DNA Sequencing- Definition, Principle,

Steps, Types, Uses

DNA sequencing refers to methods for determining the order
of the nucleotide bases adenine,guanine,cytosine, and
thymine in a molecule of DNA.
The first DNA sequence was obtained by academic researchers,
using laboratories methods based on 2- dimensional
chromatography in the early 1970s.
By the development of dye based sequencing method with
automated analysis, DNA sequencing has become easier and faster.
Chemical Cleavage Method (Maxam–
Gilbert Method)
 In 1976-1977, Allan Maxam and Walter Gilbert developed a DNA
sequencing method based on chemical modification of DNA and
subsequent cleavage at specific bases.
 The method requires radioactive labelling at one end and
purification of the DNA fragment to be sequenced.
 Chemical treatment generates breaks at a small proportions of one
or two of the four nucleotide based in each of four reactions
(G,A+G, C, C+T).
 Thus a series of labelled fragments is generated, from the
radiolabelled end to the first ‘cut’ site in each molecule.
 The fragments in the four reactions are arranged side by side in
gel electrophoresis for size separation.
 To visualize the fragments, the gel is exposed to X-ray film for
autoradiography, yielding a series of dark bands each
corresponding to a radiolabelled DNA fragment, from which the
sequence may be inferred.
Key Features
 Base-specific cleavage of DNA by certain chemicals
 Four different chemicals, one for each base
 A set of DNA fragments of different sizes
 DNA fragments contain up to 500 nucleotides
Advantages
 Purified DNA can be read directly
 Homopolymeric DNA runs are sequenced as efficiently as
heterogeneous DNA sequences
 Can be used to analyze DNA protein interactions (i.e. footprinting)
 Can be used to analyze nucleic acid structure and epigenetic
modifications to DNA
Disadvantages
 It requires extensive use of hazardous chemicals.
 It has a relatively complex set up / technical complexity.
 It is difficult to “scale up” and cannot be used to analyze more
than 500 base pairs.
 The read length decreases from incomplete cleavage reactions.
 It is difficult to make Maxam-Gilbert sequencing based DNA kits.
Chain Termination Method (Sanger
Dideoxy Method)
 The chain terminator method is more efficient and uses fewer toxic
chemicals and lower amount of radioactivity than the method of
Maxam and Gilbert.
 The key principle of the Sanger method was the use of
dideoxynucleotide triphosphates (ddNTPs) as DNA chain
terminators.
 The chain termination method requires a single-stranded DNA
template, a DNA primer,a DNA polymerase, radioactively or
fluorescently labelled nucleotides,and modified nucleotides that
terminate DNA strand elongation.
 The DNA sample is divided into four separate sequencing
reactions, containing all four of the standard deoxynucleotides
(dATP, dGTP, dCTP, dTTP) and the DNA polymerase.
 To each reaction is added only one of the four dideoxynucleotide
(ddATP, ddGTP, ddCTP, ddTTP) which are the chain terminating
nucleotides, lacking a 3’-OH group required for the formation of a
phosphodiester bond between two nucleotides, thus terminating
DNA strand extension and resulting in DNA fragments of varying
length.
 The newly synthesized and labelled DNA fragments are heat
denatured, and separated by size by gel electrophoresis on a
denaturing polyacrylamide-urea gel with each of the four reactions
run in one of the four individual lanes (lanes A, T, G, C).
 The DNA bands are then visualized by autoradiography or UV light,
and the DNA sequence can be directly read off the X-ray film or gel
image.
 A dark band in a lane indicates a DNA fragment that is result of
chain termination after incorporation of a dideoxynucleotide
(ddATP, ddGTP, ddCTP, or ddTTP).
 The relative position of the different bands among the four lanes
are then used to read (from bottom to top) the DNA sequence.
 The technical variations of chain termination sequencing include
tagging with nucleotides containing radioactive phosphorus for
labelling, or using a primer labelled at the 5’ end with a fluorescent
dye.
 Dye- primer sequencing facilitates reading in an optical system for
faster and more economical analysis and automation.

