AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Manuel d'utilisateur télécharger pdf (Page 41)

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Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays

Manually Designing Primers and Probes

34 Primer Express Software 3.0 Getting Started Guide

Notes

Manually Designing Primers and Probes

You may choose to manually design primers and probes for a various reasons:

• Automated primer/probe design did not find primers or probes.

• To design primers and probes according to your own specifications.

Creating an Allelic

Discrimination

Document

Create an MGB allelic discrimination document and load a sequence file as you would

for automatic primer/probe set design. See “Creating an Allelic Discrimination

Document” on page 29.

Manually

Designing the

Allele 1 Probe

To design the probe for Allele 1:

1. In the Sequence tab, identify the SNP site and the putative probe sequence.

2. Assign the SNP target (see “Assigning a SNP Target” on page 30).

3. Select the sequence for the probe (13 to 25 bases) then select Edit > Copy with

Allele 1.

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.

4. Select Tools > Pri mer P rob e Test Tool.

5. From the Document Type drop down menu, select the desired document type.

Verify that the Parameter field is set to Default. For more information about

parameters, see Primer Express Software Version 3.0 Online Help.

6. Paste (Ctrl+V) the annotated sequence in the Probe 1 field. The software displays

the Tm, %GC, and the oligonucleotide length to the right of the Probe 1 field (see

“Figure 13. Primer Probe Test Tool dialog box” on page 35). Note that the original

Allele 1 variant base appears in lower case on the Primer Probe Test Tool.

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AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Manuel d'utilisateur Page 41