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Multiplex PCR

Multiplex polymerase chain reaction (PCR) is a variant of PCR that enables the amplification of two or more target sequences by more than one pair of primers in the same reaction. Because of the considerable savings of time and effort, multiplex PCR has been applied to many areas of DNA testing, including gene deletion analysis, mutation and polymorphism analysis, quantitative analysis, and reverse-transcription PCR, and multiplex PCR is also a valuable tool for the identification of viruses, bacteria, and parasites.

Advantages of Multiplex PCR

Multiplex PCR can be designed either as a single-template PCR reaction using several sets of primers to amplify a specific region within a template, or as a multi-template PCR reaction using multiple templates and several sets of primers in the same reaction. The reagent cost and preparation time of multiplex PCR are less than those of systems using several tubes of single-primer-set PCR. To maximize the efficiency of preparation, reactions can be prepared in bulk, randomly tested for quality, and stored frozen without enzyme or template until use. Another advantage of multiplex PCR is that a set of primers can be used as an internal control, thereby eliminating the possibility of false positives or false negatives. In addition, multiplex PCR is ideal for conserving costly polymerases and templates in short supply.

Optimization of Multiplex PCR

The optimization of multiplex PCR should aim to minimize nonspecific interactions. When multiple targets are amplified simultaneously with multiple primer pairs in multiplex PCR, the ratio of primer to template needs to be adjusted to avoid the formation of primer dimers. Attention should be paid to the design parameters of the primer, such as homology of primers with their target nucleic acid sequences, length, GC content and concentration. Hot start PCR usually eliminates the non-specific reaction (primer dimer generation) caused by primer annealing at low temperatures (4-25 °C) before commencement of thermocycling. Ideally, all primer pairs in a multiplex PCR should enable similar amplification efficiency for their respective targets. This can be achieved by using primers that have identical optimum annealing temperatures and should not show significant homology within or between each other. The alteration of components in multiplex PCR, such as PCR buffer components, dNTPs, MgCl2, and enzyme concentrations often results in significantly increased sensitivity and specificity of the test. The optimal combination of annealing temperature and buffer concentration is the necessary condition for obtaining highly specific amplification products in PCR. Optimizing Mg2+ is critical because Taq DNA polymerase is a magnesium dependent enzyme. In addition to Taq DNA polymerase, template DNA primers and dNTPs bind Mg2+. The concentration of magnesium chloride needs to be proportional to the amount of dNTP. In multiplex PCR, adjusting primer amount for each locus is also essential.

Multiplex TEMPase 2x Master Mix

Amerigo Scientific offers Multiplex TEMPase 2x Master Mix to facilitate simultaneous amplification of multiple PCR products in a single reaction tube. Our product is designed to minimize the need for optimization, make the development of multiplex PCR assays quick and simple, and diminish formation of non-specific products. This allows the reaction to be set up at room temperature with high sensitivity and high product yield.

Multiplex TEMPase 2x Master Mix is an all-in-one 2x master mix containing TEMPase Hot Start DNA polymerase, optimized buffer system, dNTPs, and 6 mM MgCl2. TEMPase is a modified form of thermostable Taq DNA polymerase, which is activated by heat treatment. A chemical moiety is attached to the active site of the enzyme, rendering the enzyme inactive at room temperature. During setup and the first ramp of the thermal cycle, the enzyme is inactive and misprimed primers are not extended. Thus, TEMPase is particularly suitable for multiplex PCR compared to standard DNA polymerases, enabling reactions with higher specificity, increased sensitivity, and greater yields.

Contents of Multiplex TEMPase 2x Master Mix

Each reaction required 25 µl of 2x Master Mix, to which additional primers, template and water are added to a total reaction volume of 50 µl to successfully carry out multiplex PCR.

Amplification of a 5-plex and a 10-plex Reaction

Five different templates of the CFTR gene (5-plex) and ten different templates of the DMD gene (10-plex) were simultaneously amplified in one tube, respectively. M: Marker