UNIT-5

PROTEIN AND GENE MANIPULATION

CHAPTER-5.1
RECOMBINANT DNA TECHNOLOGY
MULTIPLE CHOICE QUESTIONS
1. Which Enzyme is used by bacteria for defense against viruses?
a) DNA Polymerase
b) DNA Ligase
C) Restriction Endonuclease
d) Primase
2. Which of these is called as the molecular vehicle?
a) Host
b) Restriction Enzyme
c) Vector
d) Gene of interest
3. Which vector is used for genome sequencing?
a) M13
b) YAC
c) BAC
d) Both B & C
4. By which method the recombinant vector enters into the host cell?
a) Transition
b) Transformation
c) Conjugation
d) None of the above
5. Which of these produces ssDNA?
a) pBR322
b) M13
c) BAC
d) YEp
6. Which enzyme is produced by lacZ gene?
a) Beta Galactosidase
b) Beta lactase
c) Beta permease
d) Acetylase
7. Which of these is not true for lambda phage vector:-
a) It is linear and double stranded
b) It has cos sites
c) It follows lytic pathway
d) It's insert size is 45 kbp
8. Which of these is not a selectable marker gene?
a) LEU2
b) GFP
c) ampR
d) MCS
9. Which of these gene transfer methods may need calcium
phosphate?
a) Transformation
b) Microinjection
c) Biolistics
d) Transfection
10. Which of these is a shuttle vector?
a) YEp
b) pBR322
c) pUC19
d) BAC
11. If your foreign DNA is of size 35 kbp, which vector would you use?
a) BAC
b) YAC
c) Cosmid
d) lambda
12. A vector based on which virus was used in the first cloning
experiment involving mammals?
a) Adenovirus
b) Papilloma virus
c) SV40
d) Retrovirus
13. Which microscope is required for microinjection?
a) Electron Microscope
b) Phage Contrast Microscope
c) Fluorescent Microscope
d) All can be used
14. How does a scientist select for a sensitive or negative trait
following a transformation experiment?
a) By Replica Plating
b) By Blue White Screening
c) Both can be used
15. Who found the transformation of [Link] cells upon treatment with
chilled CaCl2 solution?
a) Mandel & Higa
b) Herbert Boyer & Paul Berg
c) Watson & Crick
d) Stanley & Cohen
16. If a PCR reaction initiated with three molecules of a dsDNA
template, after 35 cycles the amount of DNA will be:
a) 3 x 35/2
b) 3 x (2 x 35)
c) 3 x (2/35)
d) 3 x 235
17. From the options given below which vector is used between two
hosts?
a) pBR322
b) pUC19
c) YEp
d) All of these
18. For performing Site directed mutagenesis, which vector will be
required?
a) phage
b) cosmid
c) M13
d) BAC
19. What is the expansion of ddNTP?
a) Dedioxynucleotide triphosphate
b) Dideoxynucleoside triphosphate
c) Dideoxynucleotide triphosphate
d) Deoxynucleotide triphosphate
20. Which of these is not present in a YAC vector?
a) Centromere
b) Telomeric Sequence
c) cos site
d) Autonomously Replicating Sequence (ARS)
21. Which of these is not an animal vector?
a) SV40
b) Gemini Virus
c) Adenovirus
d) Papilloma Virus
22. Which of these enzymes can be used to prevent selfligation?
a) EcoRI
b) DNA Ligase
c) HindIII
d) Alkaline Phosphatase
23. Which of these is used by bacteria to protect their own DNA from
the action of restriction endonucleases?
a) Alkylation
b) Phosphorylation
c) Methylation
d) Hydrolysis
24. If you are using YEp vector having LEU2 selectable marker gene,
what would be your media?
a) with/containing leucine
b) without leucine
c) None of these
d) can't say
25. Which of these vectors would be needed for template
preparation for sanger's method of DNA sequencing?
a) Lambda phage
b) pBR322
c) M13
d) BAC
26. Which of these methods is used exclusively for gene transfer in
plants?
