Bangabandhu Sheikh Mujibur

Rahman Science & Technology

University, Gopalganj

An assignment on Specific DNA Fragments Amplification using Polymerase Chain

Reaction (PCR) and
Genes Cloning

Course Title : Microbial Biotechnology Lab

Course Code: BGE-412

Submission Date: 23-08-2021

Submitted By Submitted To
Rayhan Parvej Shovon Dr.Md. Sharafat Ali

ID NO: 15BGE015 Assistant Professor,

Dept. of
4th year 1st semester Biotechnology and Genetic
Engineering ,
Dept. of Biotechnology & Genetic
Engineering Bangabandhu
Sheikh Mujibur Rahman Science
Bangabandhu Sheikh Mujibur & Technology University ,
Rahman Science & Technology Gopalganj ,8100
University , Gopalganj ,8100

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Introduction :
The polymerase chain reaction (PCR) and the cloning of expressed genes are two
biotechnological accomplishments that are still used in research today. Both technologies are
used to increase the amount of DNA in the human genome, but they do it in distinct ways.
Cloning, in specifically, is the process of making DNA from mRNA with the help of an enzyme
called reverse transcriptase. Although this technique reverses the flow of genetic information, it
successfully replicates the process by which RNA viruses "flip" the direction of transcription in
their host cells, allowing these cells to produce viral DNA despite the fact that the viruses
themselves only carry RNA. The polymerase chain reaction, on the other hand, does not need an
initial mRNA template to make DNA. PCR, on the other hand, is the process of making
numerous copies of a particular DNA fragment using an enzyme called DNA polymerase. This
technique allows for the synthesis of billions of DNA molecules in just a few hours, making it far
more efficient than cloning expressed genes. When just the mRNA template (not the DNA
template) of a sequence of interest is available, cloning remains the preferred approach for
researchers. Both technologies are used in forensics, genetic testing, and diagnostics to make
numerous copies of DNA from a particular section of DNA.

Polymerase Chain Reaction (PCR) Technique :

The Polymerase Chain Reaction (PCR) is a typical scientific technique for making numerous
copies of a specific area of DNA (millions or billions!). This DNA region may be anything the
researcher wants it to be. It might be a gene that a researcher wants to learn more about, or a
genetic marker that forensic experts use to link crime scene DNA with suspects. The objective of
PCR is usually to produce enough of the target DNA area to be examined or used in another
way. For example, PCR-amplified DNA can be sequenced, visualized via gel electrophoresis, or
cloned into a plasmid for future research.

Ingredients for PCR:

Taq polymerase:

PCR, like DNA replication in an organism, necessitates the use of a DNA polymerase enzyme to
create new DNA strands from existing ones. Taq polymerase, the DNA polymerase most
commonly employed in PCR, is named for the heat-tolerant bacteria from which it was obtained
(Thermus aquaticus). T. aquaticus is a species that may be found in hot springs and hydrothermal
vents. Its DNA polymerase is extremely heat-stable, peaking at 70 degrees Celsius (a
temperature at which a human or E. coli DNA polymerase would be nonfunctional). Taq
polymerase is suitable for PCR because of its thermal stability. As we'll see, high temperatures
are frequently employed in PCR to denature or separate the template DNA's strands.

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primers:
Taq polymerase, like other DNA polymerases, can only create DNA if it is given a primer, a
short nucleotide sequence that serves as a starting point for DNA synthesis. The experimenter
decides the area of DNA that will be replicated or amplified by the primers she or he selects in a
PCR process. PCR primers are single-stranded DNA fragments that are typically 20 nucleotides
long. In each PCR reaction, two primers are utilized, and they are intended to surround the target
area (region that should be copied). That is, they are given sequences that cause them to attach to
the template DNA's opposing strands at the boundaries of the area to be copied. By
complimentary base pairing, the primers bind to the template. The polymerase can expand the
primers when they are attached to the template, and the region between them will be replicated.
When bound, both primers point inward, in the 5' to 3' direction, towards the region to be
duplicated. Tag polymerase, like other DNA polymerases, can only synthesis DNA in the 5' to 3'
orientation. When primers are expanded, the region that sits between them is replicated as well.

