GENE CLONING

The production of exact copies of a particular gene or DNA sequence using genetic engineering
techniques is called gene cloning.

The term “gene cloning,” “DNA cloning,” “molecular cloning,” and “recombinant DNA technology” all
refer to same technique. When DNA is extracted from an organism, all its genes are obtained. In gene
(DNA) cloning a particular gene is copied forming “clones”. Cloning is one method used for isolation and
amplification of gene of interest.

DNA cloning can be achieved by two different methods:

1. Cell based DNA cloning

2. Cell-free DNA cloning (PCR)

Requirements for Gene Cloning (Cell-based)

1. DNA fragment containing the desired genes to be cloned.

2. Restriction enzymes and ligase enzymes.

3. Vectors – to carry, maintain and replicate cloned gene in host cell.

4. Host cell– in which recombinant DNA can replicate.

Principle of Gene Cloning

A fragment of DNA, containing the gene to be cloned, is inserted into a suitable vector, to produce a
recombinant DNA molecule. The vector acts as a vehicle that transports the gene into a host cell usually
a bacterium, although other types of living cell can be used. Within the host cell the vector multiplies,
producing numerous identical copies not only of itself but also of the gene that it carries. When the host
cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector
replication takes place. After a large number of cell divisions, a colony, or clone, of identical host cells is
produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule; the
gene carried by the recombinant molecule is now said to be cloned.
Steps in Gene Cloning

The basic 7 steps involved in gene cloning are:

1. Isolation of DNA [gene of interest] fragments to be cloned.

2. Insertion of isolated DNA into a suitable vector to form recombinant DNA.

3. Introduction of recombinant DNA into a suitable organism known as host.

4. Selection of transformed host cells and identification of the clone containing the gene of interest.

5. Multiplication/Expression of the introduced Gene in the host.

6. Isolation of multiple gene copies/Protein expressed by the gene.

7. Purification of the isolated gene copy/protein

A. Isolation of the DNA fragment or gene

The target DNA or gene to be cloned must be first isolated. A gene of interest is a fragment of gene
whose prod-uct (a protein, enzyme or a hormone) interests us. For example, gene encoding for the
hormone insulin.

The desired gene may be isolated by using restriction endonuclease (RE) enzyme, which cut DNA at
specific recognition nucleotide se-quences known as restriction sites towards the inner region (hence
endonuclease) producing blunt or sticky ends. Sometimes, reverse transcriptase enzyme may also be
used which synthesizes complementary DNA strand of the desired gene using its mRNA.

B. Selection of suitable cloning vector

The vector is a carrier molecule which can carry the gene of interest (GI) into a host, replicate there
along with the GI making its multiple copies.

The cloning vectors are limited to the size of insert that they can carry. Depending on the size and the
application of the insert the suitable vector is selected.

The different types of vectors available for cloning are plasmids, bacteriophages, bacterial artificial
chromosomes (BACs), yeast artificial chromosomes (YACs) and mammalian artificial chromosomes
(MACs). However, the most commonly used cloning vectors include plasmids and bacteriophages
(phage λ) beside all the other available vectors.
Fig. 1: Steps in gene cloning
C. Essential Characteristics of Cloning Vectors

All cloning vectors are carrier DNA molecules. These carrier molecules should have few common
features in general such as:

1. It must be self-replicating inside host cell.

2. It must possess a unique restriction site for RE enzymes.

3. Introduction of donor DNA fragment must not interfere with replication property of the vector.

4. It must possess some marker gene such that it can be used for later identification of recombinant cell
(usually an antibiotic resistance gene that is absent in the host cell).

5. They should be easily isolated from host cell.

D. Formation of Recombinant DNA

The plasmid vector is cut open by the same RE enzyme used for isolation of donor DNA fragment.

The mixture of donor DNA fragment and plasmid vector are mixed together.

In the presence of DNA ligase, base pairing of donor DNA fragment and plasmid vector occurs.

