Lightning-Fast PCR -- Amplifying, Detecting, and Even Quantitating at Once

Contents
Introduction
UniPrimer Fine Structure
All-In-One Tube
How to Use the Universal Amplification and Detection System

Introduction (Back to Top)
Previous articles in this series have looked at various time-saving devices for running lots of PCR reactions—from six-minute gels to rapid cycling machines. This last article deals with a product that does everything at once and can do it for any PCR reaction, time-saving of a different sort. Intergen Co.'s (Purchase, NY) Amplifluor Universal Amplification and Detection System can be used to perform quantitative or end-point PCR, and, as the name implies, can be applied with any template. While most other quantitative PCR systems require specially made hybridization probes, the beauty of Intergen's assay is that it has no such requirements.

The key to this system is something called the Uniprimer, which uses energy transfer to generate signal, similar to molecular beacons*. However, the Uniprimer is unique in having a tail through which it can engage any PCR primer with a complementary sequence. This means you only need this one primer for quantitating any PCR reaction. This is both a time and money saver, since expensive hybridization probes (TaqMan, Molecular Beacons) do not need to be synthesized for each gene of interest.

UniPrimer Fine Structure (Back to Top)
UniPrimer consists of a 3' 18 base oligonucleotide tail (Z sequence) and a 5' intracomplementary sequence labeled with a pair of energy transfer molecules (fluorescein and the acceptor 4-(4'-dimethylamino-phenylazo) sulfonic acid or DABSYL). The "Z sequence" acts as a universal PCR primer and, in addition, was designed to reduce PCR background due to heterodimer formation.

To use the UniPrimer in PCR, you must add Z sequence to the 5' end of one of the target-specific primers. As shown in the figure, the UniPrimer anneals to the complement to the Z sequence (Z' sequence) contained in an amplicon generated in the initial cycles of the reaction. As the UniPrimer is incorporated and the hairpin is unfolded, quenching is no longer possible due to the increased distance between the fluorescein and DABSYL moieties. The fluorescence signal produced with each PCR cycle directly correlates to the amount of amplified DNA generated allowing for quantitation over a wide target range.

All-In-One Tube (Back to Top)
With Amplifluor, amplification and detection can be done in the same tube, which reduces the risk of cross-contamination and eliminates the need for post-PCR processing, another time-saver. And as the fluorescent signal is incorporated into the amplicon, fluorescence is directly correlated with quantity of product. The system also has a broad dynamic range—up to 3 logs with endpoint and 5 logs with real-time—which allows for sensitive quantitation (guaranteed to detect 100 copies, though company scientists report detecting as few as 10 with real-time detection).

Finally, dust off those old spectrofluorometers, because that's all you need for analyzing your PCR reaction. In fact, the fluorescence can be measured by just about any fluorescence detection device—plate readers, fluorescence imaging systems, spectrofluorometers, and PCR machines set up for real-time quantitation. Being adaptable to a variety of instruments for both endpoint and real-time provides the ultimate in flexibility in experimental design.

How to Use the Universal Amplification and Detection System (Back to Top)
Intergen markets a generic system, The Amplifluor Universal Amplification and Detection System, with enough reagents to perform 100 PCR reactions with any gene target by simply adding a unique oligonucleotide sequence (designated Z) to one of the target-specific primers. A control set of templates and primers, for Prostate Specific Antigen (PSA), are included to aid in qualification of the PCR reagents not included with the kit (such as polymerase, dNTPs) and the fluorescence measuring device. Intergen also sells kits for measuring specific genes—apoptosis genes, bax-a, bcl-2, bcl-X, and fas, and human disease genes, HPV and PSA.

The figure presents real-time data for intergen's Amplifluor Apoptosis bcl-2 kit, run on the PRISM 7700. This kit contains the UniPrimer and two gene specific primers for bcl-2. The Amplifluor UniPrimer in this demonstration detects 10 target copies, though this is not achievable with all primers. Primer design, it turns out, is key to achieving this level of sensitivity.

For more information: Intergen Co., 2 Manhattanville Rd., Purchase, NY 10577. Tel: 800-431-4505. Fax: 914-694-1429.

By Laura DeFrancesco