Modern Genetic Technology

Modern Genetic Technology

 By using special techniques and procedures, geneticists can alter an organism’s DNA or
genome.

 These will involve gene manipulation and its field studies are called genetic engineering
or recombinant DNA technology.

Main components of Recombinant DNA technology :-

 DNA restriction fragments.
o Each fragment is a sequence of nucleotides produced by the cutting action of
restriction enzyme.
o It contains the gene of interest - gene to be manipulated through genetic
engineering.

 Restriction enzymes / restriction endonuclease enzyme.
o Enzymes that are used to cut DNA into fragments.

 DNA ligase.
o Enzyme use in joining two DNA fragments.

 Recombinant DNA (rDNA) / Recombinant Plasmid.
o It has a foreign gene (gene of interest) / DNA fragment from another source.

 Bacterial plasmid / Virus plasmid.
o Plasmid is a small circular DNA found in the protoplasm of a bacterial cell.

 Cloning vector.
o Bacterial plasmid / Virus plasmid virus which can transfer gene or DNA fragments
from a donor/source to a recipient.

 Cloning.
o Cloning is an amplification process producing many copies of a gene or DNA
fragment by using bacteria as the cloning agent.

 cDNA.
o It is a DNA produced by using mRNA as template.
o A reverse transcriptase enzyme is needed to form a cDNA.

 Genetic marker.
o A specific part of DNA which marks a particular gene.
o This part has a specific restriction sites which can be cut by specific restriction
enzymes.

 Nucleic acid probe.
o A short sequence of nucleotides used to find certain segments of DNA or genes in
a genome.

 DNA sequencing.
o It is a technique used to determine nucleotide sequence of a gene.
o This enables geneticists to synthesize a gene in vitro.

 DNA hybridization.
o It is process of attaching a probe to a DNA strand through base pairing.
o This process is useful for locating a gene in the chromosome.



About Plasmid :-

 Carry only one or a few genes; genes carried in plasmids provide bacteria with genetic
advantage such as antibiotic resistance.

 It can replicate on its own and it is not part of the bacterial chromosome.
 Have many restriction sites.
 Can easily be isolated and purified from bacterial culture.
 Easy to modify and can be inserted / reintroduce back into bacterial cell.
 R-plasmids / Resistance-plasmids contain genes that give resistance against antibiotics

like tetracycline and ampicillin or poison to bacteria.
 It can be transferred from one bacterium to other bacteria through conjugation.

 In DNA cloning - R-plasmids
are used to separate
bacteria colonies that carry
the required genes from
those that do not carry the
required genes.

 This is done by exposing the
bacteria colonies to a culture
that contains these
antibiotics.

 As a result, bacteria cells that
carry the recombinant
plasmids (contain the
required genes / gene of
interest) can easily be
identified and isolated.

About Restriction Endonuclease enzyme :-
 The action of a restriction enzyme is very specific.
 Restriction endonuclease enzyme locates the specific base sequence / genetic marker
of a gene in a DNA and then cuts at that site.

 Some RE make clean and complete cut through both the DNA strands at the restriction
sites, producing DNA restriction fragment with blunt ends. Eg. PvuII and HindII.

 Some RE make incomplete cut, producing DNA restriction fragment with sticky ends. Eg.
EcoRI and BamHI.

 Cutting a DNA molecule with BamHI produces DNA restriction fragments with sticky ends
CTAG and GATC.

 A DNA fragment with a sticky ends CTAG can join with any DNA fragment with a sticky
ends GATC.

 DNA ligase enzyme forms the covalent bonds that joined the two DNA restriction
fragments together – Recombinant DNA.


DNA Cloning :-
 It is a process of transferring a DNA fragment (containing the gene of interest) from one
organism (donor) to another organism (recipient) by a self-replicating genetic element
such as bacterial plasmid (cloning vector).