Key Features
 Uses dideoxy nucleotides to terminate DNA synthesis.
 DNA synthesis reactions in four separate tubes
 Radioactive dATP is also included in all the tubes so the DNA
products will be radioactive.
 Yielding a series of DNA fragments whose sizes can be measured
by electrophoresis.
 The last base in each of these fragments is known.
 Advantage
Chain termination methods have greatly simplified DNA sequencing.
Limitations
 Non-specific binding of the primer to the DNA, affecting accurate
read-out of the DNA sequence.
 DNA secondary structures affecting the fidelity of the sequence.
Significance of DNA Sequencing
 Information obtained by DNA sequencing makes it possible to
understand or alter the function of genes.
 DNA sequence analysis demonstrates regulatory regions that
control gene expression and genetic “hot spots” particularly
susceptible to mutation.
 Comparison of DNA sequences shows evolutionary relationships
that provide a framework for definite classification of
microorganisms including viruses.
 Comparison of DNA sequences facilitates identification of
conserved regions, which are useful for development of specific
hybridization probes to detect microorganisms including viruses in
clinical samples.
 DNA sequencing has become sufficiently fast and inexpensive to
allow laboratory determination of microbial sequences for
identification of microbes. Sequencing of the 16S ribosomal
subunit can be used to identify specific bacteria. Sequencing of
viruses can be used to identify the virus and distinguish different
strains.
 DNA sequencing shows gene structure that helps research workers
to find out the structure of gene products.
Illumina (Solexa) sequencing

Illumina sequencing
 Solexa developed a sequencing technology based on dye
terminators.
 In this method, DNA molecule are first attached to primers on a
slide and amplified. This is known as bridge amplification.
 Unlike pyrosequencing, the DNA can only be extended one
nucleotide at a time.
 A camera takes images of the fluorescently labeled nucleotides,
then the dye along with the terminal 3′ blocker is chemically
removed from the DNA, allowing the next cycle to commence.
Polymerase Chain Reaction (PCR): Principle,
Enzymes, Steps, Types, Uses
Polymerase Chain Reaction (PCR) is a nucleic acid amplification
technique used to amplify the DNA or RNA in vitro enzymatically. It
is a temperature-dependent enzymatic process where either a
specific targeted region of DNA or the whole DNA is replicated to
quickly make millions of copies of the target DNA or DNA segment. It
is also called “molecular photocopying”.

Principle of PCR
PCR combines the principle of nucleic acid hybridization with the
principle of nucleic acid replication with the temperature variations
of cyclic heating and cooling throughout the process. It is based on
the fact that double-stranded DNA can be thermally broken into
single-stranded DNA segments. In these ssDNA templates, primers
can anneal to their complementary sequences based on the nucleic
acid hybridization principle. DNA polymerase then elongates the
primer by sequentially adding the nucleotides to the 3’ end and
generates a dsDNA following the principle of DNA replication. By
continuously regulating the temperature, these 3 steps;
denaturation, annealing, and elongation, can be permitted to occur
in a cyclic manner leading to millions of copies of a single targeted
nucleic acid sequence.

Components of PCR (Enzymes used in

PCR)
1. Nucleic Acid Template (Template DNA)
Template DNA is the sample DNA that contains the selected nucleic acid sequence that needs to
be amplified. The template must be DNA only: Genomic DNA (gDNA), complementary DNA
(cDNA), and plasmid DNA. Reverse transcriptase polymerase chain reaction (RT-PCR) uses
RNAs as starting materials, but RNAs are primarily converted to complementary DNA (cDNA)
before amplification. The template DNA must be highly pure with an absorbance ratio of ~1.8. A
quantity of 0.1 to 200 μg can be used, with an ideal quantity of 30 μg to 50 μg.
Components of PCR (Enzymes used in PCR)
2. DNA Polymerase
DNA polymerases are enzymes that catalyze the synthesis of complementary DNA strands by
assembling the nucleotides sequentially according to the template strand. Simply, it is the
enzyme that synthesizes DNA; hence plays a key role in DNA replication.
Taq DNA polymerase, the DNA polymerase enzyme extracted from the bacterium Thermus
aquaticus, is the most widely and the best–known DNA polymerase used in PCR since its
establishment. Taq DNA polymerase is thermally stable and continues its activity after the
repeated heating and cooling cycle. It is stable up to 95°C and shows the most effective reaction
at around 72°C to 78°C incorporating about 60 bases per second. In a 50 L reaction mixture,
around 1 to 2 units of Taq polymerase is sufficient for amplification.
Recently, two other thermostable DNA polymerase enzymes are available, viz., the Vent
enzyme isolated from Thermococcus litoralis, and the Pfu enzyme isolated from Pyrococcus
furiosus.
3. Primers
Primers are artificially synthesized short single-stranded sequences of oligonucleotides that are
complementary to the target nucleic acid sequence in the template DNA. They are short
sequences of around 15 to 30 bases that act as starting point for DNA synthesis. They anneal at
their complementary position in a single-stranded template DNA strand. The DNA polymerase
enzyme then extends this primer from its 3’ OH- end forming a new complementary strand.
Usually, 10 to 12 pMol of each primer is sufficient for a PCR reaction.
PCR primers are of 2 types; forward and reverse primers. The forward primers are
complementary to the antisense strand (template strand from 3’ to 5’ direction), and are
responsible for the amplification of the antisense strand. They are also called 5’ primers.
The reverse primers are complementary to the sense strand (template strand from 5’ to 3’
direction) and are responsible for the amplification of the sense strand. They are also called 3’
primers.

