TYPES OF PCR

PCR entails enzymatic amplification of specific DNA sequences using two oligonucleotide primers
that flank the DNA segment to be amplified. The rapid production of large quantities of a specific DNA
sequence took a leap forward with the development of the PCR. The PCR requires two nucleotide
oligomers that hybridise to the complimentary DNA strands in a region of interest. The oligomers serve as
primers for a DNA polymerase that extends each strand. The primer must compliment to opposite strand so
that after annealing, their 3ends in effect face each other. Repeated cycling of PCR yields large amounts of
DNA molecule of interest in a matter of hours as opposed to days & weeks associated with cloning
techniques. The PCR amplification of a specific DNA sequence can be accomplished with a purified DNA
sample or a small region within a complex mixture of DNA.

The PCR procedure has three steps, which are usually repeated many times in a cyclical manner.

1. Denaturation at 94°C: During the denaturation, the double strand melts open to single stranded
DNA, all enzymatic reactions stop (for example: the extension from a previous cycle).
2. Annealing at 54°C*: The primers are jiggling around, caused by the Brownian motion. Hydrogen
bonds are constantly formed and broken between the single stranded primer and the single stranded
template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little
piece of double stranded DNA (template and primer), the polymerase can attach and starts copying
the template. Once there are a few bases built in, the hydrogen bond is so strong between the
template and the primer, which it does not break anymore.
* The exact annealing temperature varies with the length and nucleotide composition of the primers
and it has to be standardised for each PCR by trial and error. The annealing temperature is usually
3-5 ºC less than the temperature of melting (Tm) of the primers which is calculated as follows:
Tm (ºC) = (G +C) x 4 + (A+T) x 2
Annealing temperature (Ta) = Tm - (3-5ºC)
3. Extension at 72°C: This is the ideal working temperature for the polymerase. The primers, where
there are a few bases built in, have a stronger attraction to the template, created by hydrogen bonds,
than the forces breaking these attractions. Primers that are on positions with no exact match get
 loose again (because of the higher temperature) and do not give an extension of the fragment. The
bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds
dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the
template).
Each set of three steps comprises a cycle. The extension products of one primer provide a template
for the other primers in subsequent cycles so that each successive cycle essentially doubles the amount of
DNA. This result in exponential accumulation of specific target fragment by approximately to 2n, when n is
the number of cycles. This specific target fragment is called short product and each primer is physically
incorporated into one strand of the short product. Other products are synthesized during the succession of
cycles such as the large product of indefinite length, which is derived from the template molecules. The
large product only increases arithmetically during each cycle of amplification process because the
quantities of the original template remain a constant. At the end of the PCR process the short product is so
overwhelmingly abundant compared with long product that its purification is not required for most
purpose.
The PCR is commonly carried out in a reaction volume of 10–100 µl in small reaction tubes (0.2–
0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the
temperatures required at each step of the reaction. Many modern thermal cyclers make use of the Peltier
effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the
electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid
thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the
reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture
or a ball of wax inside the tube.

Amplification of Diglyceride acyl transferase-1 Gene (DGAT1)

from Genomic DNA by PCR
a) Buffers and Solutions
b) Genomic DNA template (50 ng /µl)
c) dNTP mix
d) 10X Taq polymerase buffer
e) Taq DNA polymerase
f) Forward primer
g) Reverse primer
h) Nuclease free water
Protocol
1. Label the thin walled PCR tubes suitably with a marker pen. The details may consist of the sample
number, gene name etc.
2. Preparation of the primers: The primers are obtained as lyophilized powder. Spin the tube briefly to
collect the primers at the bottom prior to opening the screw cap. Reconstitute the primer in required
volume of nuclease free water to obtain a stock concentration of 100 pM/µl. Tap the bottom of the
tube gently to dissolve the primer and keep it at room temperature for 1 hour to facilitate complete
dissolution. Spun briefly to allow undissolved particles to settle down and store the primer in aliquots
at -20 C. The working solution is prepared by dilution of the stock with nuclease free water to obtain
10 pM/ µl at the time of use.
3. The template DNA is pipetted directly to the bottom of the labeled reaction tubes.
4. Preparation of the master mix. A master mix of all components of the PCR except the template DNA is
prepared as follows:
 For 1 sample For 10
samples
†Template DNA (50 ng/ µl) 2 µl 20 µl

10X Taq polymerase buffer (without 5 µl 50 µl

MgCl2)

MgCl2 (25 mM) 3 µl 30 µl

dNTP mix (10 mM each) 1 µl 10 µl

Forward primer (40 pM/µl) 1 µl 10 µl

Reverse primer (40 pM/µl) 1 µl 10 µl

Taq DNA polymerase (5 U/µl) 0.3 µl 3 µl

Nuclease free water 35.7 µl 357 µl

Total volume of reaction 50 µl 500 µl

† Template DNA is added separately into each reaction tube and is not included in the master mix
5. The template DNA is pipetted separately into the bottom of the labeled PCR tubes. The quantity of all
other components for the required number of samples are calculated and pipetted into a sterile
microcentrifuge tube in the order of Nuclease free water, 10 X buffer, dNTP mix, primers and finally
Taq DNA polymerase and mix well by pipetting.
6. Pipette 48 µl of maser mix into each PCR tube to which the template DNA has already been added and
close the lid properly.
7. Spin the tube briefly to collect the contents at the bottom of the PCR tubes and load into PCR machine.
8. Select the desired programme and run. After the PCR is over, test the samples by electrophoresis on
1.5 % agarose gels using a molecular size ladder to confirm the size of the amplicon.

Programme for amplification of DGAT1 gene

35 Cycles of

Initial Primer
Denaturation Primer annealing Final extension
denaturation extension

94ºC for 2 min 94ºC for 30s 59ºC for 1min 72ºC for 1 min 72ºC for 7 min

2. REAL-TIME PCR (qPCR):

In molecular biology, real-time polymerase chain reaction, also called quantitative real time
polymerase chain reaction (qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a laboratory
technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA
molecule. It enables both detection and quantification of one or more specific sequences in a DNA sample.
The procedure follows the general principle of polymerase chain reaction; its key feature is that the
amplified DNA is detected as the reaction progresses in real time, a new approach compared to standard
PCR, where the product of the reaction is detected at its end. Two common methods for detection of
products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded
DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a
fluorescent reporter which permits detection only after hybridization of the probe with its complementary
DNA target.

Real-time PCR with double-stranded DNA-binding dyes as reporters:

A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the
dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is
measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsDNA dyes such
as SYBR Green will bind to all dsDNA PCR products, including non-specific PCR products (such as
primer dimer) necessitating the need for perfect standardization of PCR without the amplification of non-
specific products.

Fluorescent reporter probe method:

Fluorescent reporter probes detect only the DNA containing the probe sequence; therefore, use of
the reporter probe significantly increases specificity, and enables quantification even in the presence of
non-specific DNA amplification. Fluorescent probes can be used in multiplex assays for detection of
several genes in the same reaction, based on specific probes with different-coloured labels, provided that
all targeted genes are amplified with similar efficiency. The specificity of fluorescent reporter probes also
prevents interference of measurements caused by primer dimers, which are undesirable potential by-
products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer
dimers, which may depress accumulation of the desired products in the reaction.
The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of
fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents
detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of the Taq
polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence,
which can be detected after excitation with a laser. An increase in the product targeted by the reporter
probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of
the probe and release of the reporter.
Relative Quantification and Absolute quantification Real-time PCR data are quantified absolutely and
relatively. Absolute quantification employs an internal or external calibration curve to derive the input
template copy number. Absolute quantification is important in case that the exact transcript copy number
needs to be determined, however, relative quantification is sufficient for most physiological and
pathological studies.

Absolute quantification: For absolute quantification of a target gene mRNA, a standard curve is first
generated by plotting Ct values (Y-axis) against the log of the copy number (on the X-axis). For this known
copy numbers (from 101 to 107 copies) of the same target (in a cloned plasmid) is used as templates for
Real time PCR and the respective Ct values are recorded. A line representing the best fit is calculated for a
standard curve using the least squares method of linear regression.

y=mx+b

where y = Ct, m = slope,

x = log10 template amount,

 and b = y-intercept

For double-stranded templates, use 660 gm/mole/base. To generate a standard curve, prepare seven
10-fold dilutions, starting with 107 template copies and ending with 10 copies. For example, if the gene is
cloned in a plasmid of 7200 bp, instead of 75, write 7200.
  A line representing the best fit is calculated for a standard curve using the
least squares method of linear regression
Unknown
Standard Curve

101

102

Cycle number
103
y=mx+b
where y = Ct, m = slope,
x = log10 template amount,
Log of concentration and b = y-intercept.
Division of Veterinary Biotechnology
Indian Veterinary Research Institute

Relative quantification

Relative quantification relies on the comparison between expression of a target gene versus a
reference gene (to calculate ΔCt); and the expression of same gene in treatment sample versus control
(calibrator) samples (to calculate ΔΔCt). Since relative quantification is the goal for most of the real time
PCR experiments, several data analysis procedures have been developed. The method used is referred to as
Comparative Ct Method or ΔΔCt method. This involves comparing the Ct values of the samples of interest
(such as treated) with a control or calibrator (such as a non-treated) sample or RNA from normal tissue.
The Ct values of both the calibrator and the samples of interest are normalized to an appropriate
endogenous housekeeping gene.

