Using Taqman ® Probe-Based Reagent Configuration; Chapter 2 Designing A Plus/Minus Experiment - Applied Biosystems 7300 Getting Started Manual

Chapter 2 Designing a Plus/Minus Experiment

®
Using TaqMan Probe-Based Reagent Configuration
Using TaqMan
About the
Chemistry
Chemistry
®
TaqMan reagents or kits
Description
TaqMan probe-based
chemistry uses a
fluorogenic probe to detect
a specific PCR product as it
accumulates during PCR
cycles.
Chemistry Kits for
Plus/Minus Assay
Notes
8
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
®
Probe-Based Reagent Configuration
Plus/minus assays with an IPC use the fluorogenic 5' nuclease chemistry (also known as
®
TaqMan probe-based chemistry).
Polymerization
FORWARD
PRIMER
5'
3'
5'
Step 1: A reporter (R) and a
quencher (Q) are attached to the
5' and 3' ends of a TaqMan
probe.
Cleavage
5'
3'
5'
Step 2: During each extension
cycle, the AmpliTaq Gold
polymerase cleaves the reporter
dye from the probe.
For more information about the TaqMan probe-based chemistry, refer to the Real-Time
PCR Systems Chemistry Guide (PN 4378658).
The following reagents are available from Applied Biosystems for designing and
running plus/minus assays.
®
TaqMan Exogenous Internal Positive Control Reagents with TaqMan
Universal PCR Master Mix
®
TaqMan Universal PCR Master Mix
The IPC DNA, primers, and probe supplied in these reagents can be used with all
Note:
sample target systems. Refer to the TaqMan
(PN 4304449) for instructions on optimizing amplification of your target.
Process
Strand Displacement
R = REPORTER
R Q
PROBE
Q = QUENCHER
3'
5'
5'
3'
3'
5'
5'
REVERSE
PRIMER
Step 1 (continued): When both dyes
are attached to the probe, reporter
dye emission is quenched.
Polymerization Completed
R
Q
3' 5'
5' 3'
3' 5'
5'
Step 3: After being separated from
®
DNA the quencher, the reporter dye
emits its characteristic
fluorescence.
Kit
®
Universal PCR Master Mix Protocol
Q
3'
5'
3'
5'
Q
R
3'
5'
3'
5'
Part
Number
4308323
4304437