What are the important design considerations for molecular beacons?

We recommend the following when designing Beacons and Beacon-based assays:

Amplicon:
1) Keep Short (70 – 120 bp)
2) Cross exon boundaries for RT-PCR
3) Screen GenBank for existence of pseudogenes
4) Primer Tm 50-60oC

Probe:
1) GC Content 30-70%, ~15-25 bases
2) No runs of >4 G's
3) For High GC area, use C-rich strand
4) Positions SNPs in center
5) Hairpin Tm 5oC higher than anneal temp usually 5-6 bases long
6) Probe Tm about the same as hairpin Tm
7) Primers and probes can be close but should not overlap

In addition, you should review Dr. Russell Kramer's website (Molecular Beacons were first described by Dr. Kramer). He has extensive information on design considerations and troubleshooting.

IDT has additional information in our Technical Bulletins you may want to look at, too. Molecular Beacons, Fluorescence Resonance Energy Transfer, and Dark Quenchers.

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