AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Instrukcja Użytkownika Pobierz pdf (Strona 42)

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December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays

Manually Designing Primers and Probes

Primer Express Software 3.0 Getting Started Guide 35

Notes

Figure 13. Primer Probe Test Tool dialog box

7. If the Tm is not between 65 °C to 67 °C, highlight a section of the sequence to view

the corresponding Tm, %GC, and oligonucleotide length. Once the highlighted

region results in the desired Tm, click on Trim to delete the non-highlighted bases.

Ensure the following guidelines are met (for more information on design

guidelines, refer to Primer Express Software Version 3.0 Online Help):

• Amplicon Length – 50 to 150 bases for optimum PCR efficiency.

• Probe Length – 13 to 25 bases (13 to 30 bases if using conventional

TaqMan probes).

• Tm – 65 °C to 67 °C.

• % GC – 30% to 80%.

• 5′ end – Cannot be a G residue. A G residue adjacent to the reporter dye

will quench the reporter fluorescence somewhat, even after cleavage.

• Tm difference between probes – Not greater than 1 °C

• SNP site – Locate in the middle third of sequence or toward 3′ end but not

in the last two bases of 3′ end (see “Figure 14. SNP site in an MGB

probe” on page 36).

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AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Instrukcja Użytkownika Strona 42