AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Instrukcja Użytkownika Pobierz pdf (Strona 22)

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December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm

Chapter 2 Designing Primers and Probes for Quantification Assays

Manually Designing Primers and Probes

Primer Express Software 3.0 Getting Started Guide 15

Notes

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

will quench the reporter fluorescence somewhat, even after cleavage.

Avoid the following motifs:

• Repeating oligonucleotides– Avoid runs of identical nucleotides. If

repeats are present, there must be fewer than four consecutive G residues.

• Consecutive A residues – Avoid six consecutive A residues anywhere in

the probe.

• G residues on the 3′ end – Avoid 5′-...GGG-MGB-3′ or

5′-...GGAG-MGB-3′

• CC dinucleotides – Avoid two or more CC dinucleotides in the middle of

the probe (TaqMan MGB probes), which can sometimes reduce signal.

• FAM

™

dye-labeled probes – If ordering FAM

™

-dye labeled probes,

avoid a G in the second position on the 5′ end.

For secondary structure design considerations, see Primer Express Software Version

3.0 Online Help.

Note: If you cannot achieve the recommended Tm, you can design using the

complementary sequence. For more information, see Primer Express Software Version

3.0 Online Help.

7. Once the correct Tm is achieved, return to the Sequence tab and highlight the

sequence found in the Probe 1 field of the Primer Probe Test Tool. To manually

design primers, go to “Manually Designing the Primers” on page 16.

8. To automatically find primers after manually designing the probe, select Edit >

Annotate > Probe ( ). The selected probe sequence text is displayed in green.

For more information on annotating sequences, see Primer Express Software

Version 3.0 Online Help.

9. Select Tools > Find Primers/Probes ( ). Primer Express software performs its

calculations based on default parameter values. The status bar, located at the bottom

of the window, displays information about the progress of the calculations as the

software searches for primers based on the designed probe. If primers are found, go

to “Viewing Results” on page 16.

If primers were not found:

If the software does not find primers using default parameters, a pop-up will appear

stating that no acceptable primer pairs were found and that you can see the Interim

Results window. For more information on Interim Results, see Primer Express Software

Version 3.0 Online Help.

At this point, you can manually design primers described in “Manually Designing the

Primers” on page 16.

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