An Overview - Steps in gene cloning :-
 The DNA fragment containing the gene of interest is isolated / cut from the chromosomal
DNA by using specific restriction enzyme.
 It is then unites with a bacterial plasmid that has been cut with the same restriction
enzyme.
 The recombinant DNA / plasmid is reintroduced back to the host / bacteria.
 This is done either for one or both of the following reasons :
o To replicate the recombinant DNA / plasmid molecule by cloning in order to get
multiple copies of the gene of interest (GOI).
o To let the GOI get express and produce desired protein.

Using R-plasmid in making multiple copies of the GOI :-

Notes :
 Bacteria cells with the R-plasmid can grow in the medium treated with ampicillin or
tetracycline.

Using Ti-plasmid to alter certain trait in plant.

 Agrobacterium tumefaciens is a widespread naturally occurring soil bacterium that
causes crown gall (tumor), it has the ability to introduce new genetic material into the
plant cell (Gelvin, 2003).

 The genetic material that causes the Crown Gall disease is situated in a fragment called
the T-DNA (transferred DNA) found in Ti plasmid.

 During infestation, the Ti-plasmid injects the T-DNA fragment into the host’s nucleus and
later the bacteria’s T-DNA is incorporated into the plant’s DNA.

Agrobacterium-mediated Plant Transformation Process :-

 The Agrobacterium-mediated transformation process involves a number of steps:
o Isolation of the genes of interest from the source organism.
o Isolation of the Ti-plasmid from the bacteria.
o Insertion of the transgene (GOI) into the T-DNA segment on the Ti-plasmid.
o Introduction of the transformed Ti-plasmid into the bacteria.
o Mixture of the transformed bacteria with plant cells to allow transfer of the T-DNA
into the plant chromosome.
o Regeneration of the transformed plant’s cells into genetically modified (GM)
plants and
o Testing for trait performance or transgene expression at lab, greenhouse and field
level.

Production of human insulin by bacteria :-
 E. coli bacterium is the most common species of bacteria chosen to synthesize human
insulin.
 Insulin production by E. coli involves these following steps.

 The gene responsible for the production of insulin in human is extracted from the human
pancreatic cells by using restriction enzyme.

 Bacteria’s plasmids are isolated from the bacterial cell.
 Both are then cut open by using the same restriction enzyme.
 The DNA restriction fragment with the human insulin gene is inserted into the bacterial

plasmid forming recombinant plasmid.
 The two DNAs are joined together by DNA ligase.
 The recombinant plasmid is then reintroduced back to the bacteria forming transgenic

bacteria.
 The bacteria are given time to grow and reproduce in a culture medium.
 The insulin gene in the bacteria is expressed when the bacteria colony is exposed to high

sugar concentration in the culture medium.
 As a result, insulin is synthesized by the bacteria.
 By culturing the transgenic bacteria, limitless supplies of insulin will be available for future

use.

Forensic investigation : DNA Fingerprinting / DNA profiling.
 DNA Fingerprinting / DNA profiling methods can be applied in forensic investigation to
identify an individual’s unique DNA fingerprint.
 DNA is extracted from a biological specimen.
 It is then cut with restriction enzyme to produce smaller DNA fragments – Restriction DNA
fragments.
 Agarose gel electrophoresis is used to separate these small Restriction DNA fragments.
o Restrictions DNA fragments will move in the Agarose gel at different rate
depending on their length and size to form a pattern of DNA bands.
o Shorter DNA fragments travel through the pores of the Agarose gel matrix faster
than longer ones.
 Once separated, the Restriction DNA fragments are transferred to a nitrocellulose or
nylon filter / membrane.
 This is done by placing a nylon filter / membrane on the gel, soaking them overnight.
 This process is called Southern Blotting.


 So who is the prime suspect? #1, #2, #3?



Polymerase Chain Reaction (PCR) :-
 Polymerase chain reaction (PCR) is a common laboratory technique used to make many
copies of DNA fragment containing the GOI in a DNA / Amplifying DNA fragment by
means of in vitro (in a test tube rather than an organism).
 PCR technique requires DNA polymerase enzyme that makes new strand of DNA, using
existing DNA strand as template.
 A primer, which is a short sequence of nucleotides (single stranded DNA) is used to
provide the starting point for DNA strand synthesis.
 Two primers are used in each PCR reaction, and they are designed so that they flank the
target region with the GOI.

