Real-Time PCR 与Traditional PCR比较

The TaqMan® Probe is designed with a high-energy dye termed a Reporter at the 5 end, and a low-energy molecule termed a Quencher at the 3 end. When this probe is intact and excited by a light source, the Reporter dye’s emission is suppressed by the Quencher dye as a result of the close proximity of the dyes, Figure 15.

When the probe is cleaved by the 5 nuclease activity of the enzyme, the distance between the Reporter and the Quencher increases causing the transfer of energy to stop. The fluorescent emissions of the reporter increase and the quencher decrease.

Figure 15: Increased florescence activity due to the cleaved probe


The increase in reporter signal is captured by the Sequence Detection instrument and displayed by the software. Figure 16 shows an increase in the reporter signal over time. The amount of reporter signal increase is proportional to the amount of product being produced for a given sample.
Figure 16: Increase in Reporter Signal

The combination of FRET and the 5‵nuclease activity of AmpliTaq Gold® DNA Polymerase enables the 5‵nuclease assay and the SDS instrumentation to collect data in real time. When the fluorescent signal Reporter increases to a detectable level it can be captured and displayed as an Amplification Plot, Figure 17.

Figure 17: Amplification Curve
Rn: measure of reporter signal

The Amplification Plot contains valuable information for the quantitative measurement of DNA or RNA. The Threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background. The threshold line is set in the exponential phase of the amplification for the most accurate reading. The cycle at which the sample reaches this level is called the Cycle Threshold, Ct. These two values are very important for data analysis using the 5‵ nuclease assay.

SYBR Green Dye
SYBR Green chemistry is an alternate method used to perform real-time PCR analysis. SYBR Green is a dye that binds the Minor Groove of double stranded DNA. When SYBR Green dye binds to double stranded DNA, the intensity of the fluorescent emissions increases. As more double stranded amplicons are produced, SYBR Green dye signal will increase. Figures 18 & 19 show the entire process of each type of realtime chemistry. SYBR Green dye will bind to any double stranded DNA molecule, while the 5 Nuclease assay is specific to a pre-determined target.

Real-Time PCR Applications
Real-Time PCR can be applied to traditional PCR applications as well as new applications that would have been less effective with traditional PCR.With the ability to collect data in the exponential growth phase, the powerof PCR has been expanded into applications such as:

Viral Quantitation

Quantitation of Gene Expression

Array Verification

Drug Therapy Efficacy

DNA Damage measurement

Quality Control and Assay Validation

Pathogen detection

Genotypin

Summary
Advantages of using Real-Time PCR:

Traditional PCR is measured at End-Point (plateau), while Real- Time PCR collects data in the exponential growth phase

An increase in Reporter fluorescent signal is directly proportional to the number of amplicons generated

The cleaved probe provides a permanent record amplification of an Amplicon

Increase dynamic range of detection

No-post PCR processing

Detection is capable down to a 2-fold change

For Research Use Only. Not for use in diagnostic procedures.
Practice of the patented polymerase chain reaction (PCR) process requires a license.
The PCR process and 5’ nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.
Applied Biosystems is a registered trademark and the AB design and Applera are trademarks of Applera Corporation or its subsidiaries in the US and other countries.