DEPARTMENT OF CHEMICAL ENGINEERING

IIT BOMBAY
CL 662: Introduction to Computational Biology
Homework 1: Due on 18/01/2018; Online as PDF file.
Note: 1. Please provide concise answers. Do not submit pages after pages of output from
database searches. Provide brief summary and analysis of your search. 2. Make appropriate
assumptions and state them clearly. 3. Clearly explain the logic you used while answering
questions. No credit will be given otherwise. 4. All questions are compulsory. 5. Submit a
handwritten answer / printout as appropriate. 6. Copying will result in an FR grade.

1. The following DNA fragment was sequenced by the Sanger method. The asterisk
indicates a fluorescent label.
*5'----------3'-OH
3'----------ATTACGCAAGGACATTAGAC-5'
A sample of the DNA was reacted with DNA polymerase and each of the nucleotide
mixtures (in an appropriate buffer) listed below. Dideoxynucleotides (ddNTPs) were
added in relatively small amounts.
1. dATP, dTTP, dCTP, dGTP, ddTTP
2. dATP, dTTP, dCTP, dGTP, ddGTP
3. dATP, dCTP, dGTP, ddTTP
4. dATP, dTTP, dCTP, dGTP
The resulting DNA was separated by electrophoresis on
an agarose gel, and the fluorescent bands on the gel
were located. The band pattern resulting from
nucleotide mixture 1 is shown below. Assuming that all
mixtures were run on the same gel, what did the
remaining lanes of the gel look like?

2. DNA Cloning: The plasmid cloning vector pBR322 (See figure below) is cleaved with
the restriction endonuclease PstI. An isolated
DNA fragment from a eukaryotic genome (also
produced by PstI cleavage) is added to the
prepared vector and ligated. The mixture of
ligated DNA is then used to transform bacteria
and plasmid-containing bacteria are selected by
growth in the presence of tetracycline.

(a) In addition to the desired recombinant

plasmid, what other type of plasmids might be
found among the transformed bacteria that are
tetracycline-resistant? How can the types be
distinguished?

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(b) The cloned DNA fragment is 1,000 bp long
and has an EcoRI site 250 bp form one
end. Three different recombinant
plasmids are cleaved with EcoRI and
analyzed by gel electrophoresis, giving
the patterns shown below. What does
each pattern say about the cloned DNA?
Note that in pBR322, the PstI and EcoRI
restriction sites are about 750 bp apart.
The entire plasmid with no cloned inset is
4,361 bp. Size markers in lane 4 have the
number of nucleotides noted.

3. Visit the NCBI (or JGI) genome page for a cyanobacteria of your choice. A link has
been given in the list itself and it should work. Else, you can search on
Cyanobase. How many chromosomes (circular and linear) and plasmids are
there in this organism? See the cartoon below for the instructor's organism
Cyanothece sp ATCC 51142 as an example. Provide a statistics on genome size,
number of protein coding genes, rRNA and tRNA coding genes in this organism
(on chromosomes, plasmids, etc).

4. Extract and report the 16S rRNA (or rDNA) sequence for your organism.
5. What is the significance of 16S rRNA sequence in bioiformatic analysis?
6. BLAST the 16S rRNA sequence with the NCBI DNA sequence database and report
the 10 closest neighbours of your organism with score and E-value. Explore the

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 BLAST page, its tutorial, options, etc (you don't have to write anything on this in
the assignment).
7. Report the codon usage table for your organism. If you are curious, you can also
look up codon usage for another organism and look for differences.
8. Go to the protein table of your organism and get a sense of what percent of
genes are assigned functions and what fraction are termed as hypothetical
proteins?

*********Paper Ends*********