MULTIPLEX PCR AND ITS APPLICATION:

COMPILED BY: MS. PRITAM BAGWE AND MR. NAGENDRA P.

M.TECH. PHARMACEUTICAL BIOTECHNOLOGY, ICT,MATUNGA, MUMBAI.

DATE: 06/02/2017.1

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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• 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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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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MULTIPLEX PCR (MULTIPLE TEMPLATES)5

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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• 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. 7

• 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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NEB's Tm Calculator

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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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OVERVIEW WITH APPLICATIONAL USE

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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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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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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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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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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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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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