Gene cloning involves copying a gene and inserting it into a self-replicating vector like a bacterial plasmid. This allows copies of the gene to be made for applications like sequencing, mutagenesis, and protein expression. PCR is a newer gene cloning technique that uses thermal cycling to rapidly amplify a target gene sequence. Both techniques allow researchers to isolate genes and study them in detail. Gene cloning and PCR have many important uses in fields such as medicine, biotechnology, forensics, and more.

1. GENE CLONING
By: - Divesh

2. Introduction
 Gene cloning is a common practice in molecular biology labs
 It is used to create copies of a particular gene for downstream applications, such as sequencing,
mutagenesis, genotyping or heterologous expression of a protein.
 The traditional technique for gene cloning involves the transfer of a DNA fragment of interest from one
organism to a self-replicating genetic element, such as a bacterial plasmid.
 This technique is commonly used today for isolating long or unstudied genes and protein expression.
 A more recent technique is the use of polymerase chain reaction (PCR) for amplifying a gene of interest.
 The advantage of using PCR over traditional gene cloning, is the decreased time needed for generating a
pure sample of the gene of interest.
 However, gene isolation by PCR can only amplify genes with predetermined sequences. For this reason,
many unstudied genes require initial gene cloning and sequencing before PCR can be performed for
further analysis.

3. What is gene cloning?
 A clone is an exact copy of an organism, organ, single cell, organelle or macromolecule.
 Cell lines for medical research are derived from a single cell allowed to replicate millions
of times, producing masses of identical clones.
 Gene cloning is the act of making copies of a single gene.
 Cloning can provide a pure sample of an individual gene, separated from all the other
genes that it normally shares the cell with.
 Once a gene is identified, clones can be used in many areas of biomedical and industrial
research.
 Genetic engineering is the process of cloning genes into new organisms, or altering a
genetic sequence to change the protein product.

4. History
 In 1922, Morgan and his colleagues developed the technique for gene mapping.
 In 1903,W.sutton proposed the idea of a gene residue on chromosome.
 By 1922, they had analysis the relative positions of over 2000 genes on the 4th
chromosomes of fruit fly, drosophila melongata.
 Until 1940’s, there was no real understanding of molecular nature of gene.
 In 1944, the experiments of Avery, McLeod, and McCarty and in 1952, Hershey and
chase, stated that DNA ( deoxyribose nucleic acid) is the genetic material: up until then it
was thought that genes were made up of protein.
 Discovery in the role of DNA was tremendous stimulus to the genetic research.
 Delbruck, Chargaff, crick and monad, contributed in the second great age of genetics.

5.  Between 1952 and 1966, in this years the structure of DNA was elucidated, the genetic code cracked,
and the process of transcription and translation.
 There was a period of anti-climax, some molecular biologist was in state of frustration that the
experimental techniques of the late 1960’s were not sophisticated enough to allow the gene to
studied in any greater extends.
 During 1971-73, there was a revolution thrown back into gear by introducing completely new
methodology, recombinant-DNA technology or genetic engineering.
 This new methodology as heir core in the process of gene cloning, it sparkled as another great age of
genetics.
 It led to rapid and efficient DNA sequencing techniques that enabled the structure of individual genes
to be determined, reaching culmination with the massive genome sequencing projects including the
Human genome project which was completed in 2000.
 In 1985, Kary Mullis invented the PCR , an exquisitely simple technique that acts as a perfect
complement to Gene Cloning.
 PCR has made easier many of the technique, that were possible but difficult to carry out when gene
cloning was used on it own.
 It extended to the range of DNA analysis and enabled molecular biology to find range in the field of
medicine, agriculture, and biotechnology.
 With the invention of PCR , the Archeogenetics, molecular biology, and DNA Forensics have to
become possible.
 40 years passed since the dawning of age of gene cloning, but there is no end to the excitement in
sight.

