FCH 532 Homework 3
1. Which of restriction enzymes listed in Table 5-4 is a blunt end cutter?
AluI , EcoRV, HaeIII, PvuII
(http://rebase.neb.com can be used to help you figure it out).
Which sets are isoschizomers
(enzymes with the same recognition sequence but do not necessarily cleave the same
sites) HpaII, MspI
and which are isocaudamers (enzymes that produce identical sticky ends)
BamHI, BglII
SalI, XhoI
TaqI, MspI, HpaII
2. Translate the following sequence to a polypeptide chain in three reading frames.
Show each of the reading frames relative to the nucleotide sequence.
5’-ATGTATACGAAGATTATTGGTACTGGCAGCTATCTGCCCGAACAAGTGC-3’
10
1
20
30
40
50
ATG TAT ACG AAG ATT ATT GGT ACT GGC AGC TAT CTG CCC GAA CAA GTG C
M
Y
T
K
I
I
G
T
G
S
Y
L
P
E
Q
V
C
I
R
R
L
L
V
L
A
A
I
C
P
N
K
C
V
Y
E
D
Y
W
Y
W
Q
L
S
A
R
T
S
3. Reconstruct the following plasmid from the fragment sizes:
Restriction enzyme
EcoRI
HindIII
SalI
EcoRI + HindIII
EcoRI + SalI
HindIII + SalI
Fragment sizes (kb)
5.4
2.1, 1.9, 1.4
5.4
2.1, 1.4, 1.3. 0.6
3.2, 2.2
1.9, 1.4, 1.2, 0.9
4. The plasmid pBR322 contains the ampR and tetR genes, which respectively confer
resistance to the antibiotics ampicillin and tetracycline. The tetR gene contains a
cleavage site for SalI, the only such site in the entire plasmid. Describe how one can
select for E. coli that had been transformed by pBR322 that contains a foreign DNA
insert into its SalI site.
First grow the colonies on a plate containing ampicillin to select for the plasmid.
Then, using a sterile velvet stamp, transfer the colonies to another plate containing
both ampicillin and tetracycline. Look for a difference between the colonies grown
on the plate with ampicillin alone and the plates with ampicilin and tetracycline.
The colonies that appear on the ampicillin plate but not on the amp/tet plate are
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strains where the pBR322 has a foreign insert in the SalI site. This interrupts the
tetracycline resistance gene and therefore the cells would be sensitive to tetracycline.
5. A protein segment of sequence Phe-Cys-Gly-Val-Leu-His-Lys-Met-Glu-Thr- is
encoded by the following DNA segment:
5’-TTC TGC GGA GTC CTA CAC AAG ATG GAG ACA-3’
5’-TTC TGC GGA GTC CTA CAC AAG ATG GAG ACA-3’
F
C
G
V
L
H
K
M
E
T
Design an 18-base oligonucleotide that could be used to change the protein’s Leu-His
segment to Ile-Pro via site-directed mutagenesis.
To change the amino acids, we need to change the codons encoding Leu and His to those
for Ile and Pro. We also want to do this by changing the fewest nucleotides possible.
5’-TTC TGC GGA GTC ATA CCC AAG ATG GAG ACA-3’
F
C
G
V
I
P
K
M
E
T
6. Define the following terms as they relate to biochemistry
a. RFLP-restriction fragment length polymorphism. Restriction enzyme digests of
the corresponding segments from homologous chromosomes contain fragments of
different lengths.
b. 3’ to 5’ exonuclease-This activity degrades the new synthesized 3’ end of a
daughter strand one nucleotide at a time to edit out mistakes that are sometimes
incorporated.
c. 5’ to 3’ exonuclease - This activity degrades up to 10 nucleotides from the 5’ end
of a single-strand nick.
d. primase- initiates DNA replication by producing an RNA primer on the template
DNA from which DNA polymerase may replicate DNA.
e. DNA polymerase I-removes RNA primers and replaces them with DNA (highly
important on the lagging strand).
f. DNA polymerase III- Synthesizes the leading strand of DNA and most of the
lagging strand.
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g. X-gala colorless
chemical that
can be
cleaved by
betagalactosidase.
When it is
cleaved the
insoluble
portion can
oxidize to
produce a
blue color.
Used in blue-white screening.
h. IPTG-(isopropylthiolgalactoside) a synthetic nonmetabolizable analog to allolactose
that can be used as an inducer to activate the lac operon by binding to the lac repressor
protein and inhibiting its binding to the operator sequence.
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i. Southern Blotting-Technique
This technique takes advantage of the fact
that nitrocellulose or nylon membranes can
bind to single stranded DNA after
treatment with a base (0.5 M NaOH) and
can be used to identify a specific DNA
fragment through use of a probe. The steps
are outlined in the following figure:
f. PCR-Polymerase chain reaction. Used
to amplify a piece of DNA through a series
of denaturation, annealing, and extension
steps using a specific DNA polymerase.
g. clone-a collection of identical
organisms or nucleic acid sequences
that are derived from a single ancestor.
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h. colony hybridization
i. genome- DNA sequence from one complete set of chromosomes
j. transgenic-Describing an organism that contains a segment of DNA containing gene
sequence that has been isolated from one organism and introduced into a different
organism.
k. relaxed control plasmid – 10-700 copies per cell. If protein synthesis is inhibited by
chloramphenicol, the plasmid will continue to replicate up to 2000-3000 copies per cell.
l. stringent control plasmid- one to a few copies per cell.
m. chromosome walking-When a DNA segment is too large to sequence in one piece, it
can be fragmented and cloned. The clone is picked and DNA insert is sequenced. A
small fragment of the insert near one end is subcloned (cloned from a clone) and used as
aprobe to select a clone containing an overlapping insert which is in turn sequenced. This
is repeated until the chroosome is sequenced.
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7. Describe the lac operon and how it works. Use figures and drawings to help explain
your answer. Why is an inducible system instrumental for blue-white screening?
lac repressor
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β-galactosidase
permase
transacetylase