Multiplex PCR

Overview of Assay

Multiplex polymerase chain reaction (PCR) is a powerful and broadly used method for simultaneous amplification of two or more targets in the same reaction. Multiplex PCR increases the information content of DNA testing applications for genotyping, quantitative assays, forensics, and diagnostics.  Co-amplification of multiple targets maximizes the use of limiting amounts of starting material and saves time and reagent costs.  Multiplex PCR may be the end point of analysis, or preliminary to further analyses by targeted next-generation sequencing or microarray hybridization studies.

A major factor for implementation of multiplex PCR is the requirement for optimization. Multiplex assays can be particularly difficult, tedious, and time-consuming to establish.  The more primer pairs involved in a multiplex mixture the greater the possibility of non-specific interactions (mis-priming, primer dimerization).  The situation is aggravated by the use of less stringent conditions to ensure comparable efficiencies for annealing and extension of all multiplexed primers.  Non-specific amplification products impact the yield of less efficient amplicons by competing for available reagents.  Mispriming events also lead to imbalanced or even lack of amplification of some targets and difficult-to-reproduce assays.   Thus, minimizing or reducing nonspecific primers interactions is crucial for successful PCR multiplexing.

Common Problems

Errors in multiplex PCR can occur before the start of the reaction or during the course of the reaction.  The most useful methods for preventing these errors are good primer design and the use of hot start PCR methods.  Hot start reagents maintain PCR DNA polymerases inactive until the reaction reaches higher temperatures.  These reagents, however, represent a substantial added cost and fail to address subsequent mis-priming during the amplification reaction.

How ThermaStop and ThermaGo Overcome Common Problems

ThermaStop and ThermaGo are simple additives that successfully address many of the difficulties that hinder multiplex PCR.  These cost-effective reagents enable rapid and efficient multiplex PCR with minimal or no optimization. ThermaStop suppresses mispriming prior to PCR while ThermaGo suppresses mispriming during PCR amplification.

The following examples illustrate the advantages of ThermaStop and ThermaGo for multiplex PCR.

ThermaStop Improves Multiplex PCR

With Non-Hot Start Taq DNA Polymerases

ThermaStop + ThermaGo Improves Multiplex PCR

REACTION CONDITIONS:

25 µl PCR mixtures contained 50 ng mouse DNA, 1X PCR buffer (20 mM Tris, pH 8.4, 70 mM KCl), 0.4 mM of each dNTP, 2.5 units Invitrogen Taq DNA polymerase, 2.5 mM MgCl2, 0.2 µM of each primer, and either no additive, 2.5 units of ThermaStop or 2.5 units of ThermaStop and 0.25 units ThermaGo. Thermocycling conditions were 95°C, 10 min; 35 cycles at 95°C, 15 sec; 60°C, 30 sec; 72°C, 1 min; then extension at 72°C

for 5 min.  PCR products (5µl) were visualized on a E-Gel™ EX 2% agarose gel.

DESCRIPTION:

ThermaStop together ThermaGo effectively suppressed non-specific products in a duplex PCR test1.  White asterisks indicate non-specific products. ThermaStop reduces non-specific products and results in greater product yield.  Addition of ThermaGo to ThermaStop completely prevented non-specific products. Note: ThermaStop reagent is not visible on an agarose gel.

1Le et al., BioTechniques 47, No. 5, 972–973, 2009