Genetic Engineering 2 Questions

1. What is the basic purpose of PCR?

 is a laboratory technique used to make multiple copies, amplify of a
segment of DNA.
2. How do you specify which DNA to amplify?

by using polymerase chain reaction, or PCR in three Cycle :

 First Put DNA Templet into to tube that contains primers, free
nucleotides, DNA polymerase, and the mixture is placed in a PCR machine.
increases and decreases the temperature of the sample in automatic,
programmed steps.
 First, the genomic DNA is briefly heated to separate the DNA strands
Then, annealing, the genomic DNA is cooled to allow primers to form
hydrogen bonds with ends of the target sequence.
Finally, in extension, the DNA polymerase adds nucleotides to the 3’ end
of each primer.
3. Why is Taq polymerase used in PCR?
 U-Taq DNA Polymerase is a thermostable enzyme that can withstand
prolonged incubation at temperature up to 95℃ without significant loss
of activity. Like DNA replication in an organism

4. Why is the DNA heated to 94C?

 the paired strands will separate (denature), by breakage of hydrogen
bonding present between complimentary bases
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5. How do you determine the hybridization or annealing
temperature for the primers to attach to the template?
 Determined by calculating the melting temperature (Tm) to begin with
annealing temperature 3–5°C lower than the lowest Tm of the primers.
 The time and temperature of this step can vary depending on the nature
of the template DNA and salt concentrations of buffer.
6. After PCR, what types of procedures are done with the amplified
fragments?
Method called electrophoresis can be used to check the quantity and size
of the DNA fragments produced
7. Plasmids are important in biotechnology. Give a full &complete
definition of plasmid.
 A plasmid (extrachromosomal DNAs) is a small double-stranded unit of
DNA, usually circular but sometimes linear, is capable of self-replication.
 that is used as a tool to insert genes into bacteria to encourage their
production of therapeutic proteins such as human insulin.
8. Why are bacterial plasmids widely used as cloning vectors?
 manipulated to form recombinant plasmids by insertion of foreign DNA
and then introduced into bacterial cells.
9. Why are both the gene of interest and the plasmid cut with
the same restriction enzyme?
creating matching sticky ends.

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 10. What is the role of DNA ligase in this process?

 DNA ligase is an enzyme which can connect two strands of DNA together by forming
a bond between the phosphate group of one strand and the deoxyribose group on
another. It is used in cells to join together the Okazaki fragments which are formed
on the lagging strand during DNA replication.
11. After transformation has occurred, why are some colonies blue?
 Colonies with non-recombinant plasmids will be blue because they
cannot be insert in the beta-galactosidase
12. Why are some colonies white? Why is this important?
 it is important because they are now ampicillin resistant. inserted into the
beta-galactosidase gene will not hydrolyze X-gal and will produce white
colonies. Therefore, white colonies are the desirable ones.
Getting a cloned eukaryotic gene to function in bacterial host
cells can be difficult. What are two problems with bacterial gene
expression systems, and how is each solved?
1. Gene expression varies between eukaryotes & prokaryotes,
2. Eukaryotes have introns and this prevents correct expression
in bacteria because they do not have RNA splicing machinery.
to solve this use yeast ( eukaryotic ) ,produce quickly
14. Why are the advantages of using yeasts as host for cloning
and/or expressing genes of interest?
A) they are eukaryotic cells
B) they do not have plasmids

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 C) only yeast cells allow the gene to be cloned
D) they easily form colonies
15. (PCR) is a Nobel Prize–winning idea that is used by scientists to
amplify DNA, particularly when the quantity of DNA is very small
or contaminated. Explain the three initial steps that occur in cycle
1 of PCR.
First, the genomic DNA is briefly heated to separate the DNA strands via
denaturation.
Then, through annealing, the genomic DNA is cooled to allow primers to
form hydrogen bonds with ends of the target sequence.
Finally, in extension, the DNA polymerase adds nucleotides to the 3’ end
of each primer.
16. Why is the DNA sample to be separated by gel electrophoresis
always loaded at the cathode or negative end of the power
source?
DNA fragments are negatively charged, so they move towards the
positive electrode. Because all DNA fragments have the same amount of
charge per mass, small fragments move through the gel faster than large
ones.
17.Explain why shorter DNA molecules travel farther down the gel
than larger molecules.
As a result the molecules are separated by size.

