PCR TYPES AND ITS APPLICATIONS

COLONY PCR
Colony PCR is a convenient

high-throughput method
for determining the
presence or absence of
insert DNA in plasmid
constructs. Individual
transformants can either
be lysed in water with a
short heating step or
added directly to the PCR
reaction and lysed during
the initial heating step
 Hot start PCR
Hot start PCR is a modified form of Polymerase chain reaction (PCR) which
avoids a non-specific amplification of DNA by inactivating the polymerase at
lower temperatures.

The technique may be performed manually by heating the reaction components

to the melting temperature (eg. 95°C) Ie. Master mix, before adding the
polymerase.

DNA Polymerase-Eubacterial type I DNA polymerase , Pfu. These thermophilic

DNA polymerases show a very small polymerase activity at room temperature.

Activation of the Clean Amp dNTPs occurs not only during initial denaturation of
Hot Start PCR but also at each subsequent thermal cycle.
Each cycle, hot temperature isolates the primers stopping molecular
interactions, reinitiating the process.

Each cycle, hot temperature releases just enough actived dNTPs to allow
amplification on the newest interactions during the cooling, that are the
most stringent ones.
 Nested PCR

• The specificity of PCR is determined by the specificity of the PCR primers.

• For example, if your primers bind to more than one locus (e.g. paralog or common

domain), then more than one segment of DNA will be amplified.

• To control for these possibilities, investigators often employ nested primers to ensure

specificity.

• Nested PCR employs two pairs of primers.

• The first pair amplified the locus as seen in any PCR experiment.

• The second pair of primers (nested primers) bind within the first PCR product &produce a

second PCR product that will be shorter than the first one.

• The logic behind this strategy is that if the wrong locus were amplified by mistake, the

probability is very low that it would also be amplified a second time by a second pair of

primers.
Nested PCR
 Multiplex PCR

• Multiplex PCR is a widespread molecular biology technique for amplification of

multiple targets in a single PCR experiment.

• 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.

• Annealing temperatures for each of the primer sets must be optimized to work
correctly within a single reaction and amplicon size ie. Their base pair length,
should be different enough to form distinct bands when visualized by gel
electrophoresis.
 Multiplex PCR

WE CAN RUN DNA SEQUENCES AT

A SAME TIME AMPLIFICATION OF
MANY AT DIFFERENT ORDER.

INSIDE THE CELL, THE AMOUNT OF

AMPLICON THAT IS PRESENT AND
TYPE OF AMPLICON IS PRESENT OR
NOT

SIMULATANEOUS AMPLIFICATION
OF SEVERAL TARGETS OF INTEREST
AT A SAME TIME

4 DIFFERENT TYPES OF PRIMERS-

CROSS TEMPLATE PRIMER
BINDING SHOULD BE AVOIDED

SIMILAR DNA SEQUENCES

CANNOT BE TESTED
Applications:
Identification; Mutation analysis; Forensic science
 INVERSE PCR
Inverse PCR (ochman et al., 1988)
uses standard PCR –Primers oriented
in the reverse direction.

The template for the reverse primers

is a restriction fragment that has
been self ligated.

Inverse PCR functions to clone

sequences flanking a known
sequence. Flanking DNA sequences
are digested and then ligated to
generate circular DNA.

Application
Amplification and identification of
flanking sequences such as
transposable elements and the
identification of genomic inserts
 Long PCR
Extended or longer than standard PCR, meaning
over 5KB (frequently over 10Kb)

Long PCR is useful only if it is accurate. Thus,

special mixtures of proficient polymerases along
with accurate polymerases such pfu are often
mixed together.

Application:
To clone large genes that are not possible with
conventional PCR.
 Reverse Transcriptase PCR
Based on the process of reverse transcription, which
reverse transcribes RNA into DNA and was initially
isolated from retro viruses.

First step of RT-PCR –”first strand reaction”-synthesis of

cDNA using oligo dT primers (37°C) 1 hr.

“second strand reaction”- Digestion of cDNA:RNA

hybrid (Rnase H)-standard PCR with DNA oligo primers.

