Multiplex PCR allows for the amplification of multiple DNA templates in a single reaction by using multiple primer pairs. This technique has the potential to reduce time and costs compared to performing individual PCR reactions. Optimization is required to prevent cross-hybridization between primers and ensure even amplification of all templates. The document discusses the advantages and disadvantages of multiplex PCR as well as optimization of reaction components, primer design parameters, applications, and references.

1. MULTIPLEX PCR AND ITS APPLICATION:
COMPILED BY: MS. PRITAM BAGWE AND
MR. NAGENDRA P.
M.TECH. PHARMACEUTICAL BIOTECHNOLOGY,
ICT,MATUNGA, MUMBAI.
DATE: 06/02/2017.
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2. Multiplex PCR:
• Widespread molecular biology technique,
• Amplification of single template as well as multiple templates in a single
PCR experiment.
• By using multiple primer pairs in a reaction mixture.
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3. • ADVANTAGES:
• This technique has the potential to produce considerable savings in time and
effort within the laboratory
• Without compromising on the utility of the experiment.
• DISADVANTAGES:
• Optimization is difficult; since many sets of forward and reverse primers are
to be designed for use.
• Increases cost.
• Presence of multiple primer may lead to cross hybridization with each other
and the possibility of mis-priming with other templates.
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4. TYPES OF MULTIPLEX PCR
1. Single template PCR reaction;
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;
This technique uses multiple templates and
Several primer sets of forward and reverse primers for each template and regions
within the template; in the same reaction tube.
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5. MULTIPLEX PCR
(MULTIPLE TEMPLATES)5

6. OPTIMIZATION OF MULTIPLEX REACTION
COMPONENTS (REACTION MIX):
• Amount of Primer
• dNTP and MgCl2 Concentrations
• dNTP/MgCl2 Balance
• PCR Buffer Concentration
• Amount of Template DNA and Taq DNA Polymerase
• Use of Adjuvants: DMSO, Glycerol, BSA
REACTION
MIXTURE
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7. • Primers:
Initially, equimolar primer concentrations of 0.1–0.5 µM each are used in the
multiplex PCR.
When there is uneven amplification, with some of the products barely visible even
after the reaction was optimized for the cycling conditions, 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.
The final concentration of the primers (0.04–0.5 µM) may vary considerably among
the loci.
• dNTP and MgCl2 Concentrations:
MgCl2 concentration is kept constant (2 mM),
While the dNTP concentration is increased stepwise from 0.5–1.6mM.
Optimization of Mg2+
is critical since Taq DNA polymerase is a magnesium-
dependent enzyme.
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8. • PCR Buffer Concentration:
Raising the buffer concentration to 2X improves the efficiency of the multiplex
reaction but different concentrations of the buffer are optimised depending on the
reaction mix.
• Amount of Template DNA and Taq DNA Polymerase:
The amount of template DNA is low (optimum), efficient and specific amplification
can be obtained.
Different concentrations of Taq DNA polymerase are tested. (experimental
optimization).
• Use of Adjuvants: DMSO, Glycerol, BSA:
The most difficult multiplex PCR reactions can be significantly improved by using a
PCR additive, such as DMSO, glycerol, BSA, which relax DNA, thus making
template denaturation easier.
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9. NEB's Tm Calculator
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10. Process followed for (5X) concentration:
INITIAL DENATURATION: 95°C (1-2 min)
DENATURATION: 95°C (5-30 sec)
ANNEALING: (30 sec -1 minute)
Optimized by doing a temperature gradient PCR, starting 5°C below the
calculated tm
EXTENTION: Recommended: 68°C (1-2 minutes/kb)
Final extension: 68°C (5mins)
Cycle number: 30-35 cycles gives sufficient product.
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11. OVERVIEW WITH
APPLICATIONAL USE
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12. PRIMER DESIGN PARAMETERS FOR MULTIPLEX PCR
Design of specific primer sets is essential for a successful multiplex reaction. The important
primer design considerations described below are a key to specific amplification with high
yield.
1. Primer Length
Multiplex PCR assays involve designing of large number of primers, hence it is required that
the designed primer should be of appropriate length. Usually, primers of short length, in the
range of 18-22 bases are used.
2. Melting Temperature
Primers with similar Tm, preferably between 55°C-60°C are used. For sequences with high GC
content, primers with a higher Tm (preferably 75°C-80°C) are recommended. A Tm variation of
between 3°-5° C is acceptable for primers used in a pool.
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13. 3. Specificity
It is important to consider the specificity of designed primers to the target
sequences, while preparing a multiplex assay, especially since competition exists
when multiple target sequences are in a single reaction vessel.
4. Avoid Primer Dimer Formation
The designed primers should be checked for formation of primer dimers, with all
the primers present in the reaction mixture. Dimerization leads to unspecific
amplification.
All other parameters are similar to standard PCR primer design guidelines.
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14. ADVANTAGES OF MULTIPLEX PCR
1. Internal Controls
Potential problems in a simple PCR include false negatives due to reaction failure
or false positives due to contamination. False negatives are often revealed in
multiplex assays because each amplicon provides an internal control for the other
amplified fragments.
2. Efficiency
The expense of reagents and preparation time is less in multiplex PCR than in
systems where several tubes of uniplex PCRs are used. A multiplex reaction is
ideal for conserving costly polymerase and templates in short supply.
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15. 3. Indication of Template Quality
The quality of the template may be determined more effectively in multiplex
than in a simple PCR reaction.
4. Indication of Template Quantity
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.
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16. APPLICATIONS OF MULTIPLEX PCR
1. Pathogen Identification
2. High Throughput SNP Genotyping
3. Mutation Analysis
4. Gene Deletion Analysis
5. Template Quantitation
6. Linkage Analysis
7. RNA Detection
8. Forensic Studies
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17. REFERENCES:
• Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization
and application in diagnostic virology. Clinical microbiology reviews. 2000 Oct
1;13(4):559-70.
• Edwards MC, Gibbs RA. Multiplex PCR: advantages, development, and
applications. Genome Research. 1994 Feb 1;3(4):S65-75.
• Markoulatos P, Siafakas N, Moncany M. Multiplex polymerase chain reaction: a
practical approach. Journal of clinical laboratory analysis. 2002 Jan 1;16(1):
47-51.
• Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH. Multiplex PCR:
critical parameters and step-by-step protocol. Biotechniques. 1997 Sep 1;23(3):
504-11.
• Krause JC, Panning M, Hengel H, Henneke P. The role of multiplex PCR in
respiratory tract infections in children. Dtsch Arztebl Int. 2014 Sep 19;111(38):
639-45.
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