6. Fundamental Steps
 Identification and isolation of the desired gene or DNA fragment to be cloned.
 Insertion of the isolated gene in a suitable vector.
 Introduction of this vector into a suitable organism/cell called host.
 The vector multiplies within the host cell, producing numerous identical copies not only
of itself but also of the gene that it carries.
 During the division of the host cell, copies of the recombinant DNA molecules 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.

8. What is PCR?
 PCR is a method of copying DNA molecules. DNA replication is common in life; for
example it takes place inside your own cells every time they divide. An enzyme known as
polymerase uses one strand of DNA as a template to create a complementary strand. The
result is that one double stranded DNA molecule is converted into two, both identical to
the first.
 PCR, or the polymerase chain reaction, adds two components to this process. The initial
reaction yields twice the number of starting molecules, but then is immediately followed
by a subsequent reaction, which yields twice the molecules as the first reaction. This is
why PCR is known as a chain reaction. Commonly 25-40 reactions are chained together,
theoretically resulting in 225 – 240 more molecules of DNA then were initially present.
 Additionally, the goal of a PCR reaction is commonly to replicate only a portion of the
genome of interest. For example, somewhere between 751000 bases, instead of the
entire human genome of 3 billion bases. As PCR produces billions of copies of only the
DNA of interest, this process is known as “amplification”.

9. Why is PCR Important?
 The amplification provided by PCR is very powerful. For example, suppose we
want to detect whether a dangerous E. Coli pathogen is present in a sample of
meat. That meat sample contains a huge amount of DNA from the meat source,
and many non-pathogenic bacteria. Looking for the DNA from the pathogenic E.
Coli, is akin to searching for a needle in a haystack.
 However a PCR reaction can be designed to amplify only the DNA from a portion
of this pathogenic E. Coli. If the pathogen is present, we can make billions of
copies of its targeted DNA, which will come to outnumber the overall DNA
originally present in the sample, and allow us to easily detect it. If no such signal
is amplified by a properly controlled reaction, we can conclude the pathogen was
not present.

10. How is it used?
 PCR and related techniques have many applications. Here are just a few
 Human Diagnostics
1. Detecting viral infections (HIV, etc.)
2. Detecting bacterial infections (Tuberculosis, etc.)
3. Genotyping (detecting genetic variants, which can indicate predisposition to disease)
 Environmental Monitoring
1. Water quality monitoring
2. Food safety testing
 Scientific Research
1. Preparing DNA to sequence
2. Monitoring gene expression levels
3. Manipulating DNA in genetic engineering and synthetic biology

11. How does PCR work?
 The principles behind every PCR, whatever the sample of DNA, are the same.
 Five core ‘ingredients’ are required to set up a PCR. We will explain exactly what each of
these do as we go along. These are:
1. The DNA template to be copied
2. Primers, short stretches of DNA that initiate the PCR reaction, designed to bind to
either side of the section of DNA you want to copy
3. DNA nucleotide bases? (also known as dNTPs). DNA bases (A, C, G and T) are the
building blocks of DNA and are needed to construct the new strand of DNA
4. Taq polymerase enzyme? to add in the new DNA bases
5. Buffer to ensure the right conditions for the reaction.
 PCR involves a process of heating and cooling called thermal cycling which is carried out
by machine.

12. There are three main stages:
 Denaturing – when the double-stranded template DNA is heated to separate it
into two single strands.
 Annealing – when the temperature is lowered to enable the DNA primers to
attach to the template DNA.
 Extending – when the temperature is raised and the new strand of DNA is made
by the Taq polymerase enzyme.

13.  These three stages are
repeated 20-40 times,
doubling the number of DNA
copies each time.
 A complete PCR reaction can
be performed in a few
 hours, or even less than an
hour with certain high-speed
machines.
 After PCR has been completed,
a method called
electrophoresis can be used to
check the quantity and size of
the DNA fragments produced.

