CHG 4160 Assignment 2

Zachary Comeau, 6876641

Question 1:
a) 5ATGGCGAACCTTGGCTGGATG3
b) 3TACCGCTTGGAACCGACCTAC5
c) Transcription is the process whereby DNA is transcribed to RNA, a first step of
gene expression. RNA polymerase binds genomic DNA, with the assistance of
a variety of transcription factors, and transcribes the DNA into single
stranded RNA (with thymine being swapped for uracil). Further modifications
to the RNA may be performed in the nucleus including polyadenylation,
capping, and splicing. Now, a transcript of the gene in question the RNA exits
the nucleus to be translated into a protein.
mRNA transcript: 5AUGGCGAACCUUGGCUGGAUG3
d) Translation is the process whereby mRNA is translated, via codons, into an
amino acid sequence which, once processed, will become a functional
protein. A beginning step to this process is the assembly of ribosomes on the
mRNA at the start codon (DNA: ATG, RNA: AUG). The ribosomal subunits then
move along the mRNA reading each codon and attaching the corresponding
amino acid to a growing amino acid chain in a process known as elongation.
Once a stop codon is reached (UAA, UAG, or UGA) the chain of amino acids,
or polypeptide, is released for further processing (folding and etc. to
eventually become a protein) and the ribosomal subunits separate.
Amino acid sequence: Methionine Alanine Asparagine Leucine Glycine
Tryptophan Methionine
The stop codon used is UGA
e)

Question 3 (as theres no question 2 on the assignment handout):

a) pBluescript plasmid

b) Start with purified cell DNA, order oligos so I can perform PCR against my
gene of interest, amplify and purify my gene of interest. Find an appropriate
cut site on the plasmid that will allow for ligation of my gene of interest into
the plasmid. Cut the plasmid with (usually) two restriction enzymes, ligate in
my gene of interest, purify the resulting modified plasmid.
Use either chemically or electrically competent cells, heat shock them with
your plasmid, then grow them. Most plasmids will code for an antibiotic
resistance gene (commonly ampicillin). By growing transformed cells on the
appropriate antibiotic plate one can be sure that any colonies that grow will
have taken up the modified plasmid of interest.
c) GGGTAGAATTCAATCG
CCCATCTTAAGTTAGC

GGGTAG
CCCATCTTAA
GTTAGC

AATTCAATCG

Question 4:
One can either use oligo-nucleotides probes for cDNA clones, or anti-body probes.
The first method involves taking a known protein sequence, translating it into all
possible mRNA sequences and in turn generating radiolabeled DNA oligonucleotides
which will hybridize to the clone of interest and appear on X-ray film. The second
method involves transforming bacterial cells such that they express the protein of
interest, performing a protein-membrane absorption, and then probing the
membrane with a primary antibody (coded for the protein) and a secondary
antibody (coded for the primary antibody) which, upon binding of both, will either

produce a colored or fluorescent product, allowing for individual colonies of interest

to be identified.
Question 5:
Sequences (red represents the primers):
Forward Primer Sequence: AGTCAGTGGAACAAGCCGAG
Reverse Primer Sequence: CTGCCGAAATGTATGATGGGC
Primers bind to the red highlighted sequences
Sequence to be amplified (reverse primer binds)
5AGTCAGTGGAACAAGCCGAGTAAGCCAAAAACCAACATGAAGCACATGGCTGGTGCTGC
AGCAGCTGGGGCAGTGGTGGGGGGCCTTGGCGGCTACATGCTGGGAAGTGCCATGAGCA
GGCCCATCATACATTTCGGCAG3
Complementary sequence (forward primer binds)
5TGGGCCTGCTCATGGCACTTCCCAGCATGTAGCCGCCAAGGCCCCCCACCACTGCCCCA
GCTGCTGCAGCACCAGCCATGTGCTTCATGTTGGTTTTTGGCTTACTCGGCTTGTTCCACTGA
CT3
PCR steps:
DNA is heat denatured at 94-95C
Annealing occurs at about 68C. Primers bind to their respective sequences (red,
above)
Elongation is performed, at about 72C, as the DNA polymerase (commonly TAQ)
travels from the 5 -> 3 end of the primers, upon reaching the end of each primer
adding the appropriate nucleotides to create 2 (double stranded) strands of DNA
(from one).
The whole process is then repeated allowing for an exponential increase in the
amount of DNA strands.