 When the primers are bound to the template, both can be extended by the DNA
polymerase enzyme and the region that lies between them will get copied.

 Next : Denaturation (96oC)of the DNA strands.
 Heat is applied to separate, or denature, the DNA strands.
 This provides single-stranded template for the next step.
 Next : Annealing (55-65°C): After cooling off, the primers can bind to their

complementary sequences on the single-stranded template DNA.
 Next : Extension (72°C): The reaction temperatures is raised up ; DNA polymerase enzyme

extends the primers, synthesizing new strands of DNA.

Genetic screening :-

 Genetic screening involves detection of mutant (defective) genes in an individual.

Chorionic villus sampling (CVS) - Genetic screening for gene defect.

 Usually done between 8 -12 weeks of pregnancy.
 A (sterile hypodermic) needle is insured through abdominal wall / catheter is inserted

through cervix into the uterus of a pregnant woman.
 Position of needle/catheter and foetus is monitored by ultra sound scanner.
 A small sample of chorionic villus containing living foetal cells is removed.
 Biochemical analysis / DNA analysis is conducted to determine the karyotype of the

foetal’s cell.
 This procedure detects chromosomal abnormalities / genetic / metabolic disorder of the

foetus // to provide genetic counselling.

Gene therapy :-

 Gene therapy is a procedure in which genes within cells are removed, replaced, or
altered to produce new proteins that change the function of the cells // Gene therapy is
the technique in genetic engineering to replace a faulty gene by a normal healthy and
functional gene.

 Gene therapy is designed to introduce genetic material into cells to compensate for
abnormal genes or to make a beneficial protein.

 If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may
be able to introduce a normal copy of the gene to restore the function of the protein.

 Certain retroviruses are often used as vectors to deliver the new gene by infecting the
cell.

 These viruses are modified so they can't cause disease when used in people.
 Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but

the DNA is not integrated into a chromosome.
 These vectors can be injected or given intravenously (by IV) directly into a specific tissue

in the body, where it is taken up by individual cells.

Sample questions :
1. The summary of the procedure to clone a gene is shown in the diagram below.

What processes are represented by I, II, III and IV in the above diagram?

I II III IV

A Isolation of donor Transformation Action of restriction Insertion and

DNA and vector endonuclease ligation

DNA

B Insertion and Action of restriction Isolation of donor Transformation

ligation endonuclease DNA and vector

DNA

C Isolation of donor Action of restriction Insertion and Transformation

DNA and vector endonuclease ligation

DNA

D Action of Isolation of donor Transformation Insertion and

restriction DNA and vector ligation

endonuclease DNA

2. The replication of DNA is shown in the diagram below. Which is true of P, Q and R?

P Q R
A. Leading strand Okazaki fragment Template
B. Leading strand Template Okazaki fragment
C. Template Leading strand Okazaki fragment
D. Template Okazaki fragment Leading strand

3. Which enzyme is used to produce complementary DNA (cDNA) from mRNA?
A. Restriction enzyme
B. Reverse transcriptase
C. DNA ligase
D. RNA primase

4. Which arrangement of the following four enzymes represents the order in which they
would be used in a typical gene-cloning experiment resulting in the insertion of a cDNA
into a bacterial plasmid? Begin with the gene’s mRNA transcript.
A. reverse transcriptase, restriction enzyme, DNA polymerase, DNA ligase
B. restriction enzyme, reverse transcriptase, DNA polymerase, DNA ligase
C. reverse transcriptase, DNA polymerase, restriction enzyme, DNA ligase
D. restriction enzyme, DNA ligase, reverse transcriptase, DNA polymerase

5. The following describes the steps involved in a DNA fingerprinting process.
I DNA is treated with restriction endonuclease.
II Fragments of DNA are separated by gel electrophoresis.
III Radioactive probes with complementary base sequence are added.
IV Exposed to X-ray film.

Which of the following is the correct sequence of the steps?

