Course break down
Date
Lectures
Class activity
26-1-2015
Discuss OHTs and Quiz,
(Portein engineering and
restriction mapping)
28-1-2015
Microbial Biotechnology
( Products, Fermentation
(Principles and Applications)
2-2-2015
Microbial Biotech (Food
Biotech)
4-2-2015
Plant Biotechnology (Plant
tissue culture and
applications )
9-2-2015
GE plants and Applications
of GE plants
Problem qs.
11-2-2015
Biofertilisers
Quiz
16-2-2015
Animal cell culture and
Characterisation of cell lines
Problem qs.
18-2-2015
Applications of animal cell
culture
Quiz
problem qs.
Date
Lectures
Class activity
25-2-2015
Transgenic animal
technology
2-3-2015
Discuss OHTs
4-3-2015
Environment
Biotechnology
(Bioremediation, Phyto
remediation)
9-3-2015
Utilisation of biomass
11-3-2015
Biomining and Bioleaching
16-3-2015
Biotechnology in medicine
and Health care, (Gene
therapy)
problem qs.
18-3-2015
Xenotransplantation and
Forensic Biotechnology
Problem qs.
23-3-2015
Human Genome Project
Problem qs. Quiz
25-3-2015
Impacts of biotechnology
on Human beings:
Biotechnology Ethiccs
Solutions to OHT
Q1. Fill in the blanks.
(/6)
a. Cornerstone of biotechnology is …..DNA…………
b. Dolly was cloned in ……1996…….. by ………Ian Wilmut and collegues (Ian Wilmut,
Keith Campbell et al.)...
c. Purines are –-double---ringed heterocyclic nitrogenous bases. Two of five bases in
nucleic acids,--adenine------ and --guanine--------- are purines.
d. The restriction endonucleases which cleave the DNA with in the recognition sites are
---Type II-- restriction endonucleases.
e. Primers annealing to themselves results in to unwanted extension product called --------Primer dimer
f. Number of copies of a gene amplified in 25 cycles of PCR will be ---2(n+1)--=
67108864--------
Q2. Multiple choice questions.
(/5)
a. The enzyme DNA polymerase can only work in
a. 3’5’
b. 5’3’
c. Both directions
d. None of these
b. Problems in obtaining large amounts of proteins encoded by recombinant genes can
often be overcome by using
a. BACS
b. Expression vectors
c. YACS
d. All of these
c. Vectors are
a. Molecules that replicate DNA
b. Molecules that degrade the nucleic acids
c. Molecules that are able to covalently bond to and carry foreign DNA into the
cells
d. Molecules that protect the bacterial cells from invasion by foreign DNA
e. Which of the following can maintain the largest fragment of DNA
a. YAC
b. Cosmid
c. Plasmid
d. Phage
f. If a DNA strand has 35 nucleotides, how many phosphodiester bonds would exist?
a. 35
b. 34
c. 24
d. 70
Q3. Answer briefly to following short questions.
a. Why was the work reported by Coher and Boyer in 1973 was important?
(/2)
Stanley Cohen and Herbert Boyer develop recombinant DNA technology.
Considered to be the birth of modern biotechnology, they complete the first
successful genetic engineering experiment by inserting a gene from an African
clawed toad into bacterial DNA.
a. Compare DNA and RNA.
(/2)
b. For a gene cloning experiment, how would you protect a cleaved plasmid from selfrecirculation?
(/2)
1. Double digestion using two different restriction endonucleases.
2. Treating the plasmid with alkaline phosphatase enzyme which dephosphorylate
the 5’ end of DNA and thus it prevent recirculation /religation of linearised plasmid
a. Describe how would you identify and distinguish transformants from recombinants?
(/2)
Blue- white screening
a. Suggest different ways for introducing a recombinant plasmid in to a bacterium such
as E. coli.
(/2)
1. Chemical transformation coupled with heat shock.
2. Electroporation
a. Draw a flow diagram to explain basic steps involved in gene cloning
(/3)
Selection of Recombinant Plasmid
LacZ, White Blue Selection
• Colonies with recombinant plasmids
are white, and colonies with
nonrecombinant plasmids are blue.
• Resistant to ampicillin, has (ampr
gene)
• Contains portion of the lac operon
which codes for beta-galactosidase.
• X-gal is a substrate of betagalactosidase and turns blue in the
presence of functional betagalactosidase is added to the medium.
• Insertion of foreign DNA into the
polylinker disrupts the lac operon,
beta-galactosidase becomes nonfunctional and the colonies fail to turn
blue, but appear white.
g.
The following figure shows the restriction endonuclease digestions and
electrophoretic separation of fragments. A purified piece of DNA is cut with EcoRI and
BamHI separately (Single digestions) and then with both enzymes together (double
digestion). The horizontal lines under each digestion conditions
represent
schematically the locations of the DNA fragments (bands) in the lanes of the gel after
electrophoresis and staining of the DNA fragments. The numbers denote the length
of the digestion products (fragments) in base pairs.
Now derive a restriction endonuclease map from the digestions and
electrophoretic seperations shown in above figure.
(/3)
h. What are essential components of a PCR used to amplify a specific sequence of a
DNA?
(/3)
Solutions to Quiz
Question 1
a)
5’ G
3’
3’ CCTAG 5’
b)
5’ T
3’
3’ ACTAG 5’
c)
5’ ATTGAGGATCCGTAATGTGTCCTGATCACGCTCCACG 3’
3’ TAACTCCTAGGCATTACACAGGACTAGTGCGAGGTGC 5’
BamHI
A
5’ ATTGAG
3’
3’ TAACTCCTAG 5’
Cleavage by BamHI leaves a 5’ overhang
Cleavage by BclI leaves a 5’ overhang
B
5’ GATCCGTAATGTGTCCTGATCACGCTCCACG 3’
3’
GCATTACACAGGACTAGTGCGAGGTGC 5’
d) BclI
C
5’ ATTGAGGATCCGTAATGTGTCCT
3’
3’ TAACTCCTAGGCATTACACAGGACTAG 5’
D
5’ GATCACGCTCCACG 3’
3’
TGCGAGGTGC 5’
e) Could you cut the fragment from (d) with either BamHI or BclI? Explain.
No, the recognition sites for both BamHI and for BclI have been destroyed.
Question 2
a.
b.
You need ampicillin sensitive cells.
c.
Bacterial cells that carry a vector will be able to grow on ampicillin whereas
untransformed cells will not.
d.
Enzymes
Restriction enzymes
Ligase
DNA polymerase
RNA polymerase
Transcriptase
Reverse transcriptase
3’ to 5’ exonuclease
Cloning vector
Reagents
Size separating gel
Okasaki fragments
ATP,TTP,CTP, GTP
ddATP, ddTTP, ddCTP, ddGTP
Primers
Replication fork
Human cells
Virus
Question 3
a.
You can cut the plasmid with EcoRI and look for two fragments, one
that represents the vector and one that represents the insert. You
would not know for sure that the insert is the harE gene without
further tests. To confirm that the insert is the harE gene, you would
need to know some amino acid sequence of the harE protein. You
could then make a degenerate nucleic acid probe that would be
complementary the harE gene sequence.
b (i).
b (ii).