a) Electroporation
b) Microinjection
c) Biolistics
d) Transfection
27. What is the sequence of steps in a cycle of PCR?
a) Denaturation, Extension, Annealing
b) Extension, Annealing, Denaturation
c) Annealing, Denaturation, Extension
d) Denaturation, Annealing, Extension
28. Polymerase Chain Reaction was invented by:
a) Edward Southern
b) Kary Mullis
c) Fredrick Sanger
d) Paul Berg
29. If you are interested in amplifying a specific region of
genome of an organism, which technique would you use?
a) Sanger's dideoxy chain termination
b) Polymerase Chain reaction
c) Replica Plating
d) Blue White screening
30. If you are interested in amplifying a specific region of genome of
an organism, which of the following would actually help in such
targeted amplification?
a) Probes
b) Primers
c) dNTPs
d) ddNTPs
31. Which vector is used for blue white screening?
a) BAC
b) YAC
c) pBR322
d) pUC19
32. What was labeled with fluorescent tag in the single tube DNA
sequencing method?
a) Primer
b) dNTP mix
c) ddNTP
d) None of these
33. In DNA sequencing method, chain elongation terminates due
to lack of ____ in ddNTP.
a) 2' hydroxyl group
b) 3' hydroxyl group
c) 5' Phosphate group
d) All of these
34. In Sanger's chain termination method, lack of formation of which
bond terminates the chain elongation?
a) Phosphoester Bond
b) Phosphodiester Bond
c) Glycosidic Bond
d) Peptide Bond
35. Expand RFLP.
a) Restriction fragment length polymorphism
b) Restricted fragment linear polymorphism
c) Regenerated fragment length polymorphism
d) Regenerated fragment linear polymorphism
36. Which of these is a bacterial vector compatible with plant cells?
a) Gemini Virus
b) Cauliflower Mosaic Virus
c) Ti plasmid
d) Tobacco mosaic virus
37. If you are treating bacterial host cells with calcium chloride for
facilitating introduction of rDNA, which of these methods you are
using?
a) Transformation
b) Electroporation
c) Microinjection
d) Biolistic
38. If you are coating your DNA of interest onto the surface of
microscopic gold particles for introduction in host cell, which of these
methods you are using?
a) Transformation
b) Transfection
c) Microinjection
d) Biolistic
39. Suppose you want to insert your gene of insert in pBR322 vector
within the tetracycline resistance selectable marker gene, by which of
the following restriction enzymes you would isolate the gene of
interest?
a) AluI
b) PstI
c) EcoRI
d) BamHI
40. If you are doing replica plating using pBR322 vector for selection
of transformed recombinant cells, in which plate would the
transformed recombinant cells grow?
a) master plate
b) replica plate
c) Both the above plates
d) None of the above plates.
41. Which of the following types of restriction enzymes is used in
recombinant DNA technology?
a) Type I
b) Type II
c) Type III
d) All of these
42. Which of these is the correct sequence of steps of gene cloning?
a) Transformation, Restriction Enzyme digestion, Ligation, Selection.
b) Restriction Enzyme digestion, Selection, Ligation, Transformation
c) Restriction Enzyme digestion, Ligation, Selection, Transformation
d) Restriction Enzyme digestion, Ligation, Transformation, Selection
43. What is the advantage of MCS or polylinker site in vector?
a) increases flexibility of choice of restriction enzymes
b) decreases flexibility of choice of restriction enzymes
c) increases flexibility of choice of selectable marker genes
d) decreases flexibility of choice of selectable marker genes
44. Which of these sequences can be cut by a type II restriction
enzyme?
a) ATGCGTCGTGTCGTG
b) GTGCGATGACGTGCG
c) ATGGAATTCGTGCGTA
d) GTGATGACGTAGCGTG
45. Which of the following can be used to create a recombinant
DNA?
a) A fruitfly gene isolated by HindIII digestion and a human gene
isolated from EcoRI digestion.
b) A fruitfly gene isolated by HindIII digestion and a human gene
isolated from HindIII digestion.
c) A fruitfly gene isolated by HindIII digestion and a human gene
isolated from EcoRI digestion.
d) None of these
46. The first recombinant DNA was developed by whom?
a) Boyer, Berg, Chang, Cohen.
