Appendix B Designing Taqman Reagent-Based Assays; Assay Development Guidelines - Applied Biosystems 7900HT User Manual

Appendix B Designing TaqMan Reagent-Based Assays

Assay Development Guidelines

TaqMan Reagent-
Based Assay
Development
Program
Identify Target
Sequence(s)
Design Probes
and Primers
B-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
September 1, 2004 11:39 am, App_P&PDesign.fm
1. Identify target sequence(s) (see page B-2).
2. Design the TaqMan
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).
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
Assays" on page B-6
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
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
and 65-67 °C for allelic discrimination (using the Primer Express
• Select the strand that gives the probe with more Cs than Gs.
• For allelic discrimination (see
– Adjust probe length so that both probes have the same T
– Position the polymorphism site approximately in the center of each probe.
• For absolute or relative quantification (see
DRAFT
® probes and the forward and reverse primers (see page B-2).
for additional recommendations.
page
) in the range of 68-70 °C for quantitative PCR
m
page B-5):
"Design Tips for Quantitative PCR
B-6.
™
.
m
page B-6)
software).