AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE User Manual download pdf (Page 23)

DRAFT

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

16 Primer Express Software 3.0 Getting Started Guide

Notes

Viewing Results Primer Express software automatically displays the Primers/Probes tab, if it finds

primers. The Primers/Probes tab displays the Candidate Primers & Probes table that

contains information about the candidate primers, probes, and amplicons. The forward

primer sequences are displayed using the left-to-right 5′-to-3′ convention, and reverse

primer sequences are displayed using the right-to-left 5′-to-3′ convention.

Evaluating the candidate primer and probe sets:

The Location section of the Primers/Probes tab illustrates the location of the primers and

probes within the sequence. The number above the line is the starting base; the number

below the line is the ending base. Note that you can also see the corresponding location

of a selected candidate Primer/Probe set in the Sequence tab.

In the Sequence tab, the probe will be highlighted in pink, the forward primer in blue,

and the reverse primer in yellow. These default color designations can be changed by

clicking Tools > Options. If you place your cursor over any of these annotations, a tool

tip will appear showing the name of the annotation (Probe, Forward Primer, Reverse

Primers) start and end locations, Tm and %GC.

As a general guideline, select the primer/probe sets with a low Penalty score and a low

amplicon length (if the Penalty score and Amplicon Length fields are not displayed,

scroll to the right in the table). However, all primer/probe sets generated using default

parameters meet primer and probe guidelines. For more information regarding Penalty

scores, see Primer Express Software Version 3.0 Online Help.

Note: After the software finds primers and probes, the sequence box is locked. To edit

the sequence, click to unlock.

Manually

Designing the

Primers

To design the Forward Primer:

1. Select a sequence (at least 25 bases) to the left of the probe. The sequence should be

as close to the probe as possible without overlapping it.

2. Copy (Ctrl+C) the sequence.

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

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

oligonucleotide length to the right of the Fwd Primer field.

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AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE User Manual Page 23