Allows the detection of even rare or low copy mRNA

sequences by amplifying its complementary DNA.
• Three strategies for
synthesis of first-
strand cDNA.

(a) Random primer.

(b) oligo (dT) primer.
(c) sequence-specific
primer.
(d) Random hexamer
primer
 Reverse Transcriptase PCR

• Reverse Transcriptase PCR is employed for the

amplification of RNA molecules.

• RT-PCR is applied incase of expression profiling in order to

study the expression of a gene or to determine the
sequence of a RNA transcript

• Avian myeloblastosis virus (AMV) or Moloney murine

leukaemia virus (MuLV) reverse transcriptase are generally
used to produce a DNA copy of the RNA template.
 REAL –TIME PCR/ qPCR
Real time PCR monitors the flurescence emitted during the
rection as an indicator of amplicon production at each PCR
cycle (in real time) as opposed to endpoint detection.

Sample Reverse
preparation PCR Fluroscent Quantification
transcription
detection
Real time PCR is identical to a standard PCR except that the
progress of the reaction in monitored by a camera during each
PCR cycle or detector in “real time”.

Each technique uses some kind of fluorescent marker which

binds to the DNA.

Hence as the number of gene copies increases during the

reaction so the fluorescence increases.

Quantification is achieved by measuring the increase in

fluorescence during the exponential phase of PCR.

Quantitative PCR has a sensitivity five orders of magnitude

better than the best blotting procedures.
 Applications of Q-PCR

•Gene therapy Distribution and Main applications:

Expression assays  Are not in foresic DNA analysis
•Drug response analysis  Quantification of mRNA for
•Cell bank copy number analysis expression
•Mutation analysis /SNP detection  Detection of pathogens
•Expression system development  SNP detection
•Residual DNA analysis  GMO analysis
•Detection of pathogens
•GMO analysis
•Human and Veterinary diagnostics
•Blood products quality control
qPCR thermalcyclers detect fluorescence
energy

FRET
acceptor

fluorophore fluorophore FRET donor

+ quencher
in proximity
 PROBING ALTERNATIVES:

NON-SPECIFIC DETECTION:
Dyes: SYBR GREEN I, BEBO, BOXTO, EvaGreen

SPECIFIC DETECTION:
TaqMan Probe Primer based detection
Molecular Beacon(=) Scorpion primers
Light Up probe QZyme
Hybridization Probes Lux primers
SYBRGreen
 SYBRGreen
 DNA binding dye
 Binds to minor groove (ds DNA)
 Emits light when bound
 More ds DNA…….More binding…….more
fluorescence
 Forensically can be used to calculate how
much DNA was present before reaction
 Unspecific
 Dissociation curve
Advantages:

1. Simple to design-just need to find good specific primers for the target
sequence of interest.

2.
3. Sensitive- produces >1 reporter per amplicon

inexpensive, relative to “Taqman “ detection ,because dye labelled oligo

nucleotides are not required

Dis advantages:

4. It detects all double stranded DNA, so if PCR is poorly designed, “primer

dimer’ product will be detected and quantified.

5. Cannot multiplex SYBR Green q PCR assays.

TaqMan
Advantages:

Very specific-because combines specificity of primers and specificity of

Taqman probe-typically do not detect non specific PCR product

Can be combine to multiplex q PCR assays.

Disadvantages:

o More difficult to design because of need for efficient amplification and

efficient probe hydrolysis.

o Some Taq Man probes do not quench efficiently that leads to large
background fluorescent and lower signal to noise ratio.

o More expensive, due to cost of dual labelled oligonucleotides

Molecular beacons
Molecular probes
Different chemistries involved to
obtain quantitative signal
(a) Three phases that occur in a typical qPCR experiments
(b) PCR carriedout at different starting template concentrations

(C ) a comparison between a sample with an unknown

amount of template DNA and a standard
During the exponential phase of PCR, the amount of PCR
product is proportional to the amount of starting template
that was initally added.

