BN 212: MOLECULAR BIOTECHNOLOGY
BN 212:
MOLECULAR BIOTECHNOLOGY
FINAL EXAMINATION: 2011/2012
DATE: 31ST January 2012
TIME:
14:00-16:00h
INSTRUCTIONS: ANSWER ALL
QUESTIONS FROM SECTION A AND ANY
TWO QUESTIONS FROM SECTION B (TOTAL 60 MARKS)
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SECTION
A (30 MARKS)
1)
In the DNA
sequence below, the positions of two oligonucleotides are marked:
a)
What is the
melting temperature (Tm) for each of these oligonucleotides?
The formulas used is Tm = 4(G+C) + 2(A+T)
For the left hand primer, Tm =
4(8+3) + 2(1+7)
=44+
16
=
600C
For the right hand primer, Tm =
4(4+5) + 2(5+7)
=36 +24
=600C
b)
What length would the polymerase chain reaction product primed by these
two oligonucleotides be? (6 marks)
2)
Briefly explain how bacterial cells can be made competent for a
transformation experiment.
Ø Electroporation;
An alternative to chemical
treatment of bacterial cell to
make competent cells is called electroporation. In this process the bacteria
are harvested and washed as before but in cold distilled water or a buffer with
a very low ionic strength. A small sample of a dense suspension of these
bacteria is then
mixed with the DNA in a special
cuvette and a short pulse of a very high voltage current is passed through the
bacterial suspension. Again, it is not entirely clear why this technique works
but it is thought that the high voltage pulse makes the bacterial membrane more
permeable and possibly moves the DNA into the bacterium by a process akin to
electrophoresis (Section 3.11). Electroporation is a much more efficient
process than chemical transformation and it can be used with bacteria other
than E. coli. Electroporation can also be used to transform
mammalian and plant cells
Ø Chemical
treatment;
To prepare competent E. coli a
culture is grown and then harvested when it is in log phase, at which stage the
bacteria are dividing rapidly. The cells are harvested by centrifugation and washed
several times in a chilled buffer containing divalent cations, typically CaCl2.
The bacteria are finally suspended in a small volume of the buffer so that they
are present at a high density. To introduce DNA (for instance, a recombinant
plasmid molecule) into these cells, a small sample of this suspension is mixed
and incubated on ice with the ligation mixture; it is then heat-shocked at 42°C
for about
1 min. This technique has been
widely used for many years, although the precise mechanism by which it causes
E. coli cells to take up DNA is only now becoming clear.
Ø Gene
gun;
One of the most effective
techniques is to shoot microprojectiles coated with DNA into plant and animal
cells. The gene gun, first developed at Cornell University, operates somewhat like
a shotgun. A blast of compressed gas shoots a spray of DNAcoated metallic
microprojectiles into the cells. The device has been used to transform corn and
produce fertile corn plants bearing foreign genes. Other guns use either
electrical discharges or high-pressure gas to propel the DNA-coated
projectiles. These guns are sometimes called biolistic devices, a name derived
from biological and ballistic.
3)
Why are the following steps important in a cloning experiment?
a)
Inactivation of restriction enzymes before adding the DNA ligase.
Ø In order to avoid the cleaving the ligated sites
that are the ligation between the plasmid and the new strand as the R.E are
responsible.
b)
Inactivation of alkaline phosphatase before mixing the vector and the
fragment insert.
Ø To avoid the re-circulization
of the plasmid as the alkaline phosphate group can join with OH group (6 marks)
4)
Account for the use of the following chemicals in polyacrylamide gel
electrophoresis:
a)
β-Mercaptoethanol and Dithiothreitol
Ø The purity and molecular size of a protein
can be assessed by analyzing proteins in the presence of 0.1% SDS on
one-dimensional gels (UNIT 8.4) or two dimensional gels (UNIT 8.5).
By adding a reducing agent (e.g., 2-mercaptoethanol
or dithiothreitol) to the sample, it is possible to determine the number
and size of subunits in a pure protein. Non denaturing gels can be used to
examine and isolate the “native” protein.
b)
Sodium dodecyl sulphate (SDS)
Ø Is a detergent that coats and solubilize the
protein from quaternary structure to primary structure that is movement is according
to molecular size only (not mol. Charge)
c)
TEMED
Ø Is an enzyme that
catalyses the formation of free radical from ammonium persurphate.
d)
Ammonium persulphate
Ø Is a catalyst that
initiates the polymerization of acrylamide and bis acrylamide during PAGE(6 marks)
5) a) What is DNA profiling?
Ø Is a method of
determining the specific genome of a human individual which discriminate from
other humans.
b) What is the advantage of using short repeat
length (STRs) over long repeat length simple tandem repeats for forensic
analysis?
Ø These STR loci are
highly polymorphic with a wide variability in the number of repeats between
individuals compared to long repeats.
c) Why should STR loci selected
for forensic profiling be distributed throughout the genome?
Ø In choosing STR
loci for forensic profiling it is also important that they are distributed
throughout the genome (Table 13.1) to
ensure that each is inherited independently.