4. Nucleotides (Deoxynucleotide
triphosphates)
Deoxynucleotide triphosphates (dNTPs) are artificially synthesized nucleotides that act as
building blocks of new DNA strands. There are 4 different dNTPs used in the PCR;
deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxythymidine
triphosphate (dTTP), and Deoxycytidine triphosphate (dCTP). These four dNTPs are
sequentially added to the annealed primer by the DNA polymerase enzyme generating a new
strand of DNA complementary to the template strand.

5. PCR Buffers and Other Chemicals

The whole process needs to be carried out in a Tris – HCl based buffer system of pH 8.0 to 9.5.
The common buffer system used is a 10X buffer with additional MgCl2. Common components of
PCR buffers are dimethyl sulfoxide (DMSO), ammonium sulfate ((NH4)2SO4), nonionic
detergents, polyethylene glycon(PEG), N,N,N-trimethyl glycine, potassium chloride (KCl),
magnesium chloride (MgCl2), tetra methyl ammonium chloride, Tris – HCl,
Ethylenediaminetetraacetic acid (EDTA), 7-deaza-2′-deoxyguanosine 5’-triphosphate, glycerol,
formamide, serum albumin, etc. The buffer system increases the reaction’s efficiency and
specificity and prevents inhibition and secondary structure formation during the process.
6. Thermocycler
Also known as the PCR machine, the thermocycler is simply an electric heating device that
regulates the temperature as per need during each stage of the PCR process. This machine
increases the temperature during the denaturation step and lowers it during the annealing and
again increases it during the elongation step. This process of increasing and decreasing the
temperature occurs in a cyclic manner according to the pre-programmed setup or instruction by
the user prior to operating.

Steps of PCR
There are three core steps in any type of PCR: denaturation, annealing, and elongation. Apart
from these three primary steps, the preparation phase at the beginning and the PCR product
analysis phase at the end are also important steps. The whole steps can be summarized into three
steps; pre-preparation, amplification, and product analysis.

1. Pre-preparation
It is the initial step before the actual polymerase chain reaction takes place inside the
thermocycler. In this step, one must prepare a reaction mixture and load it on a pre-programmed
thermocycler in order to amplify the target DNA or RNA segment. Sample DNAs or RNAs are
extracted from the sample and stored (pre-extracted nucleic acids can be used). All materials are
arranged, safety measures are taken, the PCR reaction preparation area is cleaned, all the
reagents are brought to working temperature, the sample is extracted or brought from storage, the
PCR reaction mixture is prepared, the thermocycler is programmed, and the reaction mixture is
loaded on the thermocycler.
Polymerase Chain Reaction (PCR)

2. Amplification
It is the main reaction process occurring in PCR. The amplification step includes denaturation,
annealing, and elongation occurring orderly in a cyclic manner one after another for a certain
number of cycles pre-programmed by the user.
Step I: Denaturation
It is the 1st step of the amplification reaction where the double-stranded DNA is thermally
denatured into two single-stranded DNA templates. Temperature is raised to about 94°C (90 to
95°C) for about 30 to 90 seconds. At this temperature, the thermal energy overcomes the weak
hydrogen bonds holding the two DNA strands together, allowing them to separate.
dsDNA → 2 ssDNA templates
Step II: Annealing
Denaturation is followed by the annealing step, where the primer anneals the ssDNA templates at
their complementary sites. The forward primer anneals at the complementary site of the antisense
strand, and the reverse primer anneals at the complementary site of the sense strand of the
template DNA. For annealing to occur, the temperature is reduced to 55°C-70°C (the annealing
temperature differs based on the GC content of the primer). About 30 to 60 seconds are enough
for annealing in most of the PCR processes.
ssDNA + Forward and reverse primers → ssDNA with annealed primers
Step III: Elongation
It is the final step in the amplification reaction where the temperature is raised to 72°C so that the
Taq DNA polymerase enzyme begins synthesizing new DNA strands in the 5’ to 3’ direction.
The DNA polymerase enzyme adds nucleotides from the reaction mixture to the 3’ OH- end of
the annealed primer forming a new complementary strand. The time required for elongation
depends on the sample nucleic acid sequence length and the DNA polymerase activity.
Generally, elongation takes place at the rate of 1 kbp per 0.5 to 1 minute. At the end of
elongation, two new dsDNA will be formed from a single dsDNA template at the beginning of
the reaction.
2 ssDNA with annealed primers + dNTPs → 2 new dsDNAs