The result-ing DNA molecule is a hybrid of two DNA molecules – the GI and the vector. In the
ter-minology of genetics this intermixing of dif-ferent DNA strands is called recombination.

Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the technology is
referred to as the recom-binant DNA technology.

E. Transformation of recombinant vector into suitable host

The recombinant vector is transformed into suitable host cell mostly, a bacterial cell.

This is done either for one or both of the following reasons:

To replicate the recombinant DNA mol-ecule in order to get the multiple copies of the GI.

To allow the expression of the GI such that it produces its needed protein product.

Some bacteria are naturally transformable; they take up the recombinant vector automatically.

For example: Bacillus, Haemophillus, Helicobacter pylori, which are naturally competent.

Some other bacteria, on the other hand require the incorporation by artificial methods such as Ca++ ion
treatment, electroporation, etc.
F. Isolation of Recombinant Cells

The transformation process generates a mixed population of transformed and non-trans- formed host
cells.

The selection process involves filtering the transformed host cells only.

For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed.

For examples, PBR322 plasmid vector contains different marker gene (Ampicillin resistant gene and
Tetracycline resistant gene. When pst1 RE is used it knock out Ampicillin resistant gene from the plasmid,
so that the recombinant cell become sensitive to Ampicillin.

G. Multiplication of Selected Host Cells

Once transformed host cells are separated by the screening process; becomes necessary to provide
them optimum parameters to grow and multiply. In this step the transformed host cells are introduced
into fresh culture media. At this stage the host cells divide and re-divide along with the replication of the
recom-binant DNA carried by them.

If the aim is obtaining numerous copies of GI, then simply replication of the host cell is allowed. But for
obtaining the product of interest, favourable conditions must be provided such that the GI in the vector
expresses the product of interest.

H. Isolation and Purification of the Product

The next step involves isolation of the multiplied GI attached with the vector or of the protein encoded
by it. This is followed by purification of the isolated gene copy/protein.

Applications of Gene Cloning

1. A particular gene can be isolated and its nucleotide sequence determined

2. Control sequences of DNA can be identified & analyzed

3. Protein/enzyme/RNA function can be investigated

4. Mutations can be identified, e.g. gene defects related to specific diseases Organisms can be
‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.
 DNA Fingerprinting

Definition of DNA Fingerprinting

"DNA fingerprinting is a procedure that shows the hereditary cosmetics of living things. It is a strategy
for finding the distinction between the satellite DNA areas in the genome."

DNA profiling, DNA testing, DNA examination, Genetic profile, DNA distinguishing proof, genetic
fingerprinting, and genetic investigation are a portion of the mainstream names utilized for DNA
fingerprinting. This technique was invented by Alec Jeffreys in 1984.

Principle of DNA fingerprinting

The human genome consists of innumerable small noncoding sequences which are inheritable and
repeatedly present. They can be separated from the bulk DNA as satellite upon performing density
gradient centrifugation and thus known as satellite DNA. They can be categorized into either
microsatellites or microsatellites depending on the length, base composition and tandemly repetitive
units. These satellite DNAs show polymorphism and this polymorphism is the basis of DNA fingerprinting.
The repeat regions can be divided into two groups based on the size of the repeat - variable number
tandem repeats (VNTRs) and short tandem repeats. These repeats act as genetic markers and every
individual inherits these repeats from their parents. Thus, every individual has a particular composition
of VNTRs and this is the main principle of the DNA fingerprinting technique.

DNA Fingerprinting Steps

1. Collection of organic example blood, spit, buccal swab, semen, or solid tissue.

2. DNA extraction.

3. Restriction absorption or PCR intensification.

4. Agarose gel electrophoresis, slim electrophoresis or DNA sequencing.

5. Interpreting outcomes.

The Process of DNA Fingerprinting

Sample collection, DNA extraction, absorption or intensification and investigation results are significant
advances.