The comparative Ct method is also known as the 2–∆∆Ct method, where

∆∆Ct = ∆Ct,sample - ∆Ct,calibrator

∆Ct = Ct value for the gene of interest –Ct value of the endogenous control.

Here, ∆Ct,sample is the Ct value for any sample normalized to the endogenous housekeeping gene and
∆Ct,calibrator is the Ct value for the calibrator also normalized to the endogenous housekeeping gene (eg. 18 S
rRNA gene). For the ∆∆Ct calculation to be valid, the amplification efficiencies of the target and the
endogenous reference must be approximately equal. This can be established by looking at how ∆C t varies
with template dilution. Common internal controls include ß-actin and GAPDH mRNAs and 18S rRNA. If a
housekeeping gene cannot be found whose amplification efficiency is similar to the target, then the relative
standard curve method is preferred.
 Then RQ is found out by the formula, RQ = 2- ΔΔCt = 2-2

Protocol for relative quantification of gene expression

Samples : cDNA from peripheral blood leukocytes of Vechur and Crossbred cattle
Target gene : Hsp70-1
Reference gene : Beta actin

Buffers and Solutions

Maxima SYBR Green qPCR Master Mix (2X) with ROX: Maxima SYBR Green qPCR Master Mix
(Thermo Scientific make) contains all PCR reagents such as dNTPs, Taq DNA polymerase, buffer and
MgCl2. It also contains SYBR green 1 dye and ROX dye which allows for correction of well to well
variations due to pipetting inaccuracies and fluorescence fluctuations.
1. Check the concentration of the RNA isolated and use 100 ng of total RNA for cDNA synthesis (It is
important to take same of amount of RNA for cDNA synthesis from test and calibrator samples for
accurate qRT PCR analysis).
2. Set up separate PCR reactions for each Hsp70-1 and Beta actin genes. At least three biological
replicates (3 Vechur and 3 crossbred animals each) are needed for relative quantification. Each sample
is amplified in triplicate (known as technical replicates). In addition, one non-template control (NTC)
for each gene and Reverse Transcription minus (RT minus) control for each sample and a negative
control (with only nuclease free water) are also included in the reaction. In RT minus control, RNA is
loaded instead of cDNA to check for any DNA contamination in the isolated RNA. Accordingly, a
suitable plate setting is done prior to start of the experiment as shown in the figure below.

3. Prepare the master mix for required number of reactions as follows:

Master Mix for Hsp70-1 gene

For one sample 15 samples

Components
(µl) ( µl)

Template (cDNA) 1.0 15

Maxima SYBR Green qPCR

6.25 93.75
Master Mix (2X)

Forward Primer (10 pM/ µl)) 1.0 15

Reverse primer (10 pmol/µl) 1.0 15

 Nuclease free water 3.25 48.75

Total reaction volume 12.5 187.5

Master Mix for beta actin gene

For one
Components 15 samples
sample (µl)

Template (cDNA) 1.0 15

Maxima SYBR Green qPCR Master Mix

6.25 93.75
(2X)

Forward Primer (10 pM/ µl)) 1.0 15

Reverse primer (10 pmol/µl) 1.0 15

Nuclease free water 3.25 48.75

Total reaction volume 12.5 187.5

4. The Illumina Eco RT PCR system utilizes a 48 well microplates to carry out the reactions. The template
(cDNA) is loaded separately into the designated wells.
5. Make a master mix of the rest of the components as in the case of conventional PCR and load 23 µl
each in the wells.
6. For loading samples place a 48 well plate into the Eco sample loading dock, aligning the notch with the
matching indentation on the adapter. Turn on the dock light and incline the dock to a comfortable
angle for pipetting.
7. Pipette samples and qPCR reagents into the plate and seal the plate with an Eco optical seal, holding
the plate in place on the Eco sample loading dock, drag the squeegee firmly across the surface to
ensure the seal is secure.
8. Place the plate adapter with your loaded and sealed plate into a plate centrifuge along with a second
plate adapter for balance. Centrifuge the plate at 250 x g for 30 seconds.
9. Open the Eco lid and place the plate on the block, aligning the notch against the top left corner. Close
the lid and start the reaction.

PCR conditions for qRTPCR

Sl No Step Temperature Duration

1 Initial denaturation 95 °C 10 min

2 Denaturation 95 °C 15 S

3 Annealing 60 °C 60 S

Steps 2 &3 were repeated to 35 cycles & Data acquisition is performed during the annealing step.

10. Perform a melt curve analysis after the reaction for checking specificity of amplification. The
programme for melt curve analysis consists of denaturation at 95 ⁰C or 15 seconds, annealing at 55⁰C
 for 15 seconds followed by 95⁰C for 15 seconds. Data acquisition is performed during the final
denaturation step.
11. Analyse the data for relative quantification by comparative Ct Method or ΔΔCt method.

3. REVERSE TRANSCRIPTION PCR (RT-PCR):

The complementary DNA (cDNA) is DNA copy of the RNA molecules, synthesized by the enzyme
RNA dependent DNA polymerase (Reverse transcriptase) enzyme. The reverse transcriptase enzymes are
generally isolated from Avian Leukemia Virus (ALV) or from a strain of E.coli that expresses a cloned
copy of the reverse transcriptase gene of Moloney strain of murine leukemia virus (Mo-MLV) are
commercially available. cDNA synthesis is an important molecular biology step for various purposes like
construction of cDNA libraries, for PCR diagnosis of diseases caused by RNA viruses and for gene
expression studies. The first strand of the cDNA is copied from the mRNA (polyA+ RNA) by Reverse
transcriptase enzyme using oligo dT or random oligonucleotide primers. For construction of cDNA
libraries, it is necessary to synthesize the second strand to obtain the double stranded cDNA for cloning in
a suitable plasmid vector. It is synthesized using DNA polymerase enzyme, after removing the RNA from
the RNA-DNA heterodimer, using different strategies. For PCR based analysis, it is sufficient to synthesis
only the first strand of cDNA.
Synthesis of cDNA from total RNA using cDNA synthesis kit (Fermentas, K1211)

Materials supplied

a) Reverse transcriptase (M-MuLV)

b) 5X reaction buffer
c) Oligo(dT)18 and random hexamer primers
d) RiboLock RNase inhibitor (20 U/µl)
e) Control RNA (RNA template of GAPDH gene)
f) Primers for GAPDH
g) 10 mM dNTP mix
h) Nuclease free water

Precautions to be taken: Since RNA is highly prone to degradation by contaminating RNases, the
following precautions need to be taken while synthesizing cDNA from total RNA.

1. Treat all tubes with DEPC and use only RNase free pipette tips.
2. Change gloves frequently
3. Use RNAse free reagents including high quality nuclease free water.
4. Keep all kit components tightly sealed when not in use. Keep all tubes tightly closed during the
reverse transcription reaction.
Protocol for First strand cDNA synthesis
1. Mix and briefly centrifuge all components after thawing and keep in ice.
2. Add template RNA (isolated RNA), primer and nuclease free water into sterile nuclease free tubes
on ice mix gently, centrifuge briefly and incubate at 65 ⁰C for 5 minutes.
3. For positive control, use RNA template of GAPDH gene supplied in the kit instead of the isolated
RNA.
4. Chill on ice, spin down and place the vial back on ice.
5. Prepare a master mix of rest of the components for the required number of samples and centrifuge
briefly.
 Volume (µl) Volume (µl)
Components
for 1 sample for 5 samples
†
Template RNA (50 ng/µl) 2 10
†
Oligo (dT)18 primer or Random hexamer
1 5
primer
†
Water, nuclease free 8 40

5X reaction buffer 4 20

RiboLock RNase inhibitor (20 U/µl) 1 5

dNTP mix (10mM) 2 10

M-MuLV Reverse Transcriptase 2 10

(20U/ µl)
Total volume of reaction 20 100

†Added separately into each reaction tube and is not included in the master mix

6. Add 9 µl of maser mix into each tube followed by mixing and centrifuging.
7. For Oligo (dT)18 primed cDNA synthesis incubate for 60 minutes at 37 ⁰C (Oligo (dT)18 primed
cDNA is usually preferred for full length cDNA synthesis).
8. For random hexamer primed synthesis (for real time PCR or similar purposes) incubate for 5
minutes at 25 ⁰C followed by 60 minutes at 37 ⁰C.
9. Terminate the reaction by heating at 70 0C for 5 minutes.
The reverse transcription product can be directly used for PCR applications or store at
–20 ⁰C for less than one week. For longer storage, -70 ⁰C is recommended.