3. Product Analysis Phase

It is the phase after completion of the PCR where the reaction mixture subjected to PCR is
analyzed to confirm that desired amplification is achieved. For this, mostly agarose gel
electrophoresis is employed in order to check for amplified DNAs or RNAs. However, no
additional step is required in some types of PCR, like real-time PCR.

Polymerase Chain Reaction (PCR)

Applications
Since its establishment, PCR has found numerous applications in different aspects of molecular
biology. Some of them are;

1. Identification and Classification of

Organism
PCR is used widely in identifying microorganisms up to the level of subspecies and strains. This
has reduced the time required for microbial identification from days to a few hours. Additionally,
larger animals can also be identified and systematically classified using PCR. DNA isolated from
fossilized animals are also amplified and studied to relate them with animals that are still living
on the Earth.

2. Infectious Disease Diagnosis

The use of PCR in the identification of pathogens has led to the quick and accurate diagnosis of
infections. Not only diagnosis but parallel identification of antimicrobial resistant genes in the
pathogen is also possible, allowing choosing of appropriate antimicrobial treatment option.
HIV, SARS CoV – 2, human T – cell leukemia virus (HTLV type I and II), Tuberculosis,
Hepatitis Virus, Enterovirus, Sexually Transmitted Diseases (STDs), etc., are diagnosed using
PCR.

3. Detection of Gene Mutation and Genetic

Disorders
Mutation in any segment of a gene can be detected using PCR. Knowing this mutation, we can
confirm a genetic disorder. DNA polymorphism can also be analyzed, which can also relate to a
genetic disorder of some kind. In medical science, PCR is used as one of the most important
tools to diagnose congenital diseases, genetic disorders, and any mutation leading to a negative
health problem and behavioral change in the prenatal stage. Detection of cancerous cells is
another very important application of PCR in medicine.
 4. DNA Fingerprinting
In forensics, PCR is used for DNA fingerprinting. DNA fingerprinting is used for the
identification of criminals or individuals and for confirming parents.

5. Gene Sequencing
For gene sequencing, a gene must be amplified into a large number using techniques like PCR.
All the sequencing methods use PCR as their important step.

6. DNA and RNA Quantification

PCR can also be used for the quantification of sample DNA and RNA. Quantitative Real-Time
PCR (RT – qPCR) is one common type of PCR used for the quantification of sample DNA.

7. As a Tool in Genetic Engineering

PCR is used in genetic engineering for analyzing modified DNAs and amplifying target or vector
DNA. Desired genes are amplified using PCR and applied in the required process.