b) Boyer, Mullis, Sanger
c) Arber, Smith and Nathans
d) Linus Pauling, Winkler
47. The first type II restriction enzyme was isolated by whom?
a) Boyer, Berg, Chang, Cohen.
b) Boyer, Mullis, Sanger
c) Arber, Smith and Nathans
d) Linus Pauling, Winkler
48. What is the source of DNA ligase for recombinant DNA
technology?
a) T4 bacteriophage
b) Calf intestine
c) Bacteria
d) Plants
49. The natural genetic engineer of plants is:-
a) Alcaligenes eutrophus
b) Agrobacterium tumefaciens
c) Bacillus thuringiensis
d) Bacillus amyloliquefaciens
50. What is the role of GFP in recombinant DNA technology?
a) acts as Selectable marker gene
b) performs restriction digestion
c) increases rate of transformation in host cells
d) all of these
ANSWERS OF MCQs
Q. Ans Q. Ans Q. Ans Q. Ans Q. Ans
1 c 11 c 21 b 31 d 41 b
2 c 12 c 22 d 32 c 42 d
3 d 13 b 23 c 33 b 43 a
4 b 14 a 24 b 34 b 44 c
5 b 15 a 25 c 35 a 45 b
6 a 16 d 26 c 36 c 46 a
7 d 17 c 27 d 37 a 47 c
8 d 18 c 28 b 38 d 48 a
9 d 19 b 29 b 39 d 49 b
10 a 20 c 30 b 40 a 50 a

5.1.2. ASSERTION REASON QUESTIONS

Following Questions consist of two statements– Assertion (A) and
Reason (R). Answer these questions selecting the appropriate option
given below:
a. Both Assertion and Reason are true and the reason is the correct
explanation of the assertion
b. Both Assertion and Reason are true but the reason is not the
correct explanation of the assertion
c. Assertion is true but Reason is false
d. Both Assertion and Reason are false
1) Assertion: Type III restriction enzymes are used in recombinant
DNA technology.
Reason: Type III enzymes recognise and cut the DNA within a
recognition sequence.
2) Assertion: Vectors are treated with alkaline phosphatase.
Reason: This enzyme avoids self ligation of vectors by removing their
5' phosphate group.
3) Assertion: YEP is used for both prokaryotes and eukaryotes
cells.
Reason: It is a shuttle vector.
4) Assertion: PCR based diagnosis is faster, safer and more specific.
Reason: As it does not use live pathogens.
5) Assertion: PCR uses Taq DNA polymerase enzyme.
Reason: It is isolated from a thermostable bacteria.
6) Assertion: pBR322 based true recombinants will be tetracycline
sensitive if the vector was
disarmed with Bam H1 restriction endonuclease enzyme.
Reason: Insert fails to show insertional inactivation in tetracycline
resistance gene in recombinant cells.
7) Assertion: M13 is used in DNA sequencing and site directed
mutagenesis techniques.
Reason: It produces replicative forms.
8) Assertion: The ends of lambda phage DNA are sticky and
called cos sites.
Reason: 12 bases on each end are unpaired and complementary to
each other.
9) Assertion: pUC19 is an expression vector.
Reason: pUC19 has signals for transcription and translation in given
host.
10) Assertion: Agrobacterium tumifaciens is known as natural
genetic engineer.
Reason: It inserts a part of its own DNA sequence into host plant cell.
ASSERTION- REASON QUESTIONS
ANSWER KEY
[Link]. 1 2 3 4 5
ANSWER d a a a a
[Link]. 6 7 8 9 10
ANSWER c b a a a
5.1.3. VERY SHORT ANSWER QUESTIONS
1) Name any two scientists involved in designing the first recombinant
DNA molecule.
Ans. Paul Berg, Herbert Boyer, Annie Chang and Stanley Cohen.
(Any two)
2) Answer the following:-
a) Which vector was used in the first cloning experiment involving
mammalian cell?
b) Identify the vector shown below.
Ans. a) Simian Virus 40
a) pBR322
3) Answer the following:
a) Explain R-M system existing in bacteria.
b) How does a modification enzyme protect bacterial DNA from its
own restriction enzymes?