Therefore, the exponential phase of PCR is analysed to

quantitate the initial template concentration. The
commonly used method is called the cycle threshold
method (ct method)
Advantages:

Being sensitive and precision. This precision exists because quantitation of

the gene sequence is determined by the ct, which is calculated during the
exponential phase of the reaction .

High specificity is conferred by the requirement of three oligos to anneal to

the DNA before any data are collected.

Applications:
Detection of genomic or viral DNA in tissues can be valuable diagnostic tool.
Gene expression can be measured after extraction of total RNA and
preparation of c DNA by a reverse transcription (RT) step.

Setup and analysis are simple and can more easily be extended to the
clinical environment than traditional PCR techniques.
 Bridge PCR
• Opposite to conventional PCR, surface amplification
is performed on constant temperature (60°C).
• Formamide works as a denaturing agent. Normally, 35
cycles of pumping:
• formamide (at 60°C formamide melts DNA duplexes;
equivalent to "denaturation step" in normal PCR);
• Extension buffer(non-denaturating conditions,
equivalent to "annealing step" in normal PCR);
• Extension mixture (primer extension step, equivalent
to "extension step" in normal PCR);
• Two PCR
primers are
attached to
the surface of
flowcell. One
of the primers
has a cleavable
site (cross on
red primer).
• Formamide
Original
library
molecule is
denatured
and washed
away.
• Extension
buffer
Extended
molecule
bends and
hybridizes to a
second PCR
primer (forms
a bridge).
• Extension mixture
Extension of
hybridized primer.
• Formamide
washing
Two DNA
strands are
denatured.
• Extension
buffer
Extended
molecules
may
hybridize to
each other
again or find
other PCR
primers.
• After 35 cycles
cluster consists
on a number of
double-stranded
bridges.
Thank you
 Applications of PCR
• Classification • Detection of
of organisms pathogens
• Genotyping • DNA
• Molecular fingerprinting
archaeology • Drug discovery
• Mutagenesis • Genetic matching
• Mutation • Genetic
detection engineering
• Sequencing • Pre-natal
• Cancer research diagnosis
 Applications of PCR
Basic Research Applied Research

• Mutation screening • Genetic matching

• Drug discovery • Detection of pathogens
• Classification of organisms • Pre-natal diagnosis
• Genotyping • DNA fingerprinting
• Molecular Archaeology • Gene therapy
• Molecular Epidemiology
• Molecular Ecology
• Bioinformatics
• Genomic cloning
• Site-directed mutagenesis
• Gene expression studies
 Applications of PCR

Molecular IdentificationSequencing Genetic Engineering

• Molecular Archaeology • Bioinformatics • Site-directed mutagenesis
• Molecular Epidemiology • Genomic cloning • Gene expression studies
• Molecular Ecology • Human Genome Project
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
MOLECULAR IDENTIFICATION:
Molecular Identification:

Detection of Unknown Mutations

SSCP gels:
“shifts” representing a
mutation in the amplified
DNA fragment
Molecular Identification:

Classification of Organisms

1) Relating to each other* Fossils

* Trace amounts
2) Similarities * Small organisms
Insufficient data
3) Differences

! DNA !
Molecular Identification:

Detection Of Pathogens
Molecular Identification:

Detection Of Pathogens

Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul

Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul
Molecular Identification:

Genotyping by STR markers

Molecular Identification:

Prenatal Diagnosis

• Chorionic Villus
• Amniotic Fluid

644 bp
440 bp

204 bp

Molecular analysis of a family with an autosomal recessive disease.

 SEQUENCING
Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP)

NO polymerisation after a dideoxynucleotide!

Fragments of DNA differing only by one nucleotide are generated

Nucleotides are either or

Sequencing:

Classical Sequencing Gel

Sequencing:

Reading Classical Sequencing Gels

Sequencing:
 Summary

blood, chorionic
villus, amniotic fluid, 68,719,476,736 copies Gel Analysis,
semen, hair root, Restriction
saliva Digestion,
Sequencing
 Conclusion
The speed and ease of use, sensitivity, specificity
and robustness of PCR has revolutionised
molecular biology and made PCR the most widely
used and powerful technique with great
spectrum of research and diagnostic applications.