Applied Biosystems 7900HT User Manual

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September 1, 2004 11:39 am, App_P&PDesign.fm
Appendix B Designing TaqMan Reagent-Based Assays
B-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
Assay Development Guidelines
TaqMan Reagent-
Based Assay
Development
Program
1. Identify target sequence(s) (see page B-2).
2. Design the TaqMan
®
probes and the forward and reverse primers (see page B-2).
3. Order reagents.
4. Quantitate the concentrations of the probes and primers (see page B-3).
5. Prepare the master mix (see page B-3).
6. Optimize the primer concentrations (see page B-3).
7. Run the assay (see page B-4).
Identify Target
Sequence(s)
A target template is a DNA, cDNA, RNA, or plasmid containing the nucleotide
sequence of interest. For optimal results, the target template should meet the
following requirements:
• The target nucleotide sequence must contain binding sites for both primers
(forward and reverse) and the fluorogenic probe.
• Short amplicons work best. Amplicons ranging from 50–150 bp typically yield
the most consistent results.
• If designing assays for quantitative PCR, see “Design Tips for Quantitative PCR
Assays” on page B-6 for additional recommendations.
Design Probes
and Primers
The following sections contain general guidelines for designing primers and probes.
For specific design tips, refer to the appropriate section: for Allelic Discrimination
see page B-5 and for Quantitative PCR see page B-6.
Design Probe(s) for the Assay
Adhere to the following guidelines when designing TaqMan probes:
• Keep the G-C content in the range of 30–80%.
• Avoid runs of an identical nucleotide (especially guanine, where runs of four or
more Gs should be avoided).
• No G on 5´ end.
• Keep the melting temperature (T
m
) in the range of 68-70 °C for quantitative PCR
and 65-67 °C for allelic discrimination (using the Primer Express
™
software).
• Select the strand that gives the probe with more Cs than Gs.
• For allelic discrimination (see page B-5):
– Adjust probe length so that both probes have the same T
m
.
– Position the polymorphism site approximately in the center of each probe.
• For absolute or relative quantification (see page B-6)

Applied Biosystems 7900HT Specifications

General

Brand	Applied Biosystems
Model	7900HT
Category	Laboratory Equipment
Language	English