Multiplex PCR (Polymerase Chain Reaction) is a molecular biology technique that allows amplification of multiple DNA targets in a single reaction. In traditional PCR, only one specific target sequence is amplified in each reaction. However, multiplex PCR enables the simultaneous amplification of se...
ultiplex pcr enables the simultaneous amplificatio
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Molecular Diagnosis
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Content: -
• Introduction
• Principle
• Advantages & Disadvantages
• Types of Multiplex PCR
• Optimization of Multiplex Reaction Components
• NEB'S Tm Calculator
• Primer Design parameters for Multiplex PCR
• Advantages of Multiplex PCR
• Application of Multiplex PCR
• Some Multiplex PCR Kit Used in diagnosis
• Conclusion
• References
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, Introduction
In multiplex PCR, two or more primer sets designed for amplification of different targets are
included in the same PCR reaction. Using this technique, more than one target sequence in a
clinical specimen can be amplified in a single tube. As an extension to the practical use of PCR,
this technique can save time and effort. The primers used in multiplex reactions must be
selected carefully to have similar annealing temperatures and must be not complementary to
each other. The amplicon sizes should be different enough to form distinct bands when
visualized by gel electrophoresis. Multiplex PCR can be designed in either single-template
PCR reaction that uses several sets of primers to amplify specific regions within a template, or
multiple-template PCR reaction, which uses multiple, templates and several primers sets in the
same reaction tube (Fig.). Although the use of multiplex PCR can reduce costs and time to
simultaneously detect two, three, or more pathogens in a specimen, multiplex PCR is more
complicated to develop and often is less sensitive than single-primer-set PCR. The advantage
of multiplex PCR is that a set of primers can be used as internal control, so that we can eliminate
the possibility of false positives or negatives. Furthermore, multiplex PCR can save costly
polymerase and template in short supply.
Fig.- The overview of multiplex PCR.
Multiplex PCR represents a variant of PCR in which two or more DNA fragments are
simultaneously amplified within a single reaction tube. This is achieved by including more than
one primer pair to the reaction mixture. The approach is particularly relevant to food analysis,
where it is often necessary to test for the presence of a variety of toxicants in a single sample.
Multiplex reactions can usefully discriminate between real and false negative results. For this
purpose, one set of primers is targeted at a target known to be present in the sample, while the
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,second set targets the sequence of interest. The former allows the experimenter to distinguish
between a true negative and a reaction failure. Multiplex primers must be designed so that each
separate amplification product is of distinct size, to ensure that all fragments can be identified
following amplicon separation by either agarose gel or capillary electrophoresis. Illustrating
the simultaneous amplification of seven targets in a mixture of DNAs extracted from
independent transgenic events in maize and soybean and non-transgenic maize and soybean.
In this example, the primers were designed to amplify sequences within the transgenes and
targeted in addition fragments of endogenous genes (zein in maize and lectin in soybean) to
provide a negative control. Multiplex PCR – particularly when a significant number of primer
pairs is involved – can require intricate optimization, and it may be in some instances be too
difficult to achieve. Nevertheless, it represents an important technique for high-throughput
analyses in a cost-effective manner.
• Multiplex polymerase chain reaction (PCR), first reported in 1988, is a type of PCR in
which two or more target sequences can be amplified simultaneously by including more
than one pair of primers in the same reaction.
• 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.
• A multiplex PCR amplifies multiple targets within a single PCR run, in a single reaction
vessel, from a single sample aliquot - instead of performing many separate reactions.
Principle
M-PCR is the simultaneous amplification of more than one target sequence in a single reaction
tube using more than one primer pair. This co-amplification 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) so they anneal to and dissociate
from complementary DNA sequences at approximately the same temperatures, allowing each
amplification to proceed at the selected temperature. This procedure could not be done if one
primer set was annealing at the time that another primer set was dissociating from its target.
Therefore, all primers must be selected so their TmS are within a few degrees (°C) of each
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, other. Each amplification proceeds independently of the others (as long as none of the reagents
is present at rate-limiting concentrations) and each specific amplification product or amplicon
is synthesized in an unencumbered way. 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. This can be done by gel electrophoresis with visual identification of separate
amplicons of different size or by hybridization with specific DNA probes and detection using
spectrophotometry, fluorometry, autoradiography, or chemiluminescence.
Fig.- Difference Between traditional PCR Vs Multiplex PCR
Multiplexing offers some important advantages that make it worth considering. When multiple
target sequences are amplified in a single reaction, there is a substantial savings in master mix
reagents. Half as many wells or fewer means half as much total dye, dNTPs, and more to
acquire and spend – and that cost savings is appealing to many laboratories.
Additionally, pipetting issues sometimes mean that the total reaction volume isn’t the same
between all qPCR reactions. This can be a problem when, for example, comparing a target gene
to a reference gene analysed in separate single plex reactions.
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