15. What happens at each stage of PCR?
 Denaturing stage
 During this stage the cocktail containing the template DNA and all the other core
ingredients is heated to 9495⁰C.
 The high temperature causes the hydrogen bonds? between the bases in two
strands of template DNA to break and the two strands to separate.
 This results in two single strands of DNA, which will act as templates for the
production of the new strands of DNA.
 It is important that the temperature is maintained at this stage for long enough to
ensure that the DNA strands have separated completely.
 This usually takes between 15-30 seconds.

16.  Annealing stage
 During this stage the reaction is cooled to 50-65⁰C. This enables the primers to attach
to a specific location on the single-stranded template DNA by way of hydrogen bonding
(the exact temperature depends on the melting temperature of the primers you are
using).
 Primers are single strands of DNA or RNA? sequence that are around 20 to 30 bases in
length.
 The primers are designed to be complementary? in sequence to short sections of DNA
on each end of the sequence to be copied.
 Primers serve as the starting point for DNA synthesis. The polymerase enzyme can only
add DNA bases to a double strand of DNA. Only once the primer has bound can the
polymerase enzyme attach and start making the new complementary strand of DNA
from the loose DNA bases.
 The two separated strands of DNA are complementary and run in opposite directions
(from one end - the 5’ end – to the other - the 3’ end); as a result, there are two
primers – a forward primer and a reverse primer.
 This step usually takes about 10-30 seconds.

17.  Extending stage
 During this final step, the heat is increased to 72⁰C to enable the new DNA to be made by a special Taq
DNA polymerase enzyme which adds DNA bases.
 Taq DNA polymerase is an enzyme taken from the heat-loving bacteria Thermus aquaticus.
 This bacteria normally lives in hot springs so can tolerate temperatures above 80⁰C.
 The bacteria's DNA polymerase is very stable at high temperatures, which means it can withstand the
temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.
 DNA polymerase from most other organisms would not be able to withstand these high temperatures,
for example, human polymerase works ideally at 37˚C (body temperature).
 72⁰C is the optimum temperature for the Taq polymerase to build the complementary strand. It
attaches to the primer and then adds DNA bases to the single strand one-by-one in the 5’ to 3’
direction.
 The result is a brand new strand of DNA and a double stranded molecule of DNA.
 The duration of this step depends on the length of DNA sequence being amplified but usually takes
around one minute to copy 1,000 DNA bases (1Kb).
 These three processes of thermal cycling are repeated 20-40 times to produce lots of copies of the
DNA sequence of interest.
 The new fragments of DNA that are made during PCR also serve as templates to which the DNA
polymerase enzyme can attach and start making DNA.
 The result is a huge number of copies of the specific DNA segment produced in a relatively short
period of time.

18. Why gene cloning and PCR are so important?
 Obtaining a pure sample
of a gene by cloning

19.  The problem of selection.

20.  PCR can also be used to purify a gene
Gene isolation by PCR

21. Cloning applications
 Gene cloning has made a phenomenal impact on the speed of biological research and it is
increasing its presence in several areas of everyday life. One of the reasons why biotechnology
has received so much attention during the last decade is because of gene cloning.
 Production of recombinant protein
 Proteins that are normally produced in very small amounts include growth hormone, insulin in
diabetes, interferon in some immune disorders and blood clotting factor VIII in haemophilia, are
known to be missing or defective in various disorders. Prior to the advent of gene cloning and
protein production via recombinant DNA techniques, these molecules were purified from animal
tissues or donated human blood. But both sources have drawbacks, including slight functional
differences in the non human proteins and possible viral contamination. (e.g. HIV, CJD). Production
of protein from a cloned gene in a defined, non pathogenic organism would circumvent these
problems. A gene for an important animal or plant protein can be taken from its normal host,
inserted into a cloning vector, and introduced into a bacterium. If the manipulations are performed
correctly then the gene will be expressed and the protein is synthesized by the bacterial cell. Then it
is possible to obtain large amounts of the protein.
But in practice obtaining recombinant protein is not as easy as theoretically it sounds. For this
special types of cloning vectors are needed.