A. III, I, II, IV B. I, II, III, IV

C. II, I, IV, III D. I, II, IV, III

6. In the field of genetic engineering, plants can be manipulated more easily than animals
because
A. A transformed somatic cell can divide and differentiate to form a new plant.
B. Recombinant genes can be inserted into plant cells through microinjection.
C. Plant genes do not contain introns.
D. More vectors can be used to transfer recombinant DNA into plant cells.

7. The diagram below shows a procedure in human gene cloning.

a) (i) What is the role of the plasmid in this procedure ?
Plasmid acts as a cloning vector.

[ 1 mark ]

(ii) Why plasmid is suitable to play the role you mentioned above?
Plasmid is a circular DNA which can easily be inserted with human DNA.
Plasmid has a small size, so it is easy to manipulate/ modify.
Plasmid can cause transformation/ can be inserted back into the
bacterial cells for cloning process.
[ 2 marks ]

b) The plasmid used must have different markers. What is the importance of these
markers ?
With these markers, its presence in the bacterial cell can be detected.
It is important for screening process to select the bacteria which have
successfully taken in the foreign DNA.
[ 2 marks ]

c) State three other properties of the plasmid used. Explain.
Genes carried by the plasmid can express themselves, so the inserted foreign
gene can also be expressed.
It has different restriction sites which can be cut open by restriction enzymes, this
enable foreign DNA to be inserted into the opened plasmid.
Plasmid is common in bacteria // Plasmid is found in large number in bacteria, so
it is not difficult to isolate the plasmid.
[3 marks]

8. Apart from producing beneficial transgenic organisms, DNA technology is also applied in
medical procedures such as genetic screening and gene therapy.

(a) What is genetic screening?
Genetic screening involves detection of mutant (defective) genes in an
individual.
[ 1 mark ]

(b) The diagram below shows one of the methods of genetic screening, often used
to detect and identify certain serious genetic diseases in the unborn baby.

Identify the method above.
Chorionic villus sampling.

[ 1 mark ]
(c) Briefly describe gene therapy.

Gene therapy is a procedure in which genes within cells are removed, replaced,
or altered to produce new proteins that change the function of the cells.
// Gene therapy is the technique of genetic engineering to replace a faulty gene
by a normal healthy functional gene.

[ 1 mark ]
(d) The diagram below shows the steps involved in gene therapy.

Human RNA with normal allele

Step 1

Retrovirus Step 2
Step 3
Bone marrow cell
of a patient

Step 4 Bone
Bone marrow

Briefly describe the steps involved in the method above.

Step 1: Human RNA with normal allele is inserted into retrovirus RNA.

Step 2: Retrovirus is allowed to infect bone marrow cells which have been

removed from the patient and cultured.

Step 3: Bone marrow cells carrying chromosome with the Viral DNA are

injected back into the bone marrow of the patient.

[ 3 marks ]

(e) Name this type of gene therapy

Ex Vivo Gene Therapy

[ 1 mark ]

9. In a genetic engineering experiment, a synthetic gene is inserted into a bacterial host as
shown in the diagram below.

(a) What term is used to describe the function of the plasmid in this experiment?

Vector.

[ 1 mark ]

(b) State one method by which a copy of the synthetic gene can be produced.

Use of reverse transcriptase enzyme on mRNA to produce cDNA.

[ 1 mark ]

(c) (i) State the function of the marker gene in the plasmid.

To identify transformed bacterial cells.

(ii) Give an example of a marker gene.

Gene for synthesis of ampicillin // Gene for synthesis of β-galactosidase.

[ 1 mark ]

(d) Name the type of enzyme used at Z and state its function.

DNA ligase – To join the sugar-phosphate backbones in the recombinant DNA

molecule // join the two DNA fragments.

[ 2 marks ]

(e) Genetically engineered human insulin is now used in the treatment of diabetes.

State two advantages of using this type of insulin.

 The probability of rejection or dangerous side effects is low.

 Insulin can be produced in large quantities.

 The killing of large number of animals is not required.

 The cost involved is lower.

[ 2 marks ]