October 6, 2004
INTRODUCTION TO ANALYZING SEQUENCES:
A) Finding sequences in the database:
To begin our sequence analysis, let’s first look at a sequence entry from Genbank.
First go to the following webpage: http://www.ncbi.nlm.nih.gov/. This is a website
funded by the human genome project that maintains databases of sequences and
provides support programs for accessing these sequences. One very useful tool here
is PUBMED, which allows you to search the literature for any area or author you are
interested in. To use PUBMED, click next to the left SEARCH button and choose
the PUBMED option from the pulldown menu. Type in a name or subject and test it
out.
Back to our task. Now at that same SEARCH button, click on nucleotide.
Enter CB092467, the entry number for the cycad clone we are working with. Take a
look at the entry-it tells you about the tissue the ESTs were made from, the way the
cDNA was isolated, and the cloning vector. Copy the DNA sequence and let’s try a
few tests of the sequence.
B) ORFS
Now let’s see of there are any ORFs (open reading frames) encoded within this DNA.
Go to the following website: http://www.ncbi.nlm.nih.gov/gorf/gorf.html and then
paste the sequence in the big box. Push the button that says ORFfind and wait a few
seconds and your results will arrive. The largest reading frames will be in green and
ranked on the right side. 1) What is the longest reading frame (which frame and how
many base pairs of DNA)? 2) How do you think this is defined? 3) How many
amino acids is that? Now push the button that says SIX FRAMES and you will see
the positions of the stop codons and start codons. Look at your figures again. 4) How
long is the longest open reading frame?
C) DOMAINS
Now let’s look at protein domains encoded by this short EST. Go to EXPASY: http://
us.expasy.org/ to first translate your sequence. Under Tools and software packages
on the right side go to DNA to PROTEIN-translate. Then click on translate at the top
of the page. Paste your sequence in and push the translate box. Wait a few seconds
for the results. Which is the best ORF? (5) If you click on the strand and reading
frame entry in blue, you will get a 1 letter code for that sequence. Copy that and go
back to the EXPASY page: http://us.expasy.org/ or EXPASY HOME. Now go to the
left hand column and click on PROSITE, the second column on the left. Paste your
amino acid sequence in the box under TOOLS FOR PROSITE. Click on quick scan.
This will take a few minutes. 6) What domain is identified by PROSITE and