Ans. a) Restriction- Modification system: The main components
include restriction enzyme (R), which cuts specific non-methylated
DNA sequences thereby restricting the propagation of any external
DNA like that of bacteriophage inside bacterial cells, and the methyl
transferase or methylase (M), which protects bacterial own DNA
sequences from the action of its own restriction enzyme(s).
b) By methylation of host DNA so that it become resistant to the action
of restriction enzymes.
4) How can LEU2 gene be used as a selectable marker?
Ans. LEU2 gene codes for an enzyme required for the biosynthesis
of amino acid leucine in yeast cells. Yeast cells having this plasmid can
grow on a minimal medium lacking leucine and hence can be selected
out of other yeast cells which do not contain this gene.
5) Specify one advantage of developing vectors based on M13
vector.
Ans. Multiple single stranded copies of the initial double stranded
gene can be obtained while gene cloning via M13 vector.
5.1.4. SHORT ANSWER QUESTIONS
1) Listed below are four different single strands of DNA. Which
of these would you expect to be cleaved by a restriction
endonuclease? Give reason.
(a) ACTCCAGAATTCACTCCG
(b) ACTCCACTCCCGACTCCG
(c) GCCTCAAAGCTTGCCTGA
(d) GAGCGGTTTATCTGAGCAG
Ans. (a) ACTCCAGAATTCACTCCG
(c) GCCTCAAAGCTTGCCTGA
These sequences have recognition palindromic sequences for
restriction enzymes a) EcoRI and c) HindIII
2) Explain various steps involved in a recombinant DNA technology
experiment.
Ans. Steps involved: 1. Isolation of a DNA fragment containing a gene
of interest that needs to be cloned (called as insert). 2. Generation of
a recombinant DNA (rDNA) molecule by insertion of the DNA
fragment into a carrier DNA molecule called vector (e.g. plasmid) that
can selfreplicate within a host cell. 3. Transfer of the rDNA into an E.
coli host cell (process called transformation). 4. Selection of only
those host cells carrying the rDNA and allowing them to multiply
thereby multiplying the rDNA molecules.
3) Students of Class XII visited Microbial Type Culture Collection,
Chandigarh and observed microbial cultures of Providencia stuartii,
Streptomyces albus and Haemophilus aegyptus. Name the restriction
enzymes obtained from them and also specify their restriction sites.
Ans. a) Source- Haemophilus aegyptus
RE- HaeIII
Res. Site- 5' G-G-C-C 3’
b) Source- Providencia stuartii
RE- PstI
Res. Site- 5' C-T-G-C-A-G 3’
c) Source- Streptomyces albus
RE- SalI
Res. Site- 5' G-T-C-G-A-C 3’
4) Selection is an important step in genetic engineering. You are
given ampicillin and tetracycline antibiotics. Using these antibiotics,
which selection technique could be used to differentiate between
recombinant and non-recombinant cells?
Ans. Transformed host cells are first plated on an agar medium
containing that antibiotic the resistance gene for which is intact, and
the DNA fragment is not inserted, within this gene. Therefore, host
cells having the recombinant DNA are expected to be resistant to this
antibiotic. This step eliminates non-transformed cells; the resulting
colonies will possess either recombinant or unaltered vector. The
colonies so obtained are then replica-plated on agar plates containing
the other antibiotic (within the gene for which the DNA insert is
placed); all the colonies that develop on this plate will contain the
unaltered vector. Therefore, the antibiotic sensitive colonies are
identified and recovered from the master plate; these colonies will
have recombinant DNA. This entire process may take 1-2 days.
5.1.5. LONG ANSWER QUESTIONS
1) Few restriction enzymes break the phosphodiester bond in such a
manner that single stranded overhanging ends are generated in the
DNA strand. EcoRI is one such restriction enzyme.
(a) Write the sequence for restriction site for enzyme EcoRI. Give a
name to the type of ends generated here. Are all the restriction
sequences palindromic in nature?
(b) Explain about any two vector-less methods that allow DNA to
enter host cells.