8. Gene Expression Analysis and Genetic

Imprinting
PCR of RNA (Reverse Transcription PCR) is used in gene expression analysis, study genetic
imprinting, etc.
9. It is also used in drug and vaccine discovery, human genome projects, paleontology, and
evolutionary biology.
Advantages of Polymerase Chain Reaction
(PCR)
1. It is a very fast method for producing multiple copies of a nucleic acid segment. Within a few hours to
a few days, billions of copies of a specific nucleic acid sequence can be produced and analyzed.
2. It can analyze a very minute quantity, 0.1 to 5 μg, of DNA or RNA.
3. Its principle and procedure are simple to understand and apply. Being a semiautomatic technique, the
process is operated and regulated automatically by a thermocycler.
4. It has shortened the time for the diagnosis of diseases and genetic disorders.
5. It is the base for several molecular techniques like DNA fingerprinting, genetic imprinting, microarray,
gene sequencing, molecular vector production, etc.
Limitations of PCR
1. Prior information regarding the sequence of the DNA or RNA target segment is required. Only then we
can design and generate primers and selectively amplify the target.
2. Even very small contamination in a sample can be amplified, giving a false positive or false negative
result.
3. There is a chance of mutation during the process if the DNA polymerase enzyme makes any error
during replication.
4. PCR fails to detect novel mutations, multigenic disorders, and structural and numerical chromosomal
abnormalities.
5. Several organic or inorganic contaminants can disrupt the process, inhibiting the process and
producing inaccurate results.
6. It is a highly temperature-dependent process; hence there must be strict regulation of temperature.
Even a slight change in temperature can inhibit the amplification process.
7. It is complex, requiring sophisticated technology, a wide range of chemicals, and highly trained
professionals to operate.
8. It is not suitable for very long DNA molecules. Very long DNA needs to be cut into smaller fragments.
Usually, from 0.1 kbp up to 10 kbp or 40 kbp can be used.
9. RNA needs to be first converted to DNA using reverse-transcriptase enzyme before its amplification.
10. Most types of PCR processes require additional steps for product analysis.
Inverse PCR
 Inverse polymerase chain reaction (Inverse PCR) is one of the
many variants of the polymerase chain reaction that is used to
amplify DNA when only one sequence is known.
 Conventional PCR requires primers complementary to both
terminals of the target DNA, but Inverse PCR allows amplification
to be carried out even if only one sequence is available from which
primers may be designed.
 The inverse PCR involves a series of restriction digestion followed
by ligation, which results in a looped fragment that can then be
primed for PCR through a single section of known sequence.
 Then, like other polymerase chain reaction processes, the DNA is
amplified by the temperature-sensitive DNA polymerase.
 Inverse PCR is especially useful for the determination of insert
locations of various transposons and retroviruses in the host DNA.
Nested PCR
 Nested PCR is a useful modification of PCR technology where the
specificity of the reaction is enhanced by preventing the non-
specific binding with the help of the two sets of primer.
 The first set of primer binds outside of our target DNA and
amplifies larger fragment while another set of primer binds
specifically at the target site.
 In the second round of amplification, second set of primer amplifies
only the target DNA.
 Nested PCR is a helpful method for the phylogenetic studies and
detection of different pathogens.
 The technique has higher sensitivity; hence even if the sample
contains lower DNA, it can be amplified which is not feasible in the
conventional PCR technique.
 Amplified fragment length polymorphism
(AFLP) PCR
 It is a PCR-based technique that uses selective amplification of a
section of digested DNA fragments to generate unique fingerprints
for genomes of interest.
 This technique can quickly generate large numbers of marker
fragments for any organism, without prior knowledge of the
genomic sequence.
 AFLP PCR uses restriction enzymes to digest genomic DNA and
allows attachment of adaptors to the sticky ends of the fragments.
 A part of the restriction fragments is then selected to be amplified
by using primers that are complementary to the adaptor sequence.
 The amplified sequences are separated and visualized on
denaturing on agarose gel electrophoresis.
 AFLP PCR is employed for a variety of applications, as to assess
genetic diversity within species or among closely related species,
to infer population-level phylogenies and biogeographic patterns,
to generate genetic maps and to determine relatedness among
cultivars.
Asymmetric PCR
 Asymmetric PCR is a variation of PCR used to preferentially amplify
one strand of the original DNA more than the other.
 Asymmetric PCR differs from regular PCR by the excessive amount
of primers for a chosen strand.
 As the asymmetric PCR progresses, the lower concentration
limiting primer is quantitatively incorporated into newly
synthesized double-stranded DNA and used up.
 Consequently, linear synthesis of the targeted single DNA strand
from the excess primer is formed after depletion of the limiting
primer.
 It is useful when amplification of only one of the two
complementary strands is needed, such as in sequencing and
hybridization probing.
Colony PCR
 Colony PCR is a method in which, where identification of DNA of
interest inserted into the plasmid is obtained by designing the
inserted DNA specific primers.
 The bacterial colony containing the plasmid can directly be
amplified using two sets of primers.
 The first set is of the insert specific primers which amplify the
insertion sequence, and the other is of vector-specific flanking
primers, which amplifies the plasmid DNA other than the inserted
DNA.
 A bacterial colony is taken and added directly into the master mix
containing all other PCR reagents.
 The main application of colony PCR is in the identification of