Control reactions: The following positive and negative control reactions are also set up cDNA synthesis.

1. Reverse transcriptase minus (RT-) negative control. This reaction contains every reagent for the
reverse transcription reaction except for the Reverse Transcriptase enzyme.
2. No template negative control. Contains every reagent for the reverse transcription reaction except
for RNA template.
3. Positive control. This reaction uses the RNA template of GAPDH gene that generates a 496 bp RT-
PCR product.
Positive control GAPDH PCR amplification

Components Volume (µl)

cDNA from positive control reaction 2

(1:1000) dilution
10X PCR Buffer 5

dNTP mix (10 mM) 1

MgCl2 (25 mM) 3

Forward GAPDH primer (10 pM/µl) 1.5

Reverse GAPDH primer (10 pM/µl) 1.5

 Taq DNA polymerase (5 units/µl) 0.5

Water, nuclease free 35.5

Total reaction volume 50

PCR conditions for GAPDH PCR amplification

Step Temperature Time No. of cycles

Initial
94 o C 3 min 1
denaturation

Denaturation 94oC 30 sec

Annealing 58oC 30 sec 35

Extension 72oC 45 sec

Load 5 µl PCR products on 1% agarose gel. A distinct 496 bp PCR product will be visible.

4. NESTED PCR:

Nested PCR (Polymerase Chain Reaction) is a variant of the traditional PCR technique that involves two
sets of primers to amplify a target DNA sequence. This method is often used when the target DNA is
present in low concentrations or when specificity is crucial.

Materials and Reagents:

1. DNA template
2. Taq DNA polymerase
3. PCR primers (outer and inner primers)
4. dNTPs (Deoxynucleotide triphosphates)
5. PCR buffer
6. MgCl2
7. Sterile water
8. Thermal cycler
9. Microcentrifuge tubes
10. PCR tubes/strips

Procedure:

A. Primer Design:

 Design two sets of primers - outer primers and inner primers.

 Outer primers should be specific to the target region, and inner primers should be designed within
the amplicon produced by the outer primers.

B. PCR Mix Preparation:

 Prepare a master mix with the following components per reaction:

o DNA template (1-100 ng)
o PCR buffer (1x concentration)
o MgCl2 (Optimized for the specific primers, usually 1.5-2.5 mM)
 o dNTPs mix (200 µM each)
o Forward and reverse outer primers (0.2-1 µM each)
o Taq DNA polymerase (1.25-2.5 units per reaction)
o Sterile water to make up the volume

C. First Round of PCR (Outer Amplification):

20- 30 Cycles of

Initial Primer
Primer Final extension
denaturation Denaturation annealing Outer
extension
Primers

94ºC for 20- 72ºC for 30s-

94ºC for 2-5 min 59ºC for 1min 72ºC for 5-10 min
30s 2min

D. Gel Electrophoresis:

 Analyze the PCR products using agarose gel electrophoresis.

 Visualize the bands under UV light.

E. Nested PCR Mix Preparation: Prepare a new master mix using the same components as in step 2, but
with the inner primers instead of the outer primers.

F. Second Round of PCR (Inner Amplification):

 Use a small amount (1-2 µl) of the first PCR product as the template for the nested PCR.
 Set up the reaction and perform amplification as described in step 3, but using the inner primers.
 Again, analyze the products by agarose gel electrophoresis.

20- 30 Cycles of

Initial Primer
Primer Final extension
denaturation Denaturation annealing Inner
extension
Primers

94ºC for 20- 72ºC for 30s-

94ºC for 2-5 min 59ºC for 1min 72ºC for 5-10 min
30s 2min

G. Analysis:

 Compare the band patterns from the first and second rounds of PCR.
 The nested PCR should yield a more specific and intense band due to the amplification within the
first PCR product.

Notes:

 Maintain sterile techniques to avoid contamination.

 Optimize PCR conditions such as annealing temperature, MgCl2 concentration, and cycle number
for your specific primers and template.
 Always include positive and negative controls in each PCR run.
5. MULTIPLEX PCR:

 Amplifies multiple target sequences in a single reaction.

 Uses multiple primer pairs with different fluorophores for detection.

Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a
single PCR experiment. In a multiplexing assay, more than one target sequence can be amplified by using
multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has
the potential to produce considerable savings in time and effort within the laboratory without
compromising on the utility of the experiment.

Amplifying different DNA sequences or different DNA templets by using the different set of primers in a
single PCR reaction is refers to as a multiplex PCR. PCR technology is now a routine day method used in
the diagnostic industries. Because, it is efficient and accurate. However, the cost of the experiment and the
time utilised in performing the experiment creates a major setback for the technology.

The cost of a single PCR experiment is very high. Also, the entire procedure takes more than 4 hours for a
single experiment, starting from DNA extraction to the gel electrophoresis.

Types of Multiplex PCR

Multiplexing reactions can be broadly divided in two categories:

1. Single Template PCR Reaction(Uni-Template multiplex PCR)

This technique uses a single template which can be a genomic DNA along with several pairs of forward
and reverse primers to amplify specific regions within a template.

2. Multiple Template PCR Reaction

It uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primers
may lead to cross hybridization with each other and the possibility of mis-priming with other template.

Multiplex PCR facilitates multiple reactions in one single run which means we can analyse multiple
template locus in 4 hours (time required for a single experiment). The technique is first described in the
year 1988 by Jeffrey S. Chamberlain and coworkers. They had used the multiplex PCR assay to screen
the Duchenne muscular dystrophy locus.

They had demonstrated the technique is for preferentially for prenatal and postnatal diagnosis. They had
used multiple sets of primers for encountering multiple deletions in the dystrophin gene. The multiplex
PCR reaction is a technique widely used in virology and in the single gene disorders.

Developing a Multiplex PCR (MPCR) assay poses challenges compared to uniplex PCR due to the need for
high expertise and extensive trial-and-error experimentation. Multiplex PCR involves amplifying multiple
targets within a single reaction, requiring a higher amount of PCR reagents. When designing a single-
template MPCR assay, a higher concentration of template DNA is recommended, surpassing that used for
uniplex PCR. Additionally, both Taq DNA polymerase and MgCl2 concentrations should be elevated for
optimal performance.

Primer design becomes a critical aspect of MPCR as multiple primer sets are utilized, demanding
uniqueness and precision. Each primer set must avoid cross-binding with others and prevent the formation
of dimers. The GC content of primers should fall within 45% to 60%, with a melting temperature between
55°C to 60°C. It is crucial that the annealing temperatures of all primer sets are closely aligned (within a
4°C difference) for efficient amplification. Furthermore, the length of each primer set, ranging from 18 to
30 base pairs, plays a crucial role in the success of the reaction. Overall, the intricacies of primer design
and optimization are paramount for the successful development of a robust and specific MPCR assay.

The PCR reaction cycles also play a vital role in achieving amplification for each template DNA. In some
cases, if the PCR cycles are more, the amplification cannot be properly achieved because of the
unavailability of the PCR reagents or templates in later cycles.

Use proper cycling condition, not more or not less and this should be achieved by practising and
experimenting repeatedly. 25 to 30 cycles are sufficient for it.

The protocol for multiplex reaction varies from the experiment to experiments. For example, the protocol
used in the Y chromosome microdeletion multiplex PCR is almost the same as the uniplex PCR for every
single marker for Y chromosome microdeletion. While the protocol for the DMD gene is more complex,
required more amount of reagents and expertise.

Components Concentration Volume (µl)

Master mix 1X 13µL

PCR Buffer 1X If needed

Forward primer (4 sets of primers) 10pM 1 X 4= 4µL

Reverse primer (4 sets of primers) 10pM 1 X 4= 4µL

Template DNA 50ng 5µL

Water, nuclease free 4µL

Total reaction volume 30

(Note: the ready to use mastermix contains the PCR buffer, so the PCR reaction buffer is not needed).

Primer Primer
PCR Steps Initial Denaturation
annealing extension Final extension
denaturation

Temperature 94ºC 94ºC 59ºC 72ºC 72ºC

Times 2-5 min 20- 30s 1min 30s-2min 5-10 min

25-28 Cycles

To avoid the non-specific bindings, primer dimer formation, hairpin formation and unavailability of the
templates different components such as MgCl2, KCl, albumin, DMSO, betaine or Taq DNA polymerase
can be added to the reaction, However, it depends on the results, what kind of problem is arise in the
reaction.

Advantages of multiplex PCR

 The multiplex PCR assay consumes less time and manpower with greater efficiency.
  The method is actually rapid and accurate.
 By comparing different amplicons of a single template we can determine the quality of the
template.
 We can gather more information in a single run hence by utilizing fewer samples.
 Also, the chance of pipetting errors is less in multiplex PCR.
 Fewer consumables and chemicals used in the multiplex PCR.