Steps of PCR:
Extraction & Denaturation of Target Nucleic Acid :
Heat, chemical, or enzymatic techniques are used to extract (release) nucleic acid from the
organism or a clinical sample that may include the target organism. Once the target nucleic acid
has been extracted, it is added to a reaction mix comprising all of the essential PCR components
(primers, nucleotides, covalent ions, buffer, and enzyme) and put in a thermal cycler for
amplification.

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Amplification :
PCR consists of 25 to 50 cycles, each of which consists of three consecutive reactions:

1. Denaturation :
The target DNA is denatured into single strands that may function as templates for DNA
synthesis by heating the reaction mixture to 95°C for a brief time (approximately 15–30 sec).

2.Primer annealing :

The mixture is immediately cooled to a certain temperature, which allows the two primers to
bind to sequences on both strands around the target DNA. Primers are single-stranded nucleic
acid sequences (usually 20 to 30 nucleotides long) designed to accurately hybridize (anneal) to a
specific nucleic acid target, essentially serving as probes. This annealing temperature has been
precisely adjusted to guarantee that the primers only bind to the desired DNA sequences (usually
around 55oC). A single primer holds each strand together. The two parental strands do not re-
anneal with each other because the primers are in considerable excess over parental DNA.

3.Extension :

The temperature of the mixture is raised to 72°C (usually) and kept there for a pre-determined
period of time to allow DNA polymerase to lengthen each primer by replicating single-stranded
templates. Annealing primers to target sequences provides the necessary template format,
allowing the DNA polymerase to add nucleotides to the 3' terminal (end) of each primer and
extend the complementary sequence to the target template. For primer extension, which takes
place at 72°C, the enzyme Taq polymerase is commonly used. This enzyme is used because it
can operate efficiently at high temperatures and can withstand several denaturing cycles at 94°C.
The ability to anneal and extend primers at high temperatures without damaging the polymerase
improves reaction stringency, lowering the risk of non-target nucleic acid amplification (i.e.,
nonspecific amplification).
This cycle repeats 25-35 times in a typical PCR reaction, requiring 2–4 hours depending on the
length of the DNA region being copied. If the reaction is efficient, the target region can increase
from one or a few copies to billions (functions well). This is because the original DNA isn't used
as a template all of the time. Instead, the new DNA produced in one cycle can be utilized as a
template for the next round of DNA synthesis. The quantity of DNA molecules in each cycle of
cycling can virtually treble as a result of this mechanism. This exponential development
tendency is seen in the diagram below.

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Fig. Amplification of Specific DNA Fragment by PCR.

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To view the results of PCR, use gel electrophoresis:
Gel electrophoresis is commonly used to view (make visible) the results of a PCR reaction. Gel
electrophoresis is a method that separates DNA fragments by size by pulling segments of DNA
through a gel matrix with an electric current. A standard, also known as a DNA ladder, is usually
provided to determine the size of the fragments in the PCR sample. If the gel is dyed with a
DNA-binding dye, DNA fragments of the same length create a "band" on the gel that can be seen
by sight. On a gel, a PCR reaction that produces an 800 base pair (bp) fragment may look like
this:

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Advantages of PCR:

 PCR (polymerase chain reaction) is an extremely simple yet immensely powerful

technique.

 It allows enormous amplification of any specific sequence of DNA provided that short
sequences either side of it are known.

 Allow faster diagnosis and identification while enhancing sensitivity and maintaining
specificity.

Application of PCR:
A DNA sequence may be amplified millions or billions of times via PCR, yielding enough DNA
copies for further analysis. The DNA might be seen via gel electrophoresis, sequenced, or
digested using restriction enzymes and cloned into a plasmid, for example. Many research
laboratories utilize PCR, which also has uses in forensics, genetic testing, and diagnostics. For
example, PCR is used to amplify genes linked to hereditary diseases from patient DNA (or from
fetal DNA, in the case of prenatal testing). A bacteria or DNA virus can also be detected in a
patient's body using PCR: if the pathogen is present, it may be possible to amplify regions of its
DNA from a blood or tissue sample.

