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Your Name: __________________
Bio499 Bioinformatics
Homework Assignment for DNA Sequence Analysis (week 3&4)
Due October 22, 2001
SNAP-25 is a neuron-specific protein that involves in exocytosis of neurotransmitters.
You are a student working on a project to compare SNAP-25 from various organisms.
By accident, you lost the label of a tube containing a SNAP-25 cDNA clone from one of
the five organisms you are working with. You know your professor will be upset when
he finds out this. So you decide to do a quick DNA sequencing analysis to identify this
clone. Both strands of DNA are sequenced and you know that the forward strand will
read the sense strand of your cDNA clone.
Please click my link under “Homework sequences” to retrieve a pair of sequences: the
forward primer will sequence the sense strand and the reverse primer will sequence the
other strand. I will assign you a number during the lecture to work on one of the 4 pairs
of sequences listed.
Please write your assigned number here: _________
1. Copy your two sequences to Biology Workbench and create a file for each sequence.
2. A typical DNA sequencing reaction can only read as far as 400-500 bases from a
primer. Since SNAP-25 coding sequence is more than 600 bp long, you will need to
use sequences from both primers to assemble the final DNA sequence in your clone.
In order to merge the two sequences, you will need to perform reverse complement
on the sequence from _________ primer (choose one: forward or reverse). Use
Workbench to ‘reverse and complement’ and then save the sequence.
3. Use “Merger” from my link to merge the two sequences. Remember one of the
sequences has to be reverse-complement so both DNAs are in the same orientation.
Use the merger.out to view if the two sequences are merged successfully (print), then
retrieve the merged sequence from ‘outseq’. Please copy the sequence and save in
Workbench as ‘Merged unknown SNAP-25’.
4. In Workbench, prepare a restriction enzyme analysis of the ‘merged unknown SNAP25’. Print a restriction map with 6+ restriction enzyme, a list of enzymes that do not
cut, and the total number of hits per enzyme. Minimize all other report outputs.
After obtain a print of the restriction map, use the same analysis tool to translate all 6
frames. . Identify where the open reading frame is (ORF). You may also use the
GeneScan program in my web linkage to identify the exact gene.


The open reading frame starts at nucleotide # ________
and ends at nucleotide # ______
Take a closer look at your restriction enzyme map printout, choose a pair of
enzymes that will help you sub-clone the whole ORF of the ‘Merged SNAP-25’
sequence.____________________________________ (3 pts)
Lin
Cal Poly Pomona
10/12/2001
1
Your Name: __________________
5. Perform Biology Workbench/Primer3 on your ‘Merged unknown SNAP-25’
sequence. Select a PCR primer pair that will amplify the whole region including the
complete ORF. You may need to limit the regions of your forward and reverse
primer search. Use default set up for the rest of the parameters. Print a copy of your
PCR primer report with the primer pair highlighted with a marker pen.

What is the melting temperature of your primer as determined by this program?
Forward _____C Reverse _____C

What is the molecular weight of your primers? (Use other tools if needed)
Forward _________ Reverse _________

Base on the Tm of your selected PCR primer pair, which of the following
temperature should be used for your PCR cycle? (circle one, 3 pts)
a) 96C 1 min, 45C 1 min, 72C 1 min
b) 96C 1 min, 55C 1 min, 72C 1 min
c) 96C 1 min, 65C 1 min, 72C 1 min
d) 96C 1 min, 75C 1 min, 72C 1 min
6. Cut and past the CDS (i.e. ORF) of ‘Merged Unknown SNAP-25’ to a new file
named ‘Merged Unknown SNAP-25 CDS’.

What is the %GC for this SNAP-25 CDS? ____________

How many bp in this ORF? _____________________
7. Translate the ‘Merged Unknown SNAP-25 CDS’ under the restriction enzyme
analysis using only frame 1. Remove all other report outputs by selecting 0 or none
or restriction enzymes of 8+. Print the report with the DNA and protein sequence
aligned together (you may try to use other translation program as long as the report
can lay out the translated protein sequence under their DNA sequence). Highlight the
start and stop codon in this report.
8. Now, use Entrez to retrieve known SNAP-25 CDS’s for 5 organisms: human, mouse,
rat, Drosophila and zebrafish. There are sub-group of SNAP-25 called SNAP-25A or
25 B or, 23. Try to use the GenBank files with just SNAP-25 complete mRNA or
cDNA. Copy their CDS sequence to Biology Workbench and save them.
What are their scientific names?
Human ______________________
Mouse _______________________
Rat __________________________
Zebrafish _____________________
Lin
Cal Poly Pomona
10/12/2001
2
Your Name: __________________
9. In Biology Workbench, perform ClustalW on the known SNAP-25s from the 5
different organisms and your ‘Merged Unknown SNAP-25 CDS’. Print a copy of
your ClustalW report that will show the rooted tree of your sequences. Please label
the common organism names in your tree if it is not clearly labeled.

Your mystery tube is likely to be a SNAP-25 cDNA clone from:
__________________(which organism, 3 pts)
10. Perform Blast 2 analysis in Biology Workbench to align the sequences from your
‘Merged Unknown SNAP-25 CDS’ and the closest related SNAP-25 CDS (from the
organism you choose in question 9). These two closely related sequences should
align very easily. Occasionally, there may be places that exhibit polymorphisms
(SNPs) or point mutations.
 If your gene is different from the GenBank sequence, described the location (nt#)
and base changes _____________________ (e.g. nt 100, A G)
 What is the nature of this point mutation: ___________________ (transition or
transversion)
Use your translated CDS report from Question 7 and the genetic code table to answer
the following questions.


Does this point mutation result in silent, missense or nonsense mutation when
translated? ____________
What is the corresponding amino acid in the GenBank sequence _________; and
the amino acid in your clone ___________ (in the one-letter abbreviation).
Please print your Blast analysis result and highlight the mismatched bases.
Check list of your report:
 This 3-page homework instruction; all question answered.
 The merger.out report (Q3)
 Restriction enzyme map (Q4)
 DNA sequence with PCR primers marked (Q5)
 CDS with translation (Q7)
 ClustalW phylogenetic report (Q9)
 Blast alignment analysis (Q10)
Grading:
 Six analysis reports: 5 points each; please follow the instruction for detail layout
requirement for each report.
 2 points for each correct answer in this homework instruction; a few answers may
cost 3 points as indicated next to the questions.
Lin
Cal Poly Pomona
10/12/2001
3