Limitations of multiplex PCR

 Although the technique is advantageous, the multiplexing is not applicable to all types of reaction.
 For the larger amplicon such as 800bp or 1000bp, multiplex PCR might not work efficiently
always.

Application of multiplex PCR

 Virology and Pathogen Detection:

o Widely used in virology and pathogen detection.
o Offers rapid and precise identification compared to traditional microbiology culture
techniques.
 Advantages over Culture Techniques:
o Overcomes the tedious and time-consuming nature of traditional culture methods.
o Significantly reduces the risk of contamination.
 Revolutionizing PCR: Multiplex PCR revolutionizes conventional PCR by enabling the
identification of multiple pathogens or strains in a single reaction.
 Diagnostic Applications: Used in the diagnostic industry for identifying infections in various
areas, including ocular, urinogenital, lung, respiratory, neurotropic viral, and tuberculosis
infections.
 Ready-to-Use Kits: Ready-to-use standard multiplex pathogen detection kits available for
detecting pathogens like HSV strains, EBV infection, VZV, CMV, T.gondii, influenza, and
adenoviral.
 Pathogen Identification:
o Enables the identification and categorization of different viruses.
o Allows grouping of pathogens and identification of their strains.
 SNP Genotyping:
o Used for multiple SNP genotyping, allowing the detection of more than single SNPs in a
single reaction.
o Eg: Thalassemia detection with various SNPs like IVS1-1, IVS1-5, IVS (G-C), CD5, and
CD15.
 Genetically Modified Organisms (GMO) Studies: Valuable tool in studying genetically modified
organisms.
 Forensic Studies:
o Utilized in forensic studies for identifying organisms by targeting different loci.
o Multiple loci can be screened in a single experiment.
 Mutation Detection and Polymorphism Analysis:
o Rapid and cost-effective for mutation detection.
o Example: Y chromosome microdeletion studies, where more than 12 markers of the Y
chromosome are examined in a single reaction.
 DNA Deletion Studies: Enables Broda range DNA deletion studies.
 Linkage Analysis and RNA Detection: Facilitates linkage analysis and RNA detection.
 Qualitative and Quantitative DNA Analysis: Allows for qualitative and quantitative analysis of
template DNA.
 Ease of Work: Facilitates the simultaneous study of multiple markers in a single reaction,
streamlining research efforts.
  Cost-Effective Mutation Detection: MPCR significantly enhances the speed and cost-
effectiveness of mutation detection, contributing to advancements in genetic studies.

6. HOT START PCR:

 Minimizes non-specific amplification during reaction setup.

 Achieved by adding a heat-activated DNA polymerase or modified primers.

Hot Start PCR is a modification of the traditional PCR technique that helps reduce nonspecific
amplification and improve reaction specificity by minimizing primer-dimer formation and background
amplification during the reaction setup.

Materials and Reagents:

1. DNA template
2. Taq DNA polymerase or a Hot Start DNA polymerase
3. PCR primers
4. dNTPs (Deoxynucleotide triphosphates)
5. PCR buffer
6. MgCl2
7. TaqStart Antibody or other Hot Start reagents
8. Sterile water
9. Thermal cycler
10. Microcentrifuge tubes
11. PCR tubes/strips

Procedure:

1. Primer Design:

 Design primers for the target DNA sequence as per standard PCR primer design guidelines.

2. PCR Mix Preparation:

 Prepare a master mix with the following components per reaction:

o DNA template (1-100 ng)
o PCR buffer (1x concentration)
o MgCl2 (Optimized for the specific primers, usually 1.5-2.5 mM)
o dNTPs mix (200 µM each)
o PCR primers (0.2-1 µM each)
o Taq DNA polymerase (1.25-2.5 units per reaction)
o TaqStart Antibody or other Hot Start reagents, following the manufacturer's
recommendations.
o Sterile water to make up the volume

3. Hot Start Activation:

 Keep the reaction mix on ice until ready to load into the thermal cycler.
 Add the Taq DNA polymerase or Hot Start enzyme to the reaction mix just before placing it into
the thermal cycler.
 Perform an initial denaturation at 94°C for 2-5 minutes.

4. PCR Cycling:

 Follow with 20-40 cycles of:

 o Denaturation at 94°C for 20-30 seconds.
o Primer annealing at the optimal temperature for the primers for 20-30 seconds.
o Extension at 72°C for a time determined by the expected amplicon size (typically 30
seconds to 2 minutes).
 Finish with a final extension at 72°C for 5-10 minutes.

20- 40 Cycles of

Initial
Primer Primer Final extension
denaturation Denaturation
annealing extension

94ºC for 20- 72ºC for 30s-

94ºC for 2-5 min 59ºC for 1min 72ºC for 5-10 min
30s 2min

5. Gel Electrophoresis:

 Analyze the PCR products using agarose gel electrophoresis.

 Visualize the bands under UV light.

6. Analysis:

 Evaluate the specificity and yield of the PCR products.

 Compare with positive and negative controls.

Advantages of Hot Start PCR:

1. Reduced Non-Specific Amplification: Hot Start PCR reduces nonspecific amplification by

preventing premature DNA synthesis at lower temperatures during reaction setup.
2. Enhanced Specificity: Improves specificity by minimizing primer-dimer formation and
background amplification, leading to cleaner and more reliable results.
3. Minimized Contamination Risk: Reduces the risk of contamination during reaction setup, as the
enzyme is inactive until the initial denaturation step.
4. Improved Amplification of GC-Rich Templates: Facilitates the efficient amplification of GC-
rich templates, which may be challenging with standard PCR due to primer mispriming.
5. Enhanced Sensitivity: Increases sensitivity by reducing background noise, making it particularly
useful for detecting low-copy-number targets.
6. Ready-to-Use Reagents: Commercially available Hot Start PCR kits provide ready-to-use
reagents, simplifying experimental setup and reducing variability.

Limitations of Hot Start PCR:

1. Cost: Hot Start PCR reagents and enzymes may be more expensive than regular PCR components,
which can increase overall experimental costs.
2. Complexity in Enzyme Modification: Some Hot Start PCR methods involve modifying the
enzyme, which can be complex and may require additional optimization steps.
3. Requirement for Specialized Equipment: Certain Hot Start PCR techniques may necessitate
specialized equipment, such as a thermal cycler with a heated lid, which may not be available in all
laboratories.
4. Limited Enzyme Options: Limited availability of Hot Start variants for certain polymerases may
restrict the choice of enzymes, potentially limiting compatibility with specific assays.
Applications of Hot Start PCR:

1. Multiplex PCR: Valuable for multiplex PCR applications, where the specificity and efficiency of
the reaction are crucial for simultaneously amplifying multiple targets.
2. Diagnostic Assays: Used in diagnostic assays for detecting specific pathogens or mutations with
high sensitivity and reduced background noise.
3. Next-Generation Sequencing (NGS) Library Preparation: Employed in NGS library preparation
to amplify specific regions with improved specificity and reduced artifacts.
4. High-Throughput PCR: Suitable for high-throughput PCR applications, where minimizing
contamination and ensuring reliable results are essential.
5. Genetic Research: Applied in genetic research, including genotyping and mutation detection, to
enhance the accuracy and reliability of results.
6. Amplification of Difficult Templates: Useful for amplifying challenging templates, such as those
containing secondary structures or high GC content, which may impede standard PCR reactions.
7. Forensic Studies: Applied in forensic studies where the detection of trace amounts of DNA with
high sensitivity and specificity is crucial.
8. Quantitative PCR (qPCR): Utilized in quantitative PCR to improve the accuracy of quantification
by reducing background noise and increasing the reliability of measurements.

Notes:

 Hot Start PCR reagents can be in the form of antibodies, modified enzymes, or other proprietary
formulations that inhibit polymerase activity until a certain temperature is reached.
 Hot Start PCR can be particularly useful when working with high-throughput PCR or in situations
where reaction setup may introduce the risk of contamination.
 Always perform reactions in duplicate or triplicate to ensure reproducibility.
 Optimize PCR conditions based on the specific requirements of your experiment and the
characteristics of the target DNA.

7. TOUCHDOWN PCR:

 Modifies annealing temperature during cycling.

 Enhances specificity by starting with higher annealing temperatures.

Touchdown PCR is a specialized PCR technique that utilizes a temperature gradient in the initial cycles,
gradually lowering the annealing temperature. This approach enhances the specificity of amplification by
reducing nonspecific primer binding, making it particularly useful for challenging templates or reactions
with multiple products.

