How Our Testing Technology Works

The Problem of Molecular Multiplexing Using RT-PCR

The following is a list of common problems associated with multiplex PCR assay development:

Incompatible Loci. Each target in a multiplex reaction demands its own optimal condition. Therefore, increasing the number of targets in a multiplex reaction can become difficult and is often impossible to execute.

Lack of Specificity. Multiple sets of high-concentration primers in a system can generate primer dimers and non-specific, background amplification.

Lack of Sensitivity. Crowding of primers reduces amplification efficiency and wastes resources by occupying enzyme and substrates.

Uneven Amplification. Differences in amplification efficiency of each target may lead to large discrepancies in amplicon yields. In a multiplex system, some loci may amplify well, while others may amplify poorly or not at all. Uneven amplification also makes it impossible to accurately perform end-point quantitative analysis.

Lot-to-lot Variation. Due to the fact that large amounts of primers are consumed in each reaction and manufacturers can generate only a limited amount of assays per lot, quality control and assurance can be difficult.

Tem-PCR Solves the Molecular Multiplexing Problem

Tem-PCR increases compatibility among loci by increasing the number of optimal conditions for each target.

Tem-PCR increases specificity by using very small amounts of gene specific primers and, therefore, reduces the chance of primer dimer formation and background amplification.

Tem-PCR increases sensitivity by using a pair of high concentration super primers that have an elevated affinity to Taq polymerase.

Tem-PCR uses only one pair of Super Primers; therefore, all targets are amplified with a more uniform efficiency. Thus, quantitative analysis becomes feasible.

Tem-PCR uses a lower concentration of primers, with one lot providing up to 100,000 reactions. Stable performance provides a high degree of confidence.

How Does Tem-PCR Work?

For each target in the Tem-PCR reaction, nested gene-specific primers are designed. Nested primer sets for each target are combined in a single, multiplex PCR reaction. (Fo= Forward out; Fi = Forward in; FS = Forward Super Primer; Ri = Reverse in; and Ro = Reverse out; and RS = Reverse Super Primer).

These primers are used at extremely low concentrations, and are only used to enrich the targets during the first few cycles of the reaction.

Some of the gene-specific primers have tag sequences that are recognized by the Super Primers.

Only the Super Primers are included at a concentration necessary for the exponential phase of PCR amplification.

Only the reverse Super Primer is labeled.

Labeled PCR products are detected with a complimentary capture probe, which is covalently coupled onto a color-coded bead (see xMAP technology for details)

Why Is Tem-PCR Multiplexing So Accurate?

Tem-PCR Primer Nesting

Most of the problems associated with standard multiplex PCR are due to the presence of multiple sets of high concentration labeled primers. Yet, these high concentrations are only required in the last cycles of a PCR reaction. With Tem-PCR, we use only enough gene-specific primers to "enrich" the targets and introduce the Super Primer tag into the products. Then, amplification is carried out with only one pair of primers.

Incompatibility among co-amplified targets is another major roadblock to standard multiplex assay development. Usually, one pair of primers define each target; therefore, one optimal condition must be identified that can allow for the co-amplification of all targets. With Tem-PCR, a target is amplified by nested primers producing four potential products for exponential amplification, thereby creating multiple optimal conditions. This feature of Tem-PCR increases compatibility among the co-amplified loci and creates fewer restrictions for multiplex assay development.