In Molecular Biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction, is a laboratory technique used to simultaneously quantify and amplify a specific part of a given DNA molecule. It is used to determine whether or not a specific sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-time version of quantitative polymerase chain reaction (Q-PCR), itself a modification of polymerase chain reaction. Each technique uses some kind of fluorescent marker which binds to the DNA. Hence as the number of gene copies increases during the reaction so the fluorescence increases. This is advantageous because the efficiency and rate of the reaction can be seen. There is also no need to run the PCR product out on a gel after the reaction.

Real-Time chemistries allow for the detection of PCR amplification during the early phases of the reaction. Measuring the kinetics of the reaction in the early phases of PCR provides a distinct advantage over traditional PCR detection. Traditional methods use Agarose gels for detection of PCR amplification at the final phase or end-point of the PCR reaction.

• Viral Quantitation
• Quantitation of Gene Expression
• Array Verification
• Drug Therapy Efficacy
• DNA Damage measurement
• Quality Control and Assay Validation
• Pathogen detection
• Genotyping

* Generally, primers used are 18-28 nt in length. This provides for practical annealing temperatures.

* Primers should avoid stretches of polybase sequences (e.g. poly dG) or repeating motifs – these can hybridize inappropriately on the template.

* Aim for 50% GC content. High GC content results in the formation of stable imperfect hybrids while high AT content depresses the Tm of perfectly matched hybrids.

* If possible the 3ด end of the primer should be rich in GC bases (GC clamp) to enhance annealing of the end which will be extended.

* Inverted repeat sequences should be avoided to prevent formation of secondary structure in the primer which may prevent hybridization to template.

* Sequences complementary to other primers used in the PCR should be avoided so as to prevent hybridization between primers (primer dimers).

* Primer pairs should have compatible Tm (within 5 degrees). * When adding sequences to the 5ด end of the primer to create a restriction site, it is important to include a few extra bases (2-6 bases) to serve as a clamp to keep the 5ด ends from breathing during digestion.

PCR products from previous PCR reactions need to be destroyed so that they do not act as templates in new reactions. In order for selective degradation of previous carryover product to occur, a GUAC dNTP mix is employed rather than the standard GTAC mix. Resulting product then contains uracil. Our Platinum qPCR Supermix UDG systems utilize this GUAC mix along with uracil-N-glycosylase to degrade only those DNA products that contain uracil bases carried over from a previous reaction. The degradation reaction occurs at 45oC before the PCR reaction. At this point any non-specific product that is created while you set up the PCR mixes will also get degraded. The glycosylase is inactivated completely at the 94oC denaturation step at the beginning of PCR and will not affect your desired PCR products.

Primers must generate a single band with no primer dimer formation (other than in the no-template control). For RT-PCR they should not amplify genomic DNA or processed pseudogenes. The general consensus for product size is from 80 - 200 bp. Shorter PCR products are more efficiently amplified, however PCR products as long as 500 bp can still be used. When comparing the relative expression of multiple genes in a single experiment, it is crucial that each gene is amplified with the same efficiency, therefore the product sizes should not be drastically different. Sometimes it is also necessary to vary the ratio of forward and reverse primers to equalize the efficiencies of different sequences. With SYBR Green I detection, larger products will fluoresce more intensely than smaller ones.

When you add SYBR Green I to your PCR reactions, you will need to add more magnesium. Typically, an increase from 1.5 mM to 4 mM magnesium is required for 1x SYBR Green I, but it may be ueful to titrate in the amount your reactions need.

LUX™ Primers are oligonucleotides labeled with a single fluorophore, custom-synthesized according to the DNA/RNA of interest. Typically 20-30 bases in length, they are designed with a fluorophore close to the 3’ end in a hairpin structure. This configuration intrinsically renders fluorescence quenching capability; no separate quenching moiety is needed. When the primer becomes incorporated into double-stranded PCR product, the fluorophore is dequenched, resulting in a significant increase in fluorescent signal. This signal increase is the basis for the LUX™ detection platform. They differ from TaqMan probes in 1) Cost: With LUX you need two primers, only one of which is labeled. With TaqMan you need two primers. TaqMan also requires a third probe that is labeled with both a dye and a quencher. Therefore LUX primers are less expensive. 2) Melting Curve analysis: Because the LUX primer fluorophore gets incorporated into the PCR product, you can do a melting curve analysis after the PCR while the TaqMan fluorophore is not incorporated into the PCR product so a melting curve cannot be performed.