Procedure:

1. Primer Design: Design specific primers for the target DNA sequence following standard PCR
primer design guidelines.
2. PCR Mix Preparation:
o Prepare a master mix with the following components per reaction:
 DNA template (1-100 ng)
 PCR buffer (1x concentration)
 MgCl2 (Optimized for specific primers, usually 1.5-2.5 mM)
 dNTPs mix (200 µM each)
 Forward and reverse primers (0.2-1 µM each)
 Taq DNA polymerase (1.25-2.5 units per reaction)
 Sterile water to make up the volume
3. Touchdown PCR Cycling:
 10 cycles with a touchdown annealing

Initial
Primer Final extension
Denaturation Denaturation Primer annealing
extension

higher
temperature (5-
10°C above the 72ºC for 30s-
94ºC for 2-5 min 94ºC for 20- 30s 72ºC for 5-10 min
calculated Tm) 2min
and decreasing by
1-2°C per cycle

Continue with 20-30 cycles a touchdown annealing

Initial Denaturation
Primer Primer Final extension
Denaturation
annealing extension

a constant lower
temperature
94ºC for 20- 72ºC for 30s-
94ºC for 2-5 min (optimal for 72ºC for 5-10 min
30s 2min
primers) for 20-
30 seconds

4. Gel Electrophoresis:
o Analyze the PCR products using agarose gel electrophoresis &Visualize the bands under
UV light.

Concentration Limitations:

 The concentration of template DNA, primers, and other reagents should be optimized based on the
specific requirements of the experiment.
 Template DNA concentration can typically range from 1-100 ng, while primer concentrations may
vary between 0.2-1 µM.

Limitations

1. Complex Optimization: Despite its benefits, the Touchdown PCR method requires careful
optimization of various parameters, including the initial annealing temperature, the rate of
temperature reduction, and the total number of cycles. This complexity can make the optimization
process time-consuming.
2. Template Dependency: The effectiveness of Touchdown PCR may vary depending on the
template and primer characteristics. Some templates may not show significant improvements with
this approach, and optimization might be required for each new template.
3. Increased Reaction Time: Touchdown PCR typically involves a higher number of cycles,
especially during the initial touchdown phase, which can increase the overall reaction time. This
may be a limitation in situations where a rapid turnaround is essential.
4. Sensitivity to Cycling Conditions: Sensitivity to cycling conditions means that small variations in
the cycling parameters, such as annealing or extension times, can impact the specificity and
efficiency of the reaction. This sensitivity may necessitate fine-tuning for optimal results.
5. Potential Primer Design Challenges: Primer design becomes crucial in Touchdown PCR, and the
method might be more sensitive to primer characteristics. Designing primers with appropriate
melting temperatures and avoiding secondary structures can be challenging.
 6. Not Universally Applicable: While Touchdown PCR is effective for many applications, it may not
be universally applicable to all PCR reactions. Some reactions may not benefit significantly from
the touchdown approach, and traditional PCR methods might be more suitable.
7. Limited Commercial Availability: Commercially available Hot Start PCR kits are more prevalent
than Touchdown PCR kits. This limitation may influence the convenience and accessibility of
reagents, especially for researchers who prefer ready-to-use solutions.
8. Risk of Over-Optimization: Excessive optimization attempts may lead to over-optimization,
where the reaction becomes highly specific for a particular set of conditions. This can make the
assay less robust when applied to different experimental setups.

Advantages of Touchdown PCR:

1. Increased Specificity: Gradual lowering of annealing temperature in initial cycles reduces

nonspecific binding, enhancing the specificity of amplification.
2. Improved Amplification of Difficult Templates: Particularly effective for challenging templates
or those prone to nonspecific amplification.
3. Reduced Primer-Dimer Formation: Minimizes the formation of primer-dimers by starting with a
higher annealing temperature.
4. Enhanced Sensitivity: Allows for efficient detection of low-copy-number targets.
5. Time Efficiency: Achieves specificity without the need for extensive optimization, saving time in
assay development.

Applications of Touchdown PCR:

1. Multiplex PCR: Useful in multiplex PCR reactions where specificity is crucial for amplifying
multiple targets.
2. Challenging Templates: Applied to amplify templates with secondary structures, high GC content,
or regions prone to nonspecific amplification.
3. SNP Genotyping: Employed in single nucleotide polymorphism (SNP) genotyping studies to
achieve precise and specific amplification.
4. Gene Expression Studies: Useful in gene expression studies where specificity is essential for
accurately quantifying mRNA levels.
5. Forensic Studies: Applied in forensic studies for reliable and specific detection of trace amounts of
DNA.
6. Diagnostic Assays: Used in diagnostic assays for detecting specific pathogens or mutations with
high sensitivity and specificity.

Touchdown PCR provides a flexible and robust approach to achieve high specificity in various PCR
applications. Adjustments to cycling conditions may be required based on the characteristics of the
template and primers used.

8. LONG PCR:

 Amplifies long DNA fragments (up to several kilobases).

 Requires special DNA polymerases with proofreading capabilities.

Long PCR is a technique designed for amplifying DNA fragments longer than the typical limit of
conventional PCR. This method involves using specialized DNA polymerases, buffer conditions, and
cycling parameters to achieve efficient amplification of longer templates.

Procedure:

1. PCR Mix Preparation:

o Prepare a master mix with the following components per reaction:
 DNA template (100 ng to 1 µg, or more, depending on template length)
  Long PCR buffer (optimized for longer fragments)
 dNTPs mix (200 µM each)
 Forward and reverse primers (0.2-1 µM each)
 Long-range DNA polymerase (e.g., Taq DNA polymerase with proofreading
activity)
 MgCl2 (Optimized for specific primers, usually 1.5-2.5 mM)
 Sterile water to make up the volume
2. Long PCR Cycling:
o Perform an initial denaturation at 94-98°C for 2-5 minutes.
o Followed by 25-40 cycles of:
 Denaturation at 94-98°C for 20-30 seconds.
 Annealing at the optimal temperature for the primers for 20-30 seconds.
 Extension at 68-72°C for a time determined by the expected amplicon size (1 minute
per kb).
o Finish with a final extension at 68-72°C for 5-15 minutes.
3. Gel Electrophoresis:
o Analyze the PCR products using agarose gel electrophoresis.
o Visualize the bands under UV light.

Advantages of Long PCR:

1. Amplification of Longer Fragments: Enables the amplification of DNA fragments longer than
those achievable by standard PCR, often up to several kilobases or even megabases.
2. Reduced Need for Subcloning: Minimizes the need for subcloning when working with long DNA
fragments, as longer regions can be amplified in a single reaction.
3. Efficient Amplification of Genomic Regions: Suitable for efficient amplification of long genomic
regions, including full-length genes, promoters, and other large genetic elements.
4. Reduced Chances of Breakpoints: Decreases the likelihood of introducing breakpoints or
artificial rearrangements during the amplification of large genomic sequences.
5. Seamless Cloning: Facilitates seamless cloning of long DNA fragments for subsequent
applications, such as gene cloning and expression studies.
6. Reduced Template Requirements: Requires lower template concentrations compared to other
long DNA amplification methods like inverse PCR.

Limitations of Long PCR:

1. Higher Sensitivity to Reaction Conditions: Long PCR is more sensitive to reaction conditions,
and optimization may be required for each target region, making it more challenging to develop
standardized protocols.
2. Increased Susceptibility to Errors: The longer the fragment, the higher the chance of introducing
errors during DNA synthesis, such as point mutations or polymerase errors.
3. Reduced Specificity: Longer amplicons may suffer from reduced specificity due to increased
opportunities for nonspecific primer binding.
4. Specialized DNA Polymerases: Requires specialized DNA polymerases with proofreading
activity, which can be more expensive than regular DNA polymerases.
5. Limited Applicability to All Templates: While suitable for many templates, not all DNA
templates respond well to long PCR, and certain challenging sequences may require alternative
methods.

Applications of Long PCR:

1. Genomic Walking: Used for genomic walking, allowing the amplification of unknown sequences
adjacent to known regions.
2. Gene Cloning and Expression Studies: Applied in gene cloning and expression studies, especially
for amplifying full-length genes and regulatory regions.
 3. Molecular Pathology: Used in molecular pathology for the amplification of large genomic regions
associated with diseases or genetic disorders.
4. Molecular Evolution Studies: Applied in molecular evolution studies, enabling the amplification
and analysis of large genomic regions to study genetic diversity.
5. Library Construction: Utilized in the construction of DNA libraries for genomic sequencing and
other large-scale genomic projects.
6. Ancient DNA Studies: Valuable for ancient DNA studies, where degraded DNA templates may
require longer fragments for analysis.
7. Structural Genomics: Used in structural genomics for amplifying large segments of genomic
DNA to study the structure and function of genes.
8. Bacterial Artificial Chromosome (BAC) Cloning: Applied in BAC cloning, allowing the
amplification of large DNA fragments for the construction of BAC libraries.

9. FAST PCR:

 Reduces overall reaction time.

 Achieved by optimizing buffer conditions, using specialized enzymes, etc.

Fast PCR is a modification of the traditional PCR technique aimed at reducing the overall reaction time
while maintaining the specificity and efficiency of DNA amplification. The method involves optimizing
various parameters, including reaction components and cycling conditions, to achieve faster results.