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 II. Select the best answer
1. Polymerase chain reaction basically consists of
two steps three steps four steps five steps
2. The original enzyme(s) used in PCR reaction is/are known as
A. E. coli DNA polymerase
B. E. coli RNA polymerase
C. Taq polymerase
D. all of these
3. What would the expected effect be on a PCR reaction if the primers used were
slightly shorter and more variable than the intended oligonucleotide sequences?
A. The PCR reaction would not commence
B. The PCR reaction would end after one cycle
C. The reaction would generate a single short PCR product
D. The reaction would yield a mixture of non-specific products
4. Which of the following does not act as a restriction enzyme?
EcorI BamHI HindIII polydeoxyribonucleotide synthase
5. E.cor1 is a
a) DNA ligase enzyme
b) Restriction endonuclease
c) A vector used for insulin synthesis
d) A plasmid used as a vector

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6. In which stage of genetic engineering a probe is used?
a) Cleaving DNA
b) Recombining DNA
c) Cloning
d) Screening
7. A DNA library is a
a) A DNA fragment inserted into a vector.
b) A general collection of all genes sequenced thus far.
c) All DNA fragments identified with a probe.
d) A collection of DNA fragments that make up the entire genome of a particular
organism.
8. Which of the following enzyme is required for end to end joining of DNA?
a) DNA ligase
b) Restriction endonuclease
c) RNA polymerase
d) DNA polymerase
9. Repressor molecules bind to
Operator Promoter Enhancer Hormone response element
10. Which of the following is untrue about DNA sequencing methods?
a) Purified fragments of DNA cut from plasmid/phage clones or
amplified by polymerase chain reaction (PCR)

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b) Clones of DNA fragments are denatured to single strands, and one of
the strands is hybridized to an oligonucleotide primer
c) Taq polymerase is quite heat sensitive
d) New strands of DNA are synthesized from the end of the primer
11. When setting up a Sanger sequencing reaction, each reaction should include
template DNA, nucleotides, dideoxynucleosides, buffer, DNA polymerase, and
a) Forward and reverse primers
b) Forward or reverse primers
c) Forward or reverse probes
d) Forward and reverse probes
12. Which of the following process is initiated by the promoter?
a) Translation
b) Replication
c) Apoptosis
d) Transcription
13. The promoter is recognized by……….
a) RNA polymerase DNA polymerase Replicas Helicase
14. Cloning vectors that can be used for recombinant protein production are

a) Expression vectors
b) Hybrid vectors
c) Hosts
d) Advanced vectors
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15. Placing a foreign gene under the control of expression signals will lead to its
a) Activation
b) Expression
c) Inactivation
d) Knockout
16. A recombinant DNA molecule is produced by joining together
1. one mRNA with a DNA segment
2. one mRNA with a tRNA segment
3. two mRNA molecules
4. Two DNA segments
17. Restriction endonucleases have the ability of cutting
1. DNA at random sites
2. DNA at specific sites
3. Both a and b
4. DNA and RNA at random sites
18. Before the production of recombinant insulin, insulin for the treatment of
diabetes in human was obtained from
1. healthy humans
2. dead human body
3. cows and pigs
4. dogs and cats

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19. Prior to the production of recombinant insulin, insulin obtained from cows and
pigs were given to patients. Some of the problems faced by this treatment was
1. the insulin was not active
2. in some humans it induced antibody production
3. it reduces the weight of patients
4. loss of memory power
20.A clone is a group of organisms produced by
1. asexual method and genetically similar
2. asexual method and genetically dissimilar
3. sexual method and genetically similar
4. sexual method and genetically dissimilar
21. Transgenic organisms are
1. produced by gene transfer technology
2. extinct organisms
3. naturally occurring and endemic
4. produced by traditional plant breeding technique
22. Which of the following statement about a vector is correct?
1. all vectors are plasmids only
2. plasmids, phages can be used as vectors
3. fungi can also be used as vectors
4. cyanobacteria can also be used as vectors

