh itself until Blast is ready with the output.. |
| When the output arrives, scroll down to the box below Distribution of n Blast Hits... for results in graphical form and further down for results in text form. | Same graphical and text formats as with BlastN. |
| Congratulate self | You're done! |