(c) Why is small size desirable in a cloning vehicle?
Ans. (a) Restriction site of EcoRI is 5’-GAATTC-3’
The ends generated will be called sticky/cohesive ends.
No, all the Restriction sequences may not be palindromic.
(b) Microinjection method can be used to inject foreign DNA into
plant and animal cells’ nucleus. Biolistic method makes use of
particle gun to bombard gold/tungsten bullets coated with DNA so as
to deliver DNA into host cells.
(c) Small size of vector facilitates entry of recombinant molecules into
the host cells. Moreover, small vectors are more stable and less likely
to break during various manipulations taking place in gene cloning
process.
2) Given below is the autoradiogram obtained as a result of Sanger's
method of DNA sequencing. Based on this answer the following
questions.
(a) Read and write the original/template DNA sequence from the
autoradiogram below.
(b) Define the principle and steps of this technique.
Ans. (a) 3’ AGCTTCAGTC 3’
(b)Principle – When a ddNTP gets incorporated in the growing DNA
chain during DNA replication, the extension stops as due to non-
availability of 3’ hydroxyl group on the ddNTP present at the growing
end, incoming nucleotide cannot join.
Steps-
This method makes use of the mechanism of DNA synthesis by DNA
polymerase. (a) DNA polymerase requires both a primer (a short
oligonucleotide strand), to which nucleotides are added, and a
template strand to guide selection of each new nucleotide. In cells,
the 3’-hydroxyl group of the primer reacts with an incoming
deoxynucleoside triphosphate (dNTP) to form a new phosphodiester
bond. (b) The Sanger sequencing procedure uses dideoxynucleoside
triphosphate (ddNTP) analogs to interrupt DNA synthesis. (The
Sanger’s method is also known as the dideoxy method.) When a
ddNTP is inserted in place of a dNTP, strand elongation is halted after
the analog is added, because it lacks the 3’-hydroxyl group needed
for the next step. (c) The DNA to be sequenced is used as the
template strand, and a short primer, radioactively or fluorescently
labeled, is annealed to it. By addition of small amounts of a single
ddNTP, for example ddCTP, to an otherwise normal reaction system,
the synthesized strands will be prematurely terminated at some
locations where dC normally occurs. Given the excess of dCTP over
ddCTP, the chance that the analog will be incorporated whenever a
dC is to be added is small. However, ddCTP is present in sufficient
amounts to ensure that each new strand has a high probability of
acquiring at least one ddC at some point during synthesis. The result
is a solution containing a mixture of labeled fragments, each ending
with a C residue. Each C residue in the sequence generates a set of
fragments of a particular length, such that the different-sized
fragments, separated by electrophoresis, reveal the location of C
residues. This procedure is repeated separately for each of the four
ddNTPs, and the sequence can be read directly from an
autoradiogram of the gel. Because shorter DNA fragments migrate
faster, the fragments near the bottom of the gel represent the
nucleotide positions closest to the primer (the 5’ end), and the
sequence is read (in the 5’ to 3’ direction) from bottom to top. Note
that the sequence obtained is that of the strand complementary to
the strand being sequenced (template strand).
3)Expand PCR. What are the three basic steps of a typical
PCR reaction? Explain in detail along with a suitable diagram.
Ans. PCR- Polymerase Chain Reaction Process:-
The process of PCR consists of a series of reaction/amplification
cycles and it is carried out in vitro. It utilizes
the following : (1) a DNA preparation containing the desired DNA
segment to be amplified (target sequence), (2) two oligonucleotide
primers (about 20 bases long) specific, i.e., complementary, to the
two 3’- borders (the sequences present at the 3’-ends of the two
strands) of the desired segment, (3) the four deoxynucleoside
triphosphates, viz., dTTP (deoxythymidine triphosphate), dCTP
(deoxycytidine triphosphate), dATP (deoxyadenosine triphosphate)
and dGTP (deoxyguanosine triphosphate), and (4) a heat stable DNA
polymerase, e.g., Taq polymerase (isolated from the bacterium
Thermus aquaticus).