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23. Which of the following statement about plasmids is correct?
1. plasmids are present in bacteria only
2. plasmids are present in all organisms
3. plasmids present in bacteria and phages
4. plasmids present in plants and animals
24. Which one of the following statement are not attributed to plasmids
1. they are circular DNA molecule
2. they have antibiotic resistant genes
3. they have the ability of autonomous replication
4. they have DNA that is as long as chromosomal DNA
25. Which one of the following statements about plasmids is correct?
1. plasmids are mobile
2. plasmids are made up of RNA and proteins
3.plasmids are present in eukaryotes
4. Plasmids are present in fungi
26. DNA finger printing was first developed by
David Suzuki Khorana Alec Jaffreys Gilbert
27. Electrophoresis, a technique used in DNA fingerprinting helps to separate
1. DNA segments
2. cells from DNA
3. Tissues
4. RNA from DNA
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28. Dolly, the first animal produced through cloning is
Camel rat cow sheep
29. The main aim of human genome project is
1. to identify and sequence of all the genes present in the human body
2. to introduce new genes to human beings
3. to remove disease causing genes from humans
4. to improve techniques of finger printing
30. In biotechnology, mass culturing of cells / microbes can be achieved by using
1. Test tube culture
2. Bioreactor
3. Autoclave
4. electrophoresis
31. Bioreactors are used for
1. large scale production of desired substances by using cells / microbes
2. kill bacteria
3. to store viruses
4. to get chemicals
32. The basic components of tissue culture media are
1. micro and macro nutrients, glucose
2. micro and macro nutrients, vitamins, agar
3. micro and macro nutrients and growth regulators, glucose
4. micro and macro nutrients, growth regulators, agar, vitamins, glucose
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33. A transgenic plant “Golden rice” consists of foreign genes that product
1. β – Carotene niacin biotin nicotinic acid
34. The “Golden rice”, aimed at curing
1. vitamin b deficiency
2. vitamin a deficiency
3. vitamin k deficiency
4. zinc deficiency
35. The cloned sheep “Dolly” had a genotype which is
1. haploid and identical to that of the mothers egg cell
2. diploid and identical to that of the mothers somatic cells
3. diploid with the haploid set of chromosomes from the father and other from the
mother
4. diploid and identical to that of the donors somatic cells
36. In an experiment, recombinant DNA bearing ampicillin-resistance gene is
transferred into E.coli cells. The host cells are then cultured on a medium containing
ampicillin. The result will be
1. both transformants and non-transformants cannot survive.
2. both transformants and non-transformants can survive.
3. transformants only and not the non-transformants can survive.
4. transformants cannot survive, but non-transformants can survive.
37- A multiclonic site (MCS)
1- a DNA sequence composed of recognition motifs for a number of restriction
endonucleases
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2- a DNA sequence composed of recognition motifs for a number of restriction
exonucleases
3- a DNA sequence composed of recognition motifs for a number antibiotic
4- a protein sequence composed of recognition motifs for a number of restriction
endonucleases
38- Ti plasmid is found in
1- E. coli
2- Agrobacterium
3- Fungus
4- Bacillus
39- A gene delivery method
1- Micro projectile bombardment
2- Recombination
3.E. coli
4.Fangus
40- A reporter gene for transformed plants
GFP IPTG Xgal ABC transporter
41- Eco friendly insecticide
1- Bacillus thuringiensis BT, CP
2- Pseudomonas
3- Rhizobacteria
4- DTT

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42- BR322 is a
1- cloning vector
2- expression vector
3- promoter
4- operator
43- bla gene, encoding beta-lactamase, confers resistance to
1- kanamycin
2- ampicillin
3- gentamycin
4- octamycin
44- The Gateway Technology cloning system is a universal cloning method based on
the
1- site-specific recombination
2- endonucleases
3- exonucleases
4- ligase enzyme
III. Complete the components of the standard protocol of:
A. PCR reaction
TAQ polymerase PCR Buffer - MgCl2 dNTPs -Target DNA
H2O - Primers
B. EcoR1 digestion o
DNA
Restriction enzyme

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 10X buffer
Sterile water
C. Ligation reaction
T4 DNA Ligase
Buffer (10X)
Vector DNA
Insert DNA
Nucleus free water
T4 DNA Ligase
A) Gateway cloning system
1. BP Reaction
2. attl
3. Entry clone
4. destination vector
5. LR clonase

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 B) E. coli Expression vector

1. T7
2. GCT
3. ampicillin

Define:
1) 35S promotor
The promoter used in the Roundup Ready gene is the 35S promoter expressed in all
plant cells
2) Glyphosate
Glyphosate is a unique molecule. There’s no other herbicide like it.
It inhibits an essential plant enzyme called EPSPS ,prevents production of aromatic
amino acids required for protein synthesis.
Glyphosate enters a plant through Flagella ; the amount of glyphosate and speed of
entry depend on plant species and glyphosate delivery system.

