Multiplex PCR ppt , its types and their applications along with advantages and disadvantages

multiplex pcr

1. Multiplex PCR
By- Jasman, Ruchika and Shimukh

2. What is Polymerase Chain Reaction (PCR) ?
●PCR is an in vitro enzymatic reaction to amplify a defined DNA region.
●Any region of any DNA molecule can be chosen. The only requirement is that
the sequences at the borders of the region are known
●PCR is widely used due to its ability to cause exponential increase in the
amount of template DNA.

3. WHY PCR ?
●The ability of PCR to amplify DNA exponentially makes its use relevant to numerous
fields.

1.DNA Amplification:
2.Gene Cloning:
3.DNA Sequencing:
4.Genetic Testing and Diagnostics:
5.Forensic Analysis:
6.Infection disease identification
7.DNA Fingerprinting
8.Ancient DNA studies

4. Various types of PCR
●PCR with the basic design , has undergone many modifications during the advancement of
molecular biology in order to serve specific purposes. Some of them are listed below:

. . .
.
. .
.



Conventional
(Qualitative)PC
R
Multiplex PCRNested PCR Quantitative
PCR.
RT-PCR and
qRT-PCR
Hot-start PCRTouchdown
PCR.
Assembly PCR
Colony PCR Methylation-s
pecific PCR.
LAMP assay.

5. LETS FOCUS ON
MULTIPLEX PCR
It is a variant of PCR in which two
or more target sequences can be
amplified by including more than
one pair of primers in the same
reaction.

6. A BRIEF HISTORY
Multiplex-PCR was first described in 1988 by Chamberlain et al. while
working on the dystrophin gene.
He was screening Deletion of the Duchenne muscular dystrophy locus via
multiplex DNA amplification.
Duchenne Muscular Dystrophy (DMD) is a genetic disorder characterized by
progressive muscle degeneration and weakness due to the alterations of a
protein called dystrophin that helps keep muscle cells intact.

7. In this method, sequence flanking six deletion prone exons of the gene are
simultaneously amplified by the PCR.
Any of these regions absent from patient DNA fail to amplify and are readily
identified via agarose gel electrophoresis .
The failure of any region to be amplified indicates that there is a deletion
mutation in the gene and thus leading to the disease.

8. PRINCIPLE
●M-PCR is based on the principle of conventional PCR. However
This involves coamplification of two or more targets in a single
reaction is dependent on the compatibility of the PCR primers
used in the reaction.

●All primers in the reaction must have similar melting
temperatures (Tm) . This procedure could not be done if one
primer set was annealing at the time that another primer set
was dissociating from its target.

●Primers should also be chosen that define amplicons of
approximately the same size range (100-500 bp), so each is
synthesized efficiently and at equal rates. Each M-PCR assay
must also have a detection step capable of identifying each
amplicon.

9. Developing a multiplex PCR assay
1.)Choosing nucleic acid targets
●In general the targets should be similar in size so that a much smaller target does not out
compete the larger targets for amplification reagents.
●However the PCR targets should be designed so that the various amplicons are easily
discernible by whatever the preferred method of detection (i.e., agarose or
polyacrylamide gel etc).
2.) PRIMERS
●The most important step in M-PCR assay is choosing primers that have similar conditions
for optimal amplification.
●The annealing temperatures for the primer sets should be similar (within 2-3 *C).
●Primers should be selected that have similar GC contents and melting temperatures (Tm)
so a single annealing temperature can be found that works optimally for both primer sets.
●It is imperative that there must be no base pair homology between any of the primers to
prevent primer dimer formation.

10. Primer design - work plan
Find
Sequence
Design
Primers
Check
Primers
Satisfactory?

Run PCR
YES
NO
Post PCR
analysis

11. 3.) Optimizing conditions

●Initially equimolar amount of concentrations (0.1-0.5μM) are used in M-PCR.
When there is uneven amplification,changing the proportions of various
primers in the reaction is required, with an increase in the amount of primers
for the “weak” loci and a decrease in the amount for the “strong” loci.
●Optimization of Mg2+ is critical since Taq DNA polymerase is a
magnesium-dependent enzyme. In addition to Taq DNA polymerase, the
template DNA primers and dNTPs bind Mg 2+.
●dNTP concentration is increased stepwise from 0.5–1.6 mM. The best results
are between 200 and 400 μM each dNTP values.
●Raising the buffer concentration to 2X improves the efficiency of the multiplex
reaction.
●The most difficult multiplex PCR reactions can be significantly improved by
using a PCR additive, such as DMSO, glycerol, formamide, and betaine, which
relax DNA, thus making template denaturation easier.

12. 4.) Amplicon analysis
●Gel electrophoresis followed by hybridization with labeled DNA probes has been used to
verify specific amplification products.
●If primer pairs are chosen so amplicons differ in size by 50-100 bp, agarose gel
electrophoresis can be used to resolve M-PCR amplicons.
●During the development of the assay, hybridization with labeled probes should be
included to verify specific amplification products. Southern blotting followed by
hybridization is necessary for reactions that give many nonspecific products.
●Solid-phase hybridization using 96-well microtiter plates coated with avidin to capture
biotin-containing amplicons, or complementary capture probes, has been used by many
researchers and represents an easy way to assay PCR products for specific sequences.
●Simple M-PCRs with only two or three amplicons can be analyzed with oligonucleotide
probes labeled with different enzymes and appropriate substrates giving absorbance at
different wavelengths.