DNA cloning or Gene Cloning :

The technique of generating numerous, identical copies of a specific portion of DNA is known as
DNA cloning. A gene or other DNA fragment of interest (for example, a gene encoding a
medically essential human protein) is first placed into a circular piece of DNA called a plasmid
in a conventional DNA cloning method. The insertion is carried out by enzymes that “cut and
paste” DNA, resulting in a molecule of recombinant DNA, or DNA made up of fragments from
several sources. After that, the bacteria are given the recombinant plasmid. Bacteria that contain
the plasmid are chosen and cultivated. They duplicate the plasmid and transmit it on to their
progeny when they reproduce, creating copies of the DNA it contains.

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Steps of Genes Cloning :

I. Production of cDNA:

To begin, Template DNA is transcribed, resulting in various types of RNA. The

mRNA is then separated from the overall RNA. The reverse transcriptase enzyme
then reverses the mRNA to generate cDNA. Additionally, this cDNA is utilized to
create numerous copies of a gene.

Fig. Production of cDNA from mRNA.

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 II. Cutting and pasting DNA:

A restriction enzyme is a DNA-cutting enzyme that identifies and chops DNA into two pieces at
or near a specified target sequence. Cut ends with short, single-stranded overhangs are produced
by several restriction enzymes. Two molecules can base-pair and cling together if their
overhangs are the same. They won't connect to create an unbroken DNA molecule unless DNA
ligase, which closes gaps in the DNA backbone, joins them.

III. Bacterial transformation and selection:

In a procedure known as transformation, plasmids and other DNA may be introduced into
bacteria such as the lab-safe E. coli. During transformation, carefully prepared bacterial cells are
subjected to a shock (such as a high temperature) to induce them to accept foreign DNA.

Antibiotic resistance genes are commonly found on plasmids, allowing bacteria to survive in the

presence of a specific antibiotic. On nutrition plates containing the antibiotic, bacteria that picked

up the plasmid may be chosen. Bacteria without plasmids will perish, but bacteria with plasmids
will be able to live and multiply. Each bacterium that survives will produce a tiny dot-like clump
of identical bacteria that all contain the same plasmid.

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Not all colonies will possess the appropriate plasmid. That's because DNA pieces don't always
get "pasted" in the way we want them to during ligation. Instead, we'll need to gather DNA from
a number of colonies to determine if each one has the correct plasmid. Plasmids are routinely
checked using methods like as restriction enzyme digestion and PCR.

Uses of Gene Cloning :

DNA molecules built through cloning techniques are used for many purposes in molecular
biology. A short list of examples includes:

 Biopharmaceuticals. Human proteins having medicinal uses, such as insulin, may be

produced through DNA cloning. Human growth hormone, which is administered to
individuals who are unable to produce the hormone, and tissue plasminogen activator
(tPA), which is used to treat strokes and prevent blood clots, are two examples of
recombinant proteins. These types of recombinant proteins are frequently produced in
bacteria.

 Gene therapy. Patients with some genetic diseases lack the functional version of a gene.
Gene therapy aims to give a normal copy of a gene to a patient's body's cells. DNA
cloning, for example, was employed to create plasmids containing a normal form of a
gene that is inactive in cystic fibrosis. Lung function decreased less quickly when the
plasmids were given to cystic fibrosis patients' lungs.

 Gene analysis. • In basic research laboratories, scientists frequently employ DNA cloning
to create fake, recombinant copies of genes to better understand how regular genes operate
in an organism.

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 These are just a few examples of how DNA cloning is used in biology today. DNA
cloning is a very common technique that is used in a huge variety of molecular biology
applications.

Conclusion :
As a result, both cloning of expressed genes and PCR remain important techniques for
geneticists. When scientists are unable to isolate the DNA template of a sequence of interest,
cloning—which entails the synthesis of DNA from mRNA and so constitutes a reversal of the
fundamental dogma—comes in handy. Furthermore, because this approach uses mRNA rather
than DNA, it's a great way to look at how gene expression differs in various cells at different
stages of development. PCR, on the other hand, is more like "conventional" DNA synthesis in
that it requires a DNA (rather than RNA) template to begin with. The main benefit of PCR is its
speed; even if researchers start with just a single segment of DNA, they may generate billions of
molecules in just a few hours. Scientists continue to enhance PCR and the cloning process, as
they do with all technologies, guaranteeing that these procedures will continue to play a part in
genetic advances for years to come.

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