Stage 1: Sample Collection

DNA can be acquired from any bodily sample or liquid. Buccal smear, salivation, blood, amniotic liquid,
chorionic villi, skin, hair, body liquid, and different tissues are significant kinds of samples utilized.
Stage 2: DNA Extraction

We need to initially get DNA. To play out any genetic applications, DNA extraction is one of the most
significant advances. Great quality and amount of DNA expands the conceivable outcomes of getting
better outcomes.

You can utilize DNA extraction strategies enrolled beneath,

1. Phenol-chloroform DNA extraction strategy

2. CTAB DNA extraction strategy

3. Proteinase K DNA extraction strategy

In any case, we emphatically prescribe utilizing a ready to go DNA extraction unit for DNA fingerprinting.

Filter the DNA utilizing the DNA sanitization unit, if necessary.

From that point onward, measure the DNA utilizing the UV-Visible spectrophotometer. Furthermore,
perform one of the accompanying strategies recorded underneath.

DNA Fingerprinting Strategies

Stage 3: Restriction Absorption, Enhancement or DNA Sequencing

Three regular strategies are utilized:

RFLP based STR investigation

PCR based investigation

Real-time PCR investigation

Stage 4: Analysis of Results

As we examined, utilizing the southern blotting, agarose gel electrophoresis, narrow electrophoresis,
ongoing intensification, and DNA sequencing, the outcomes for different DNA profiling can be gotten in
which rt-PCR and sequencing are much of the use in forensic science.

Stage 5: Interpreting Results

By looking at DNA profiles of different examples, varieties and likenesses between people can be
distinguished. Outstandingly, the whole procedure is presently nearly automatic. We don't need to do
anything, the computer gives us conclusive outcomes.
Applications of DNA Fingerprinting

Utilizing the DNA fingerprinting strategy, the natural personality of an individual can be uncovered. For
approving one's character, there is no other preferable alternative over DNA fingerprinting.

Gravely harmed dead bodies can be distinguished.

It is utilized to detect maternal cell contamination.

One of the significant downsides of pre-birth determination is maternal cell tainting. The amniotic liquid
or CVS test contains the maternal DNA or maternal tissue, once in a while. Contamination expands the
opportunity of false-positive outcomes, particularly on account of carrier recognition. Utilizing VNTRs
and STRs markers with PCR-gel electrophoresis, maternal cell tainting can be recognized during
pregnancy hereditary testing.

One of the most significant uses of the current strategy is in the crime scene examination and criminal
check. The example is gathered from the crime site which could be salivation, blood, hair follicle, or
semen. DNA is removed and investigated against the suspect, utilizing the two markers we clarified
previously. By coordinating DNA band designs criminal's connected to wrongdoing can be built up.

Utilizing Blood-Typing in Paternity Tests

The procedure of DNA fingerprinting was discovered by Alec Jeffreys in 1984, and it originally opened up
for paternity testing in 1988. Before this kind of DNA investigation was accessible, blood classifications
were the most widely recognized calculation considered human paternity testing. Blood bunches are a
mainstream case of Mendelian hereditary qualities at work. All things considered, there are various
human blood bunches with numerous alleles, and these alleles display a scope of predominance designs.

DNA Fingerprinting and Farming

A few DNA minisatellite tests have yielded piece profiles that show up valuable for plant reproducing
work. These part profiles show no variety when vegetative spread material is broken down. So also,
examples obtained through self-inbreeding species show indistinguishable profiles. Interestingly,
hereditary recombination in cross-pollinating species brings about exceptionally factor, normally
singular, explicit piece profiles. Along these lines various cultivars can be recognized, as additionally can
genotypes of wild species in characteristic populaces. These piece profiles can likewise be used in
parentage examination, as has just been led in rice and apples, in this way empowering us to explain the
source of deficiently recorded cultivars. Also, evaluations of hereditary variety dependent on similitude
lists determined from section profiles show a nearby relationship with known degrees of hereditary
relatedness.