Procedure:

1. PCR Mix Preparation:

o Prepare a master mix with the following components per reaction:
 DNA template (1-100 ng)
 Fast PCR buffer (optimized for faster reactions)
 dNTPs mix (200 µM each)
 Forward and reverse primers (0.2-1 µM each)
 Fast DNA polymerase (designed for rapid extension)
 Sterile water to make up the volume
2. Fast PCR Cycling:
o Perform an initial denaturation at 94-98°C for 2-5 minutes.
o Followed by 20-40 cycles of:
 Denaturation at 94-98°C for 1-10 seconds.
 Annealing at the optimal temperature for the primers for 1-10 seconds.
 Extension at 68-72°C for a time determined by the expected amplicon size (1
minute per kb).
o Finish with a final extension at 68-72°C for 5-10 minutes.
3. Gel Electrophoresis:
o Analyze the PCR products using agarose gel electrophoresis.
o Visualize the bands under UV light.

Advantages of Fast PCR:

1. Rapid Turnaround Time: Significantly reduces the overall reaction time, enabling faster results.
2. Increased Throughput: Facilitates higher throughput by allowing more reactions to be performed
in a shorter period.
3. Reduced Template and Primer Requirements: Requires lower template and primer
concentrations compared to standard PCR.
4. Compatibility with Real-time PCR: Can be easily adapted for real-time PCR applications,
allowing for rapid quantitative analysis.
5. High Sensitivity: Maintains high sensitivity, making it suitable for applications where low-copy-
number targets need to be detected.
Limitations of Fast PCR:

1. Reduced Specificity: Higher cycling speeds may compromise the specificity of the reaction,
leading to increased nonspecific amplification.
2. Increased Risk of Primer-Dimer Formation: The rapid cycling conditions may increase the
likelihood of primer-dimer formation, impacting the specificity of the reaction.
3. Optimization Challenges: Achieving optimal conditions for fast PCR can be more challenging due
to the increased sensitivity to reaction parameters.
4. Compatibility with Some DNA Polymerases: Not all DNA polymerases are suitable for fast PCR,
and using inappropriate enzymes may result in suboptimal amplification.

Applications of Fast PCR:

1. Screening Assays: Rapid screening of samples, particularly in high-throughput settings.

2. Diagnostic Testing: Quick detection of pathogens or genetic markers in diagnostic applications.
3. Forensic Studies: Expedited DNA analysis in forensic studies where quick results are essential.
4. Mutation Detection: Fast identification of mutations or polymorphisms in genetic studies.
5. Real-time Quantitative PCR: Adaptation for real-time quantitative PCR applications, enabling
rapid and accurate quantification.
6. Point-of-Care Testing: Suitable for point-of-care testing scenarios where rapid turnaround is
critical.
7. Environmental Monitoring: Swift detection of specific DNA targets in environmental samples for
monitoring purposes.
8. Pathogen Detection: Rapid identification of pathogens in food, water, or clinical samples.

10. INVERSE PCR:

 Amplifies DNA fragments adjacent to known sequences.

 Useful for identifying unknown sequences flanking known regions.

Inverse PCR is a specialized PCR technique used for amplifying unknown sequences flanking a known
DNA fragment. This method involves digesting genomic DNA with a restriction enzyme, circularizing the
resulting fragments, and then using PCR to amplify sequences adjacent to the known region. Inverse PCR
is particularly useful for the study of regions with unknown flanking sequences.

Procedure:

1. Genomic DNA Digestion: Digest genomic DNA with a restriction enzyme that cuts within the
known DNA fragment, creating linear fragments.
2. Circularization: Ligase is used to circularize the linear DNA fragments, resulting in closed
circular DNA molecules.
3. Inverse PCR Mix Preparation:
o Prepare a master mix with the following components per reaction:
 Circularized DNA template
 Inverse PCR primers (designed to anneal outward from the known region)
 DNA polymerase
 dNTPs mix (200 µM each)
 Buffer optimized for DNA polymerase used
 Sterile water to make up the volume
4. Inverse PCR Cycling:
o Perform an initial denaturation at 94-98°C for 2-5 minutes.
o Followed by 20-40 cycles of:
 Denaturation at 94-98°C for 20-30 seconds.
 Annealing at the optimal temperature for the primers for 20-30 seconds.
 Extension at 68-72°C for a time determined by the expected amplicon size (1
minute per kb).
o Finish with a final extension at 68-72°C for 5-10 minutes.
5. Gel Electrophoresis:
o Analyze the PCR products using agarose gel electrophoresis.
o Visualize the bands under UV light.

Advantages of Inverse PCR:

1. Amplification of Unknown Flanking Sequences: Enables the amplification of sequences flanking

a known region, even when the surrounding sequence is unknown.
2. Genome Walking: Useful for genome walking, allowing the identification of adjacent genomic
regions.
3. Study of Insertion Sites: Applicable for studying insertion sites of transposons, retroviruses, or
other genetic elements.
4. Identification of Integration Sites: Used to identify integration sites of foreign DNA in transgenic
organisms.
5. Characterization of Genomic Rearrangements: Facilitates the characterization of genomic
rearrangements, such as deletions or insertions.

Limitations of Inverse PCR:

1. Primer Design Challenges: Designing inverse PCR primers can be challenging, and optimization
may be required to achieve specific and efficient amplification.
2. Sensitivity to DNA Quality: Sensitivity to DNA quality can affect the success of inverse PCR, and
high-quality DNA is crucial for optimal results.
3. Potential for Nonspecific Amplification: The technique may be prone to nonspecific
amplification, particularly when using suboptimal primer designs.
4. Limited to Circularized DNA: Inverse PCR is limited to the amplification of circularized DNA
fragments, which may not be suitable for linear DNA fragments.

Applications of Inverse PCR:

1. Genome Walking: Used for walking along the genome to identify unknown sequences adjacent to
known regions.
2. Transposon Insertion Site Identification: Identification of insertion sites of transposons or other
mobile genetic elements.
3. Characterization of Genomic Rearrangements: Characterization of genomic rearrangements,
including deletions, insertions, or inversions.
4. Mapping of Transgene Integration Sites: Identification and mapping of integration sites of
transgenes in genetically modified organisms.
5. Identification of Unknown Genomic Sequences: Discovery and amplification of unknown
genomic sequences adjacent to known regions.

11. QUANTITATIVE PCR (QPCR):

 Measures the amount of PCR product in real-time.

 Utilizes fluorescent dyes or probes for quantification.

Quantitative PCR, or qPCR, is a widely used technique for the quantitative measurement of DNA. It allows
the determination of the initial amount of a target DNA sequence in a sample.

Practical Manual for Quantitative PCR (qPCR):

 1. Design Primers and Probes: Design specific primers and probes targeting the region of interest.
Probes may be labeled with fluorescent dyes and quenchers.
2. Prepare cDNA (if using reverse transcription qPCR): If starting with RNA, perform reverse
transcription to convert RNA to complementary DNA (cDNA) using a reverse transcription kit.
3. Prepare qPCR Reaction Mix:
o Prepare a master mix with the following components per reaction:
 DNA template or cDNA
 Primers and probes
 qPCR buffer
 dNTPs mix (usually 200 µM each)
 DNA polymerase (usually a hot-start polymerase)
 Passive reference dye (for normalization)
 Nuclease-free water to make up the volume
4. Set Up qPCR Plates: Dispense the qPCR reaction mix into the wells of a qPCR plate, making sure
to include standards of known concentrations for creating a standard curve.
5. Run the qPCR Instrument:
o Run the qPCR instrument according to the manufacturer's instructions.
o Follow a thermal cycling protocol that includes denaturation, annealing, and extension steps.
The number of cycles and temperatures depend on the primers and probes used.
6. Analyze qPCR Data:
o Analyze the qPCR data using the instrument's software.
o Calculate the threshold cycle (Ct) values for each sample.
7. Create a Standard Curve:
o Use the Ct values from the standards to create a standard curve.
o Determine the efficiency of the qPCR reaction from the slope of the standard curve.
8. Quantification:
o Quantify the amount of target DNA in the samples by comparing their Ct values to the
standard curve.
o Normalize the data using an appropriate reference gene or internal control.
9. Data Interpretation: Interpret the results and analyze the fold change or relative expression of the
target gene across different samples or conditions.

Advantages of qPCR:

1. High Sensitivity: Detects and quantifies low amounts of DNA with high sensitivity.
2. High Specificity: Provides high specificity due to the use of sequence-specific primers and probes.
3. Quantitative Measurement: Allows accurate quantification of the initial amount of target DNA.
4. Wide Dynamic Range: Has a wide dynamic range, covering several orders of magnitude.
5. Real-time Monitoring: Enables real-time monitoring of the amplification process, allowing for
precise detection of the exponential phase.
6. Multiplexing Capability: Can be used for multiplexing, allowing the simultaneous quantification
of multiple targets in a single reaction.
7. Versatility: Applicable to various DNA sources, including genomic DNA, cDNA, and plasmid
DNA.