correct ligation and insertion of inserted DNA into bacteria as well
as yeast plasmid.
Hot start PCR
 Hot start PCRis a novel form of conventional polymerase chain
reaction (PCR) that reduces the occurrence of undesired products
and formation of primer-dimers due to non-specific DNA
amplification at room temperatures.
 The basic principle of hot-start PCR is the separation of one or
more reagents from the reaction mix until the mixture reaches the
denaturation temperature upon heating.
 Hot start PCR significantly reduces non-specific binding, the
formation of primer-dimers, and often increases product yields. It
also requires less effort and reduces the risk of contamination.
Real-Time PCR (Quantitative PCR (qPCR))
 Quantitative PCR (qPCR), also called real-time PCR or
quantitative real-time PCR, is a PCR-based technique that couples
amplification of a target DNA sequence with quantification of the
concentration of that DNA species in the reaction.
 Conventional PCR is a time-consuming process where the PCR
products are analysed through gel electrophoresis. qPCR facilitates
the analysis by providing real time detection of products during the
exponential phase.
 The principle of real-time PCR depends on the use of fluorescent
dye.
 The concentration of the nucleic acid present into the sample is
quantified using the fluorescent dye or using the fluorescent
labelled oligonucleotides.
 q-PCR is applied in genotyping and quantification of pathogens,
microRNA analysis, cancer detection, microbial load testing and
GMOs detection.
 Reverse Transcriptase PCR (RT-PCR)
 Reverse transcription PCR (RT-PCR) is a modification of
conventional PCR, whereby RNA molecules are first converted into
complementary DNA (cDNA) molecules that can then be amplified
by PCR.
 In RT-PCR, the RNA template is first converted into a
complementary DNA (cDNA) using reverse transcriptase. The cDNA
then acts as a template for exponential amplification using PCR.
 RT-PCR can be conducted either in a single tube or as two steps in
different tubes. The one-step method is more effective with fewer
chances of contamination and incorporation of variations.
 RT-PCR is used in research methods, gene insertion, genetic
disease diagnosis and cancer detection.
Touch down PCR
 Touch Down PCR is a modification of PCR in which the initial
annealing temperature is higher than the optimal Tm of the
primers and is gradually reduced over subsequent cycles until the
Tm temperature or “touchdown temperature” is reached.
 Touchdown PCR increases the specificity of the reaction at higher
temperatures and increases the efficiency towards the end by
lowering the annealing temperature.
Colony PCR
 Colony PCR is a method in which, where identification of DNA of
interest inserted into the plasmid is obtained by designing the
inserted DNA specific primers.
 The bacterial colony containing the plasmid can directly be
amplified using two sets of primers.
 The first set is of the insert specific primers which amplify the
insertion sequence, and the other is of vector-specific flanking
primers, which amplifies the plasmid DNA other than the inserted
DNA.
 A bacterial colony is taken and added directly into the master mix
containing all other PCR reagents.
 The main application of colony PCR is in the identification of
correct ligation and insertion of inserted DNA into bacteria as well
as yeast plasmid.
Hot start PCR
 Hot start PCRis a novel form of conventional polymerase chain
reaction (PCR) that reduces the occurrence of undesired products
 and formation of primer-dimers due to non-specific DNA
amplification at room temperatures.
 The basic principle of hot-start PCR is the separation of one or
more reagents from the reaction mix until the mixture reaches the
denaturation temperature upon heating.
 Hot start PCR significantly reduces non-specific binding, the
formation of primer-dimers, and often increases product yields. It
also requires less effort and reduces the risk of contamination.
Real-Time PCR (qPCR): Principle, Process,
Markers, Uses
Real-Time PCR is a technique used to monitor the progress
of a PCR reaction in real-time.
At the same time, a relatively small amount of PCR product
(DNA, cDNA or RNA) can be quantified.
Real-Time PCR is based on the detection of the fluorescence
produced by a reporter molecule which increases, as the
reaction proceeds.
 Real-Time PCR is also known as a quantitative polymerase
chain reaction (qPCR), which is a laboratory technique of
molecular biology based on the polymerase chain
reaction (PCR).
 qPCR is a powerful technique that allows exponential amplification
of DNA sequences.
 A PCR reaction needs a pair of primers that are complementary to
the sequence of interest. Primers are extended by the DNA
polymerase.
 The copies produced after the extension, so-called amplicons, are
re-amplified with the same primers leading thus to exponential
amplification of the DNA molecules.
 After amplification, however, gel electrophoresis is used to analyze
the amplified PCR products and this makes conventional PCR time
consuming; since the reaction must finish before proceeding with
the post-PCR analysis. Real-Time PCR overcomes this problem.
 The term “real-time” denotes that it can monitor the progress of
the amplification when the process is going on in contrast to the
conventional PCR method where analysis is possible only after the
process is completed.
 Principle of Real-Time PCR
This same principle of amplification of PCR is employed in real-time
PCR. But instead of looking at bands on a gel at the end of the
reaction, the process is monitored in “real-time”. The reaction is
placed into a real-time PCR machine that watches the reaction occur
with a camera or detector.
Although many different techniques are used to monitor the
progress of a PCR reaction, all have one thing in common. They all
link the amplification of DNA to the generation of fluorescence which
can simply be detected with a camera during each PCR cycle.
Hence, as the number of gene copies increases during the reaction,
so does the fluorescence, indicating the progress of the reaction.
Steps of Real-Time PCR (Protocol)

