AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Manuel d'utilisateur télécharger pdf (Page 45)

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Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays

Manually Designing Primers and Probes

38 Primer Express Software 3.0 Getting Started Guide

Notes

To design the Reverse Primer:

1. In the sequence tab, select a sequence (at least 25 bases) to the right of the

probe.The sequence should be as close to the probe without overlapping it.

2. Select Edit > Copy Complement.

IMPORTANT! The Primer Probe Test Tool eliminates non-ATCG bases. Before copying

a sequence, change any non-ATCG bases, or select a different region of the sequence.

3. On the Primer Probe Test Tool dialog box, paste (Ctrl+V) the sequence into the

Rev Primer field. The Primer Probe Test Tool displays the Tm, %GC, and the

oligonucleotide length to the right of the Fwd Primer field.

4. If the Tm is not between 58 °C to 60 °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.

Be sure to keep the above guidelines in mind.

Note that you can further customize your primer and probe set by editing the default

parameter values found under the Parameters tab. For more information on editing

parameters, see Primer Express 3.0 Software Online Help.

Saving Primer

and Probe

Sequences

Copy and paste the primer and probe sequences into a text document, then save for

future reference.

Ordering Primers

and Probes

To order primers and probes, see Chapter 4, “Ordering Primers and Probes.”

IMPORTANT! Before running your samples, Applied Biosystems recommends that you

run control samples to verify the performance of the selected primers and probes.

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AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Manuel d'utilisateur Page 45