FAQs

Plexor™ qPCR and qRT-PCR Systems

See Technical Resources for more information.

1. How do the Plexor™ qPCR and qRT-PCR Systems work?
2. Which real-time PCR instruments can I use with the Plexor™ Systems?
3. Do I need to use the Plexor™ Analysis Software? Why can’t I analyze the results using my instrument software?
4. Does the Plexor™ Analysis Software replace my instrument software?
5. What primer design software can I use to design primers for use with the Plexor™ Systems?

1. How do the Plexor™ qPCR and qRT-PCR Systems work?
   This new real-time PCR method takes advantage of the specific interaction between two modified nucleotides to achieve quantitative analysis. Two novel bases, isoguanine (iso-dG) and 5′-methylisocytosine (iso-dC), form a unique base pair in double-stranded DNA. One primer is synthesized with an iso-dC residue as the 5′-terminal nucleotide and a fluorescent label at the 5′-end; the second primer is unlabeled. During PCR, this labeled primer is annealed and extended, and during subsequent rounds of amplification, the complementary iso-dGTP pairs preferentially with iso-dC in the labeled primer. When the dabcyl-iso-dGTP is incorporated, the close proximity of dabcyl and the fluorescent label on the opposite strand effectively quenches the fluorescent signal. The initial fluorescence level of the labeled primers is high in Plexor™ System reactions. As amplification product accumulates, the fluorescent signal decreases. For more information, see the Promega Notes article entitled Plexor™ Technology: A new chemistry for real-time PCR.

2. Which real-time PCR instruments can I use with the Plexor™ Systems?
   The Plexor™ Analysis Software has been developed to analyze amplification data from a variety of real-time instruments, plot standard curves and calculate DNA concentrations of unknown samples. The software, which is distributed free-of-charge, allows freedom of choice for instrument use. Currently, data can be imported from the ABI PRISM^® 7000 and 7700 sequence detection systems, Applied Biosystems 7900HT real-time PCR system, Applied Biosystems 7300 and 7500 real time PCR systems, Roche LightCycler^® 1.0 and 2.0 instruments, Bio-Rad iCycler^® thermal cycler, MJ Research DNA Engine Opticon^® 2 fluorescence detection system, Cepheid SmartCycler^® system and the Stratagene Mx3000P^® and Mx3005P^® real-time PCR systems and Mx4000^® multiplex quantitative PCR system. For protocol information, see the Plexor™ Instrument Setup and Data Analysis Technical Manuals.

3. Do I need to use the Plexor™ Analysis Software? Why can’t I analyze the results using my instrument software?
   Standard real-time PCR analysis methods are based on an increase in fluorescence as amplification product is accumulated. Because fluorescence decreases during Plexor™ amplification, most real-time PCR instruments cannot interpret the data. The Plexor™ Data Analysis Software is designed to analyze amplification data, where fluorescence decreases in intensity as product accumulates. The software can analyze data generated by many different real-time PCR instruments and allows flexibility in the choice of instrument used for amplification.

4. Does the Plexor™ Analysis Software replace my instrument software?
   No. Your instrument software is necessary to run the real-time instrument. The Plexor™ Analysis Software is needed only for data analysis after the runs are completed. The raw data generated using the Plexor™ chemistry on the real-time PCR instrument are exported from the instrument software and then imported into the Plexor™ Analysis Software for analysis of the results.

5. What primer design software can I use to design primers for use with the Plexor™ Systems?
   We highly recommend using the Plexor™ Primer Design Software. This primer designer is based on a DNA Software engine that has been optimized to design primers for multiplex PCR and takes into account possible primer-primer matches to reduce the possibility of primer-dimer formation and misannealing.