SECTION B (30 MARKS)
6)
Describe how
successful ligation assay may be detected by insertional inactivation using:
a)
Plasmids like pBR322 with more than one antibiotic resistance gene.
Ø A plasmid like
pBR322 has both ampicillin resistance gene and tetracycline resistance gene
that allow the growth of bacteria when grown on them respectively. In this
technique only one of these is used for instance if ampicillin resistance gene
is used, the new insert is inserted on the ampicillin gene that interfere the
resistance of the bacteria when grown on the ampicillin hence cause a bacteria
not to grow, therefore those which fail to grow means insertional inactivation
is successful. And when tetracycline is used the new insert is inserted at the
tetracycline resistance gene that destroys its resistance that cause the death
of the bacteria when grown on the tetracycline therefore those dies are the
that are successful transformed.
b)
Plasmid like
pUC18 and its derivatives which make use of a gene called lacZ.
Ø Within the lac Z gene of a pUC18 plasmid there is multiple cloning site (MCS)
which codes for an enzyme β-galactosidase. β-galactosidase converts a substrate
known as X-gal/IPTG to bright blue product that is when cells are grown on an
agar plate they produce a blue colonies
therefore when the new insertion is successful it disrupts the lac Z that it becomes unable to code for
the X-gal/IPTG hence it do not produce the blue colonies but the normal color
that is white is produced. Therefore in order to know that the insertional
inactivation is successful white colonies are produced when grown on an agar
plate. (15 marks)
7)
There are many
ingenious ways of identifying the clones that contain the cDNA of interest from
a cDNA library. Explain how this can be done if:
o
We will
describe two ways of selecting cDNA clones from a library. One method simply
detects the presence of the foreign DNA attached to the plasmid vector, and the
second detects the protein encoded for by the foreign DNA. We call the process
of selecting specific clones “screening the library”.
Ø Preparation of the cDNA Library for Screening.
a)
The clone is to
be selected by virtue of its DNA sequence.
Ø Bacterial colonies are plated onto agar
plates, and the colonies are then replica-plated onto a nylon membrane, which
is then treated with detergent to burst (or lyse) the bound cells. If
the clone is to be selected by virtue of its DNA sequence (Fig. 7.7), the nylon
membrane is processed with sodium hydroxide. This is necessary to break all
hydrogen bonds between the DNA strands bound to the nylon membrane and ensures
that the DNA is single-stranded. The processed nylon membrane is an exact
replica of the DNA contained within each bacterial colony on the agar plate.
b)
The clone is to
be selected by detecting the protein encoded by the foreign DNA. (15 marks)
Ø If the clone is to be selected from the
library by detecting the protein encoded by the foreign DNA (Fig. 7.8), then
colonies are again replica-plated on to a nylon membrane. This time, however,
the nylon membrane is processed to produce an exact copy of the proteins synthesized
by each bacterial colony.
8)
a) What must be
done in order for a cloned gene to be expressed in a host bacterial cell?
Ø An expression vector must be used.
Ø The recombinant gene must have a promoter
that is recognize by the host RNA polymerase.
Ø The prokaryotic leader must be provided to
synthesize eukaryotic protein in a bacterium.
Ø Introns in the eukaryotic gene must be
removed because the prokaryotic host will not excise them after transcription
of mRNA.
b) Outline the procedure for Somatostatin
cloning and production. (15 marks)
Ø Somatostatin, the 14-residue hypothalamic
polypeptide hormone that helps regulate human growth, provides an example of
useful cloning and protein production. The gene for somatostatin was initially
synthesized by chemical methods. Besides the 42 bases coding for somatostatin,
the polynucleotide contained a codon for methionine at the 5’ end (the
N-terminal end of the peptide) and two stop codons at the opposite end. To aid
insertion into the plasmid vector, the 5’ ends of the synthetic gene were
extended to form single-stranded sticky ends complementary to those formed by
the EcoRI and BamHI restriction enzymes. A modified pBR322
plasmid was cut with both EcoRI and BamHI to remove a part of the
plasmid DNA. The synthetic gene was then spliced into the vector by taking
advantage of its cohesive ends. Finally, a fragment containing the initial part
of the lac operon (including the promoter, operator, ribosome binding site, and
much of the β-galactosidase gene) was inserted next to the somatostatin gene.
The plasmid now contained the somatostatin gene fused in the proper orientation
to the remaining portion of the β-galactosidase gene.
After
introduction of this chimeric plasmid into E. coli, the somatostatin gene was
transcribed with the β-galactosidase gene fragment to generate an mRNA having
both messages. Translation formed a protein consisting of the total hormone
polypeptide attached to the β-galactosidase fragment by a methionine residue.
Cyanogen bromide cleaves peptide bonds at methionine residues. Treatment of the
fusion protein with cyanogen bromide broke the peptide chain at the methionine
and released the hormone. Once free, the polypeptide was able to fold properly
and become active. Since production of the fusion protein was under the control
of the lac operon, it could be easily regulated.