Steps of PCR:-
At the start of PCR, the DNA from which a segment is to be amplified,
an excess of the two primer molecules, the four deoxynucleoside
triphosphates and the DNA polymerase are mixed together to form a
reaction mixture that has appropriate quantities of Mg2+.
A single PCR amplification cycle involves the following
three basic steps (Fig. 13):
a) Denaturation
The reaction mixture is first heated to a temperature between 90-
98⁰C
(commonly 94⁰C) that ensures DNA denaturation (separation of both
of the strands of DNA). This is the denaturation step. The duration of
this step in the first cycle of PCR is usually 2 minutes at 94⁰C.
b) Annealing
The mixture is now cooled to a temperature (generally 40-60⁰C) that
permits annealing of the primer to the complementary sequences in
the DNA; these sequences are located at the 3’-ends of the two
strands of the desired segment. This step is called annealing. The
duration of annealing step is usually 1 minute during the first as well
as the subsequent amplification cycles of PCR. Since the primer
concentration is kept very high relative to that of the template DNA,
primer-template hybrid formation is greatly favoured over
reannealing
of both the template strands.
c) Primer Extension/Polymerisation
The temperature is now so adjusted that the DNA polymerase
synthesizes the complementary strands by utilizing 3’-OH of the
primers; this reaction is the same as that occurs in vivo during
replication of the leading strand of a DNA duplex. The primers are
extended towards each other so that the DNA segment lying
between
the two primers is copied; this is ensured by employing primers
complementary to the 3’-ends of the segment to be amplified. The
duration of primer extension is usually 2 minutes at 72⁰C.
The completion of the extension step completes the first
amplification
cycle of PCR. Each cycle of PCR ordinarily takes 4-5 minutes. To
begin the second cycle, the DNA is again heated to convert all the
newly synthesized DNA into single strands, each of which can now
serve as a template for synthesis of more new DNA. Thus the
extension products of one cycle can serve as a template for
subsequent cycles and each cycle doubles the amount of DNA from
previous cycle. As a result, from a single template DNA molecule, it is
possible to generate 2n molecules after n number of cycles. Usually,
20-30 cycles are carried out in most PCR experiments. In case of
automated PCR machines, called thermal cyclers, the researcher has
to only specify the number
Fig. A schematic representation of the three steps performed during
each reaction cycle of PCR. and duration of cycles, etc. after placing
the complete reaction mixture for incubation, and the machine
performs the entire programme of operations precisely.
5.1.6 CASE BASED QUESTIONS
1) Forensics, short for forensic science, is the application of science
in a legal setting. Biotechnology is used by forensic scientists to
collect or process trace evidences such as hair, skin, blood or semen
samples, which are found at crime scene(s). An important aspect of
modern forensics is the use of DNA profiling, or genetic
fingerprinting. Sources of DNA include blood, hair, semen, saliva,
bone and tissue. Every person has a unique DNA profile. The only
exception to this are monozygotic twins. The chemical structure of
everyone’s DNA is the same. There are so many millions of base pairs
in each person’s DNA that every person has a different sequence.
Using these sequences, every person could be identified solely by the
sequence of their base pairs. However, because there are so many
millions of base pairs, the task would be very time-consuming.
Instead, scientists are able to use a shorter method, because of
repeating patterns in DNA. With the help of a technique that
amplifies DNA, which is used to make millions of exact copies of DNA
from a biological sample, it is possible to execute DNA fingerprinting
even analysing a very small quantity of DNA.
(i) PCR is a:-
(a) DNA degradation technique
(b) DNA sequencing technique
(c) DNA amplification technique
(d) All of these
Ans. (c) DNA amplification technique
(ii) How many DNA duplexes are obtained from one DNA duplex
after 5 cycles of PCR?
(a) 25
(b) 10
(c) 32
63
(d) 5
Ans. (c) 32
(iii). Primers used for the Polymerase chain reaction are: -
(a) Single stranded DNA oligonucleotide
(b) Double stranded DNA oligonucleotide
(c) Single stranded RNA oligonucleotide
(d) Double stranded RNA oligonucleotide
Ans. (a) Single stranded DNA oligonucleotide
(iv). What is the function of primer?