Limitations of qPCR:

1. Inhibitors: Presence of inhibitors in the sample can affect the accuracy of quantification.
2. Reference Gene Selection: Selection of a suitable reference gene for normalization is crucial and
requires careful consideration.
3. Design Challenges: Designing primers and probes can be challenging, and optimization may be
required for robust results.
4. Cost: High initial equipment costs and reagent expenses can be a limitation for some laboratories.
5. Susceptibility to Contamination: Like other PCR techniques, qPCR is susceptible to
contamination, and precautions should be taken to prevent it.
Applications of qPCR:

1. Gene Expression Analysis: Quantify gene expression levels under different conditions.
2. Pathogen Detection: Detect and quantify pathogenic DNA in clinical samples.
3. Microbial Load Determination: Determine microbial loads in environmental or food samples.
4. Mutation Detection: Detect and quantify specific mutations or polymorphisms.
5. Viral Load Measurement: Measure viral loads in viral infections.
6. Copy Number Variation Analysis: Analyze copy number variations in the genome.
7. Clinical Diagnostics: Used in clinical diagnostics for disease monitoring and prognosis.
8. Pharmacogenomics: Study the expression of genes related to drug metabolism.

12. DIGITAL PCR:

 Divides the PCR reaction into many individual reactions.

 Provides absolute quantification of target DNA molecules.

Digital PCR is a powerful technique that enables absolute quantification of nucleic acid targets with high
precision and sensitivity. Unlike traditional quantitative PCR (qPCR), dPCR partitions a sample into
numerous individual reactions, allowing for the absolute quantification of target molecules without the
need for a standard curve.

Practical Manual for Digital PCR:

1. Primer and Probe Design: Design primers and probes specific to the target DNA or RNA
sequence using standard guidelines for primer design. Probes may be labeled with fluorescent dyes.
2. Sample Preparation: Extract and purify nucleic acids from the sample using standard procedures.
3. Digital PCR Reaction Mix Preparation:
o Prepare a master mix with the following components per reaction:
 DNA template or RNA (reverse-transcribed to cDNA)
 Digital PCR primers and probes
 dPCR buffer
 dNTPs mix (usually 200 µM each)
 DNA polymerase (specific for digital PCR)
 Fluorescent dyes (if using dye-based detection)
 Passive reference dye (for normalization)
 Nuclease-free water to make up the volume
4. Partitioning: Partition the reaction mix into numerous individual reactions. This can be achieved
using droplet-based systems, microfluidics, or chip-based technologies.
5. Thermal Cycling: Perform thermal cycling according to the instrument's specifications, including
denaturation, annealing, and extension steps. Digital PCR systems often involve an initial
partitioning step and a subsequent thermal cycling step.

 Initialization: Denaturation of the template DNA or RNA

o Temperature: 95°C for 10 minutes (1 cycle)

 Digital Partitioning: Partition the sample into individual reactions (e.g., droplets, wells, or
chambers).

o Temperature: Varies by platform (this step is specific to certain digital PCR technologies)
o Time: Varies by platform

 Amplification Cycling:

o Denaturation: Denature the DNA strands

  Temperature: 95°C
 Time: Typically 15-30 seconds
o Annealing: Allow primers to anneal to the target sequence
 Temperature: Varies based on primer design (typically 55-65°C)
 Time: Typically 15-30 seconds
o Extension:
 Temperature: 68-72°C
 Time: Varies based on the expected amplicon size (typically 1-2 minutes per kb)
 Purpose: Synthesize complementary DNA strands.
o Number of Cycles:
 Varies but is often between 30 and 40 cycles.

 Detection and Analysis: Detect and analyze the fluorescence signals in each partition to
determine the presence or absence of the target.

o Temperature: Varies by detection method

o Time: Varies by detection method
6. Detection: After thermal cycling, detect and count the number of positive and negative partitions
for each fluorescent dye. The presence or absence of the target molecule in each partition is
determined.
7. Data Analysis: Analyze the data to determine the absolute quantity of the target molecule in the
original sample. This involves counting positive partitions, calculating concentrations, and
accounting for any normalization factors.
8. Interpretation: Interpret the results in terms of target molecule concentration per unit volume of
the original sample.

Advantages of Digital PCR:

1. Absolute Quantification: Provides absolute quantification of target molecules without the need for
a standard curve.
2. High Precision: Offers high precision and sensitivity, particularly for low-abundance targets.
3. Reduced Sensitivity to PCR Inhibitors: Less sensitive to PCR inhibitors present in the sample
compared to traditional PCR.
4. Improved Accuracy in Low-Abundance Samples: Particularly useful for accurately quantifying
low-abundance targets in complex samples.
5. Single Molecule Resolution: Can resolve and quantify individual molecules, allowing for the
detection of rare mutations or variants.
6. Reduced Amplification Bias: Minimizes amplification bias, as each molecule is individually
amplified and detected.
7. Increased Dynamic Range: Offers an extended dynamic range compared to some other
quantitative methods.

Limitations of Digital PCR:

1. Equipment Cost: Digital PCR systems and consumables can be more expensive compared to
traditional PCR and qPCR.
2. Throughput: Lower throughput compared to some other quantitative methods, making it less
suitable for high-throughput applications.
3. Complexity of Data Analysis: The analysis of digital PCR data can be complex, requiring
specialized software and expertise.
4. Limited Availability of Assays: Limited availability of commercially validated assays compared
to traditional PCR.

Applications of Digital PCR:

 1. Rare Mutation Detection: Detection and quantification of rare mutations or variants in a
background of wild-type DNA.
2. Absolute Gene Expression Analysis: Absolute quantification of gene expression levels without
the need for reference genes.
3. Copy Number Variation Analysis: Accurate determination of copy number variations in the
genome.
4. Viral Load Quantification: Measurement of viral loads in clinical samples.
5. Molecular Diagnostics: Clinical diagnostics, particularly in applications where high precision is
crucial.
6. Environmental Monitoring: Quantification of specific DNA targets in environmental samples.
7. Circulating Nucleic Acids Analysis: Analysis of circulating nucleic acids in liquid biopsies for
cancer diagnostics.

13. ALLELE-SPECIFIC PCR:

 Amplifies only one allele at a polymorphic site.

 Used for genotyping and identification of single nucleotide polymorphisms (SNPs).

Allele-Specific PCR is a molecular biology technique used to selectively amplify a specific allele of a
gene. It relies on the use of primers that are designed to be specific to the sequence of interest, allowing
discrimination between alleles based on single nucleotide polymorphisms (SNPs) or other sequence
variations.

Practical Manual for Allele-Specific PCR:

1. Primer Design: Design two allele-specific primers, each specific to one allele (wild-type or
mutant), and a common reverse primer. The 3' end of the allele-specific primers should perfectly
match the target sequence.
2. DNA Extraction: Extract DNA from the biological sample of interest using standard DNA
extraction methods.
3. AS-PCR Reaction Mix Preparation:
o Prepare a master mix for the AS-PCR reaction with the following components per reaction:
 DNA template
 Allele-specific forward primers (one for each allele)
 Common reverse primer
 dNTPs mix (usually 200 µM each)
 DNA polymerase
 Buffer optimized for the DNA polymerase
 MgCl2 (if required)
 Sterile water to make up the volume
4. AS-PCR Cycling Conditions:
o Perform thermal cycling using the following conditions:
 Initial denaturation: 95°C for 2-5 minutes (1 cycle)
 Denaturation: 95°C for 20-30 seconds
 Annealing: Temperature optimized for allele-specific primers, typically 55-65°C for
20-30 seconds
 Extension: 72°C for a time determined by the expected amplicon size (1 minute per
kb)
 Final extension: 72°C for 5-10 minutes (1 cycle)
5. Gel Electrophoresis:
o Analyze the AS-PCR products using agarose gel electrophoresis.
o Visualize the bands under UV light.
6. Result Interpretation: Presence of a band indicates the amplification of the corresponding allele,
allowing for genotyping based on the presence or absence of specific bands.
Advantages of Allele-Specific PCR:

1. High Specificity: Provides high specificity for the targeted allele due to the design of allele-specific
primers.
2. Simple Design: Relatively simple design compared to other genotyping methods, such as
sequencing.
3. Cost-Effective: Generally, it is a cost-effective genotyping method, especially for detecting known
mutations.
4. High Throughput: Can be adapted for high-throughput genotyping of multiple samples
simultaneously.
5. Rapid Results: Generates results quickly and is suitable for routine genotyping assays.
6. Applicability to Various Samples: Applicable to various sample types, including blood, tissue, or
cultured cells.

Limitations of Allele-Specific PCR:

1. Requires Prior Knowledge: Requires prior knowledge of the specific SNP or mutation for primer
design.
2. Challenging for Complex Loci: Challenging for genotyping in complex loci with multiple
mutations or insertions/deletions.
3. Sensitivity to Primer Mismatches: Sensitivity to mismatches at the 3' end of allele-specific
primers, which may affect specificity.
4. Limited to Known Variants: Limited to detecting known variants; discovery of novel mutations is
not feasible.

