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.
  1. 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.
  1. 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 SNAP-25’. 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)

  1. 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

  1. 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? ______

  1. 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.
  1. 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 ______

  1. 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)

  1. 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, Aà 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:

q  This 3-page homework instruction; all question answered.

q  The merger.out report (Q3)

q  Restriction enzyme map (Q4)

q  DNA sequence with PCR primers marked (Q5)

q  CDS with translation (Q7)

q  ClustalW phylogenetic report (Q9)

q  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 1