(e) To identify the particular region of DNA to be
copied by PCR.
(f) To copy DNA.
(g) To create DNA nucleotides.
(h) To maintain the temperature of the PCR
reaction
Ans. (a) To identify the particular region of DNA to be
copied by PCR.
OR
Choose the right combination of components required to set up PCR
reaction-
(a) Template DNA, two primers, dNTPs and DNA ligase
(b) Template DNA, two primers, NTPs and DNA ligase
(c) Template RNA, two primers, NTPs and DNA polymerase
(d) Template DNA, two primers, dNTPs and DNA polymerase
Ans. (d) Template DNA, two primers, dNTPs and DNA
polymerase
2. Advantage of using E. coli as host cells is that under optimal
conditions the cells divide every 20 minutes making it possible
to clone large amounts of foreign DNA and if
the appropriate signals are incorporated into the vector large
amounts of recombinant proteins are available for therapeutics and
other uses.
For the expression of eukaryotic proteins, eukaryotic cells are often
preferred because, to be functionally active, proteins require proper
folding and post translational modifications such as glycosylation
which is not possible in prokaryotic (E. coli) cells. Even cloned
eukaryotic genes containing introns cannot be processed in
E. coli thereby necessitating the use of only eukaryotic host cells.
Yeast cells have been used extensively for functional expression of
eukaryotic genes because of several features. Yeasts are the simplest
eukaryotic organisms (unicellular) and like E. coli have been
extensively characterized genetically, easy to grow and manipulate
and large amounts of cloned genes or recombinant proteins can be
obtained from yeast cultures grown in fermentors (large culture
vessels). Plant and animal cells may also be used as hosts in
rDNA experiments and cells can be grown in tissue culture or
can be induced and manipulated to form whole organisms
(creation of transgenic animals and plants).
i) Calculate the total number of E. coli cells formed from an initial
population of 10 cells incubated in optimum conditions for 04 hours.
Ans: 10 x 212 cells
ii) Mr. Raj attempted to clone and express erythropoietin gene in E.
coli. The gene was cloned successfully but could not express itself.
What could be the reason(s)?
Ans: a) Erythropoietin, being a eukaryotic gene, has both coding
and non-coding region within it. After transcription, the primary
transcript has to be modified post transcriptionally via intron
splicing, capping and tailing. E. coli, being a prokaryote, is not able to
perform such complex post transcriptional modifications.
b) Erythropoietin also needs enormous post translational
modifications, i.e., glycosylation and protein folding, which is not
possible in a prokaryotic system like E. coli.
iii) What control elements or regulatory sequences are
required in an expression vector so as to drive
expression of desired gene successfully in the host?
Ans: Promoter, Operator, Ribosome Binding Site
iv) Which device is used for large scale production of
recombinant eukaryotic protein through microbial/host
culture?
Ans: Bioreactor
v) Are genetically modified organisms (GMOs) and transgenic
organisms are same? Justify.
Ans: No, the terms are often used interchangeably but the key
difference between GMO and transgenic organism is that GMO is an
organism that has an artificially altered genome, while the transgenic
organism is a GMO that has an altered genome containing a DNA
sequence or gene from a different species.
3. The variation in size (length) of the restriction enzyme generated
fragments among individuals within a given species is termed RFLP. A
schematic representation of how RFLPs are generated is given in the
figure.
(i) The variation in size (length) of the restriction enzyme generated
fragments among individuals within a given species is due
to…………….. ?
a) Usage of different restriction enzymes
b) Variation in charge between the DNA of two individuals
c) Size of their genome.
d) Difference in their DNA sequence and position of Restriction site
Ans: d)
(ii) Which among is not a property of Restriction site?
a) Palindromic
b) Generally 4-8 bp long
c) Restriction sites are universal
d) They always get cleaved via even cuts
Ans: d)
(iii) The individuals that have same RFLP are -
a) No identical wins
b) Brothers and sisters
c) Mother and daughter
d) Cloned animals
Ans: d)
(iv) RFLP technique is used ina)
Parental diagnosis
b) Strain differentiation
c) Forensic science
d) All of these
Ans: d)