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.
AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems inc.
SYBR Green is a registered trademark of Molecular Probes, Inc.
