Molecular assays target nucleic acid (DNA or RNA) sequences to demonstrate the presence of a particular target(s) within a sample, in contrast to immunoassays, which target proteins. Most commercial molecular assays are based on PCR (the Polymerase Chain Reaction), an enzymatic amplification method that amplifies the target sequence to improve the limit of detection and time to detect. The PCR process involves forward and reverse primers, which define the target region.

Real-Time PCR assays include a third nucleic acid sequence, termed a probe, which binds a region between the amplification primers and is tagged with a fluorescent molecule and a fluorescence quencher. Thermal cycling is key to the PCR process and involves repeated rounds of heating and cooling. The reaction tube is heated to dissociate the double-stranded DNA template. As the reaction tube is cooled, the primers and probe bind to the single stranded DNA. The PCR enzyme synthesizes a new second DNA strand based on the primer “start button” and removes any DNA in it’s path as it proceeds down the strand, thereby dissociating the probe and releasing the fluorescent molecule into solution where it is “seen” by the PCR instrument’s detection element. Changes in the fluorescence properties of the reaction tube indicate that the particular target sequence is present in the sample and is being amplified. 

Direct hybridization assays employee coded bead sets, in which each bead set is functionalized with a capture oligo specific for a nucleic acid sequence (referred to as the capture oligonucleotide) in the target that binds the amplicon product of the PCR reaction, thereby demonstrating the presence of the target nucleic acid sequence.

An advantage of real-time PCR is a relatively short time to detect, typically in 15-120 minutes (instrument dependent). An advantage of bead-based assays is that a sample can be interrogated for more targets in a single tube; up to 50 vs. 4 to 6 targets for real-time PCR, which is constrained by overlap in the fluorescence spectrum of available probes.

Q. What are the differences and similarities between real time and end point PCR? 

A. Both methods use forward and reverse primers and a DNA polymerase enzyme for amplification.  Real-time PCR uses a fluorescent probe that binds one strand in between the primers.  As new strands are synthesized by the polymerase, the exonuclease activity of the polymerase chops the bound probe into single bases, thereby changes the fluorescence properties.  The change is measured by the instrument, which generates data after every cycle.  The end point method typically incorporates a biotin into one of the primers .  At the end of the PCR amplification process, the PCR product is hybridized with a capture oligo attached to a surface.  Bound PCR produce is visualized with a reporter attached to streptavidin, which binds to the biotin-labeled primer.

Q. Can real-time and end point PCR methods be multiplexed to look for multiple targets?

A.  Yes, both methods can be multiplexed.  The plexity is determined by the instrument.  Real time PCR multiplexing is limited by overlap in the emission spectrum of the fluorescent probes used to visualize amplification of the target. Many real time PCR instruments can support a four-plex, but can range from one to seven colors.  End point platforms are limited to the number of distinguishable immobilized locations; up to 500 have been demonstrated and field-ready instruments that can distinguish 50 PCR products are available.

Q. Which method is more sensitive? 

A. Both methods have demonstrated detection limits of less than five copies of a specific PCR target.  It stands to reason that a sample containing a copy number close to the limit of detection could fall below the detection limit if the sample must be divided to interrogate it for more targets than can be identified in a single reaction.

Q. Are both methods semi-quantitative?

A. No.  Real time PCR generates data at every cycle, which allows for an estimate of the copy number based on the number of cycles needed to reach a specified level of fluorescence.  For the end point PCR method, data are generated at the end of all amplification cycles, which makes it more difficult to extrapolate back to the target copy number in the original sample.

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