The working procedure can be divided into two steps:

A. Amplification
1. Denaturation
High temperature incubation is used to “melt” double- stranded DNA
into single strands and loosen secondary structure in single-
 stranded DNA. The highest temperature that the DNA polymerase
can withstand is typically used (usually 95°C). The denaturation
time can be increased if template GC content is high.
2. Annealing
During annealing, complementary sequences have an opportunity to
hybridize, so an appropriate temperature is used that is based on
the calculated melting temperature (Tm) of the primers(5°C below
the Tm of the primer).
3. Extension
At 70-72°C, the activity of the DNA polymerase is optimal, and
primer extension occurs at rates of up to 100 bases per second.
When an amplicon in real-time PCR is small, this step is often
combined with the annealing step using 60°C as the temperature.
B. Detection
 The detection is based on fluorescence technology.
 The specimen is first kept in proper well and subjected to thermal
cycle like in the normal PCR.
 The machine, however, in the Real Time PCR is subjected to
tungsten or halogen source that lead to fluoresce the marker
added to the sample and the signal is amplified with the
amplification of copy number of sample DNA.
 The emitted signal is detected by an detector and sent to
computer after conversion into digital signal that is displayed on
screen.
 The signal can be detected when it comes up the threshold level
(lowest detection level of the detector).
 There are many different markers used in Real Time PCR but the
most common of them include:
1. Taqman probe.
2. SYBR Green.
Taqman Probe
 It is a hydrolysis probe which bear a reporter dye, often fluorescein
(FAM) at its 5’ end and a quencher tetramethylrhodamine
(TAMRA), attached to the 3’ end of the oligonucleotide.
 Unders normal conditions, the probe remain coiled on itself
bringing the fluorescence dye near the quencher, which inhibits or
quenches of fluorescent signal of the dye.
 The oligonucleotide of the Taqpolymerase has a homologous
region with the target gene and thus when the target sequence is
present in the mixture, it bind with the sample DNA.
 As the taqpolymerase start to sunthesize new DNA strand in the
extension stage, it causes degradation of the probe by 5’ end
nuclease activity and the fluorescein is separated from the
quencher as a result of which fluorescence signal is generated.
 As this procedure continues, in each cycle the number of signal
molecule increases, causing the increase in fluorescence which is
positively related with the amplification of the target.
SYBR Green
 This is a dye that emits prominent fluorescent signal when it binds
at the minor groove of DNA, nonspecifically.
  Other fluorescent dyes like Ethidium Bromide or Acridine Orange
can also be used but SYBR Green is better used for its higher signal
intensity.
 SYBR Green is more preferred than the Taqman Probe as it can
provide information about each cycle of amplification as well as
about the melting temperature which is not obtained from the
Taqman probe.
 However, its disadvantage is the lack of specificity as compared to
Taqman Probe.
Advantages of Real-Time PCR
It has many advantages over the normal PCR:
 It gives a look in to the reaction that is help to decide which
reactions have worked well and which have failed.
 The efficiency of the reaction can be precisely calculated.
 There is no need to run the PCR product out on a gel after the
reaction as the melt curve analysis serve the purpose.
 The real-time PCR data can be used to perform truly quantitative
analysis of gene expression. In comparison, old fashioned PCR was
only ever semi-quantitative at best.
 Faster than normal PCR.
 Less complexity at the quantification of [Link].
Thus, unlike the ordinary preparative PCR, Real Time PCR allows the
success of multiple PCR reaction to be determined automatically
after only a few cycles, without separate analysis of each reaction,
and avoids the problem of “false negatives”.
Applications of Real-Time PCR
 Gene expression analysis
 Cancer research
 Drug research
 Disease diagnosis and management
 Viral quantification
 Food testing
 GMO food
 Animal and plant breeding
 Gene copy number

RAPD
RAPD (Random Amplified Polymorphic DNA) is a PCR-based DNA marker technique used to detect
genetic polymorphisms (variations) across the genome. It uses short, random primers to amplify different
regions of genomic DNA. These amplified fragments form unique banding patterns on agarose gel, which
help in identifying genetic differences.
What is RAPD?