Applications of Allele-Specific PCR:

1. SNP Genotyping: Commonly used for single nucleotide polymorphism (SNP) genotyping.
2. Mutation Detection: Detection of specific mutations or known sequence variations.
3. Genetic Disease Diagnosis: Used in genetic disease diagnosis where known mutations are
associated with the disease.
4. Pharmacogenomics: Genotyping of specific alleles related to drug response in pharmacogenomic
studies.
5. Plant and Animal Breeding: Applied in plant and animal breeding programs for selecting
individuals with desired genetic traits.
6. Forensic Analysis: Genotyping for forensic analysis and human identification.

14. MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA):

 Amplifies multiple targets using probe hybridization and ligation.

Multiplex Ligation-Dependent Probe Amplification (MLPA) is a molecular technique used for the
detection of copy number variations (CNVs) in genomic DNA. MLPA allows for the simultaneous
amplification of multiple target sequences, providing information about the copy number of specific
genomic regions. Here is a detailed practical manual for MLPA, along with its applications, advantages,
and limitations.

Materials and Reagents:

1. MLPA probemix containing oligonucleotide probes for the target regions

2. Genomic DNA samples
3. MLPA buffer
4. Ligase-65 enzyme
5. Polymerase chain reaction (PCR) reagents (including DNA polymerase, dNTPs, and buffer)
6. Electrophoresis equipment and reagents
 7. Capillary electrophoresis system or DNA sequencer
8. Analysis software for MLPA data interpretation

Procedure:

1. Sample Preparation:
o Extract genomic DNA from the samples using a standard DNA extraction protocol. Ensure
the DNA is of high quality and concentration.
2. Probe Hybridization:
o Mix the genomic DNA with the MLPA probe mix and MLPA buffer.
o Denature the DNA by heating to 98°C for 1-5 minutes, then cool it to the hybridization
temperature (usually 60-65°C).
o Add the Ligase-65 enzyme to the hybridization mix and incubate for several hours to allow
the specific probes to hybridize to their target sequences.
3. Ligation:
o Activate the Ligase-65 enzyme by heating the reaction to 54-65°C for 10 minutes.
o Inactivate the ligase by heating to 98°C for 5 minutes.
4. Amplification (PCR):
o Add PCR reagents, including DNA polymerase, dNTPs, and buffer, to the ligated DNA.
o Perform PCR using a thermal cycler with the following conditions:
 Initial denaturation: 95°C for 1-2 minutes
 Cycling: 30-40 cycles of denaturation at 95°C for 30 seconds, annealing at 60-65°C
for 30 seconds, and extension at 72°C for 1-2 minutes.
 Final extension: 72°C for 5-10 minutes.
5. Electrophoresis: Analyze the PCR products by agarose gel electrophoresis to check for the
presence of amplified fragments.
6. Capillary Electrophoresis: Quantify and size the PCR products using a capillary electrophoresis
system or a DNA sequencer.
7. Data Interpretation:
o Import the data into MLPA analysis software.
o Analyze the peak patterns to determine the relative copy number of the target regions.
8. Normalization: Normalize the data using internal control probes or a reference sample to correct
for variations in sample concentration and amplification efficiency.
9. Results Interpretation:
o Interpret the MLPA results by comparing the peak patterns of the test samples to a reference
or control sample.
o Peaks corresponding to the target regions provide information about the copy number
variations.

Notes:

 Ensure that the primer pairs in the MLPA probemix are specific to the target regions and do not
cross-hybridize with other genomic regions.
 Proper negative controls and reference samples should be included in each MLPA experiment for
accurate data interpretation.

Advantages of MLPA:

1. Multiplexing Capability: Simultaneously amplifies multiple targets in a single reaction, allowing

for the detection of numerous genomic regions.
2. High Sensitivity: Can detect copy number variations with high sensitivity.
3. Quantitative Information: Provides quantitative information about the copy number of specific
genomic regions.
4. No Requirement for Reference DNA: Reference DNA is not always required, as internal controls
can be included in the assay.
 5. Suitable for Various Samples: Applicable to various sample types, including DNA extracted from
blood, tissues, or formalin-fixed paraffin-embedded (FFPE) samples.

Limitations of MLPA:

1. Limited to Known Targets: Limited to detecting variations in the copy number of known target
sequences; it may not identify novel mutations.
2. Dependency on Probe Design: The success of MLPA is highly dependent on the design and
specificity of the probes.
3. Complex Data Interpretation: Data interpretation can be complex, especially in the case of a
large number of targets.
4. Cost and Time: The assay can be relatively expensive and may take more time compared to some
other methods.

Applications of MLPA:

1. Genetic Disease Testing: Used in clinical genetics for the detection of copy number variations
associated with genetic diseases.
2. Cancer Research: Applied in cancer research to identify genomic alterations associated with tumor
development.
3. Prenatal Diagnostics: Used in prenatal diagnostics to identify chromosomal abnormalities in fetal
DNA.
4. Pharmacogenomics: Applied in pharmacogenomic studies to investigate genetic variations related
to drug response.
5. Plant and Animal Genetics: Utilized in plant and animal genetics for breeding and research
purposes.
6. Forensic Genetics: Applied in forensic genetics for human identification and determination of
kinship.

MLPA is a powerful technique for detecting copy number variations in specific genomic regions. It is
particularly valuable in the clinical diagnosis of genetic disorders and cancer research. The choice of
MLPA over other methods depends on the specific goals of the study and the type of genetic variations
being investigated.

15. COLONY PCR: Identifies recombinant colonies after transformation.

Colony PCR is a molecular biology technique used to amplify DNA directly from bacterial colonies. It is a
rapid method for screening bacterial colonies to identify recombinant plasmids or confirm the presence of a
specific DNA fragment.

Materials and Reagents:

1. Sterile water
2. PCR reaction buffer
3. dNTPs mix
4. Forward and reverse primers
5. DNA template (bacterial colony or plasmid DNA)
6. Taq DNA polymerase
7. MgCl2
8. PCR tubes
9. Thermal cycler

Procedure:
 1. Colony Pick: Using a sterile toothpick or pipette tip, pick a small bacterial colony from the culture
plate.
2. Colony Suspension: Suspend the picked colony in a PCR tube containing 20-50 μl of sterile water.
Mix by pipetting up and down.
3. DNA Denaturation: Heat the tube to 95°C for 5 minutes to denature the bacterial cells and release
genomic DNA.
4. PCR Setup:
o Prepare the PCR reaction mix on ice by combining the following components for each
reaction:
 5 μl PCR reaction buffer
 2 μl dNTPs mix
 1 μl forward primer (10 μM)
 1 μl reverse primer (10 μM)
 0.25 μl Taq DNA polymerase
 1-2 μl of the colony suspension (DNA template)
 Adjust the total volume to 25 μl with sterile water
5. PCR Cycling Conditions:
o Perform PCR with the following cycling conditions:
 Initial denaturation: 95°C for 2-5 minutes (1 cycle)
 Denaturation: 95°C for 30 seconds
 Annealing: 55-65°C for 30 seconds (optimize based on primer Tm)
 Extension: 72°C for 1 minute per kb of the expected product size
 Final extension: 72°C for 5-10 minutes (1 cycle)
 Hold at 4-10°C
6. PCR Analysis:
o Analyze the PCR products by running them on an agarose gel alongside a DNA size marker.
o Visualize the gel under UV light to confirm the presence and size of the amplified product.
7. Verification: Optionally, confirm the identity of the PCR product by DNA sequencing.

Advantages of Colony PCR:

1. Rapid Screening: Provides a quick and efficient method for screening bacterial colonies.
2. Cost-Effective: Cost-effective compared to plasmid DNA extraction for multiple samples.
3. Minimizes Handling: Minimizes handling steps, reducing the risk of contamination.
4. No Need for DNA Extraction: Eliminates the need for time-consuming DNA extraction steps.

Limitations of Colony PCR:

1. False Positives: Potential for false positives due to the presence of non-specific amplification or
contamination.
2. Colony Size: Limited by the size of the bacterial colony; small colonies may not provide enough
DNA.
3. Low Concentration: Low DNA concentration in the colony suspension may affect sensitivity.
4. Primer Design Sensitivity: Sensitivity to primer design; nonspecific amplification may occur with
poorly designed primers.

Applications of Colony PCR:

1. Plasmid Verification: Confirm the presence of an inserted gene or verify plasmid constructs.
2. Library Screening: Screen bacterial libraries for the presence of specific DNA fragments.
3. Mutation Confirmation: Confirm the presence of mutations or genetic modifications.
4. Genomic Library Screening: Identify clones containing specific genomic DNA fragments.
5. Quality Control: Rapid quality control for cloning experiments.