3) Price look up (PLU) codes

commonly called PLU codes, are identification stores and supermarkets to make
check-out easier. The code may be a four or five number.

4) enolpyruvyl-shikimate synthase
EPSPS is an enzyme that catalyzes a reaction in plant and bacterial cells that is
necessary for the synthesis of some amino acids.

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5) Monsanto
Monsanto Company is American multinational agrochemical and agricultural
biotechnology. producer of genetically engineered (GE) seed and Roundup, a
glyphosate-based herbicide.
6) Expressing vectors
Are vectors enables a particular gene to be expressed in a host cell which contain
regulatory sequences required to achieve gene expression e.g. strong promoter,
ribosome binding site, transcriptional terminator.

7) T0 transgenic lines
the Arabidopsis plants that are generated directly from transformation either by
agrobacterium or gene gun.

8) Binary vectors
the Binary vectors is a shuttle vector, so called because its able to replicate in
multipule hosts (pro & Eu) ex: (E. coli and Agrobacterium tumefaciens)
9) Inducible promotor
Are promoters which can be induced by the presence or absence of certain factors
e.g. lac operon is induced in the presence of lactose or Round up genes in plants

10) Signal peptide

A signal peptide is a short peptide present at the N-terminus of the majority of newly
synthesized proteins, it is essential because it directs the protein where it should go
e.g. chloroplast transit peptide (CTP). They direct the EPSPS protein to go to the
chloroplasts in the plant cell)

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2- Write a strategy for the following famous GMOs
1- Insulin production from bacteria
 There are several strategies for producing insulin from bacteria, but the most
successful is to synthesize the A and B separately and then join them together
• The gene sequence of determining the A chain has been fused to the ß
galactosidase gene (lac Z) of E. coli. The whole Lac-Z-A chain fusion is cloned into
pBR322. Bacteria with this plasmid synthesize ß-galactosidase with the insulin A
chain.
• The B chain is produced in an identical manner.
• After purification of the two chains they are mixed, oxidized and then reduced
which allows the disulfide bridges to form and active insulin to be produced.

 Steps of human insulin production by E. coli

1) isolate human cells and grow in tissue culture.

2) isolate RNA from the human cells and converted to cDNA.
3) isolate plasmid DNA from bacteria.
4) Use the same restriction enzyme to cut the plasmid DNA
5) Mixing the recombinant plasmid with bacteria.
6) Allow new bacteria to incorporate the recombinant plasmid into the bacterial cell,

2- Producing plants with glyphosate tolerant gene

• Glyphosate inhibits EPSPS, and it is a non-selective systemic herbicide

The version of EPSPS made in a special strain of Agrobacteria has resistance to
Roundup. The steps Producing plants with glyphosate tolerant gene:

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1) Isolate the EPSPS bacterial gene (which has resistance to roundup).
2) Construct the vector which consists of the EPSPS and the 35s promotor and the
CTP coding segment to direct the EPSPS protein to the chloroplasts.
3) Transform the vector to the agrobacterium.
4) Infect the target plant with the agrobacterium which has the recombinant.
5) Apply the glyphosate to the target plants to find out the resistant plants.

3- What are the risks of having GMO products?

1) GMOs could be unhealthy.
2) Antibiotics for selection.
3) Promoters of recombinant genes.
4) GMOs contaminate.
5) GMOs increase herbicide use.
6) Genetic engineering creates dangerous side effects

4- What are the main aspects of plant binary vectors?

The binary vector is a shuttle vector, so-called because it is able to replicate in
multiple hosts (E. coli and Agrobacterium tumefactions).
This plasmid typically contains:
1) foreign DNA in place of T-DNA
2) the left and right T-DNA borders
3) markers for selection and maintenance in both E. coli and A. tumefaciens.
4) a selectable marker for plants. (antibiotic or herbicide resistance).

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