 Definition: RAPD markers are DNA fragments generated by PCR amplification of

random genomic regions using single primers of arbitrary sequence.
 Key Feature: Unlike traditional PCR, RAPD does not require prior knowledge of the
DNA sequence.
 Primer: Typically short (10-mer) primers that bind randomly across the genome.

🧪 Steps in RAPD Technique

1. DNA Extraction – Isolate genomic DNA from the organism.

2. Reaction Setup – Mix DNA with arbitrary primers, nucleotides, and polymerase.
3. PCR Amplification – Random DNA segments are amplified depending on primer
binding sites.
4. Gel Electrophoresis – Separate amplified fragments on agarose gel.
5. Visualization – Banding patterns are observed; differences indicate polymorphisms

RFLP (Restriction Fragment Length Polymorphism) is a molecular marker technique used to

identify variations in DNA sequences by analyzing patterns of DNA fragments produced after
digestion with restriction enzymes. These variations occur due to mutations such as base
substitutions, insertions, or deletions that alter restriction enzyme recognition sites.

Key Concept

When genomic DNA is cut with specific restriction endonucleases, differences in the DNA
sequence lead to fragments of different lengths. These fragments are separated by gel
electrophoresis and visualized, often after Southern blotting with a labeled probe.

Applications of RFLP

 Genetic mapping and linkage analysis

 Identification of alleles and genotypes
 Marker-assisted breeding in crops and animals
 Forensics and individual identification
 Evolutionary and phylogenetic studies
 Diagnosis of genetic disorders (e.g., screening for mutation-linked alleles)

AFLP

AFLP (Amplified Fragment Length Polymorphism) is a highly sensitive, PCR-based DNA

marker technique that detects genomic polymorphisms by combining restriction digestion of
DNA with selective PCR amplification. It is more reproducible and informative than RAPD
and faster than traditional RFLP.
Applications
 Genetic diversity analysis
 Genome mapping & QTL studies
 Plant and animal breeding programs
 Phylogenetic studies & species identification
 Fingerprinting of cultivars, microbes, & populations

Advantages
 Highly reproducible and reliable
 No prior sequence information needed
 Detects many loci across the genome simultaneously
 Suitable for complex genomes (plants, polyploids)

Principle of AFLP
AFLP works by:

1. Cutting genomic DNA with restriction enzymes (commonly EcoRI and MseI).
2. Ligating adapters to the sticky ends of the fragments.
3. Selective PCR amplification using primer pairs complementary to the adapters + a few
selective nucleotides.
4. Separation of fragments on polyacrylamide or agarose gels to observe polymorphic
band pattern

Principle

1. Extract genomic DNA

2. Digest DNA with selected restriction enzyme(s)
3. Separate fragments on agarose gel
4. Transfer DNA to membrane (Southern blotting)
5. Hybridize with labeled probe
6. Detect banding pattern → Compare polymorphisms

Applications
 Genetic diversity analysis
 Genome mapping & QTL studies
 Plant and animal breeding programs
 Phylogenetic studies & species identification
 Fingerprinting of cultivars, microbes, & populations
Advantages
 Highly reproducible and reliable
 No prior sequence information needed
 Detects many loci across the genome simultaneously
 Suitable for complex genomes (plants, polyploids)

SNP (Single Nucleotide Polymorphism) is the most common type of genetic variation found in
genomes. It refers to a change in a single nucleotide base (A, T, C, or G) at a specific position in
the DNA sequence. These variations occur naturally among individuals and are widely used as
molecular markers in genetics, breeding, and disease studies.

Definition

A SNP is a single base change at a specific location in the genome, such as:

A T G C T A → A T A C T A

Here, G is replaced by A at one position — this forms a SNP.

Type Description
Transition Purine↔Purine (A↔G) or Pyrimidine↔Pyrimidine (C↔T)
Transversion Purine↔Pyrimidine (A↔C, A↔T, G↔C, G↔T)
Coding region SNP May affect gene/protein structure
Non-coding region SNP May affect regulation or have neutral effect

Applications of SNPs

 Genetic diversity & evolutionary studies

 Genome-wide association studies (GWAS)
 Disease gene mapping in humans
 Marker-assisted selection in plants/animals
 Personalized medicine & drug response prediction

Advantages

 Extremely abundant and widespread in the genome

 Co-dominant — identifies homozygotes vs heterozygotes clearly
 High reproducibility and accuracy