13. Types of Multiplex-PCR
1.Multiple primers pairs on a single template: this involves a single template with several pairs of
primers to amplify specific regions within a template.
2.Multiple primers pairs on multiple templates: this technique involves multiple templates with
several primer sets in the same reaction tube.

14. 3.) Multiplex Ligation-Dependent Probe Amplification (MLPA) :

The MLPA is a type of one primer on multiple templates MPCR. The key difference is
the use of probes, which hybridise with DNA and are amplified instead of the sample
DNA. These probes are constituted by two probe oligonucleotides, which must
hybridize adjacently in order to be ligated. The probes can then be conventionally
MPCR amplified using primers that target said probes. This technique, being a one
primer multiple template MPCR, does not have the disadvantages of primer
dimerization or competition for resources

15. Advantages of M-PCR
●The obvious advantage of M-PCR is the ability to detect more than one agent in a
single test. For specimens such as respiratory tract secretions, from which several
different viruses can be recovered, this ability offers potential cost savings.
●Multiplexing is more efficient due to the conservation of PCR reagents, less
preparation time and overall effort. This is especially relevant in situations where
the template is in short supply.
●M-PCR has high degree of sensitivity and ability to detect both non-cultivable
virus and neutralized virus present in antigen-antibody complexes.
●The use of multiple amplicons provides internal controls for each amplified
fragment, allowing for a better recognition of artifacts and false negatives, which
can arise due to reaction failure. The amplification of some of the fragments
indicates that the reaction didn’t fail.

16. Disadvantages
●The weaknesses of M-PCR are relatively minor in nature. Cost, which was once the
major drawback due to expensive thermal cyclers, is now decreasing in importance
as more laboratories acquire instruments and competition plays an expanding role
in the market.
●Since multiplexing frequently requires the use of many different primers, this
increases the risk of primer dimerization resulting in non-specific amplification.
Consequently, when compared to standard ones, multiplex PCR primers must be
more specific.
●Each amplicon must be a different size (for electrophoresis analysis) or labelled
with a fluorescent dye that is spectrally distinct from the others used in the
reaction.
●In addition, different templates compete for resources leading to the detection of
highly abundant templates in detriment of less abundant ones, which may fade
into the background.

17. APPLICATIONS
●Gene Deletion and Mutation Detection:
The exquisite sensitivity of this method can also be exploited to demonstrate the presence or absence
of specific DNA sequences in a sample. This aspect of the procedure has led to the development of
assays that can eliminate the need for Southern analysis when screening for DNA deletions that lead to
genetic disease like in case of Duchenne Muscular Dystrophy.
●Polymorphic Repetitive DNA
1) STRs are convenient for multiplexing because they are numerous, highly polymorphic (vary between
individuals), and can be co-amplified without overlapping size ranges.
2) Repetitive DNA loci, including Y-specific STRs, are incorporated into gender-determining multiplex
assays. These assays can be used to sex embryos in families with X-linked diseases. The Y-specific STR is
co-amplified with X-specific STRs in forensic specimens for gender confirmation.
3) STRs, particularly (CA)n repeats, have been multiplexed for the diagnosis of genetic disorders such as
myotonic dystrophy, Duchenne muscular dystrophy (DMD), cystic fibrosis, and Prader-Willi/Angelman
syndromes.

18. ●Pathogen Detection
Multiplex PCR aids in pathogen detection by simultaneously amplifying multiple target DNA sequences
specific to different pathogens in a single reaction. This enables the rapid identification of diverse
pathogens within a single sample, enhancing the efficiency of diagnostic processes.
●Forensic Analysis
MPCR is employed in forensic science for simultaneous amplification of multiple DNA markers from
forensic samples, aiding in criminal investigations and identification of individuals.
●Template quantitation
The exponential amplification and internal standards of multiplex PCR can be used to assess the amount
of a particular template in a sample. To quantitate templates accurately by multiplex PCR, the amount of
reference template, the number of reaction cycles, and the minimum inhibition of the theoretical
doubling of product for each cycle must be accounted.

19. Questions For You :)
1)In multiplex PCR, why is it important to use multiple primer sets?
A) To confuse the DNA
B) To simplify the reaction
C) To amplify different DNA targets simultaneously
D) To reduce reaction time

20. Questions For You :)
2) Which of the following is a common application of multiplex PCR?

A. Determining protein structure

B. Identifying genetic mutations

C. Purifying DNA samples

D. Studying RNA processing

21. Questions For You :)
3) Non specific products can be eliminated by :-
A) Decreasing annealing Temperature
B) Using Hot Start Method
C) Increasing DNA concentration
D) Increasing Primers Concentration

22. THANK YOU