AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Instrukcja Użytkownika Pobierz pdf (Strona 43)

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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

36 Primer Express Software 3.0 Getting Started Guide

Notes

Figure 14. SNP site in an MGB probe

Avoid the following motifs:

• Repeating oligonucleotides– Avoid runs of identical nucleotides. If

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

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

5′-...GGAG-MGB-3′

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

the probe.

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

the probe, 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, or probe allele 1 is no longer within

the guidelines, you can design using the complementary sequence. For more

information, see Primer Express Software Version 3.0 Online Help.

Manually

Designing the

Allele 2 Probe

To design the probe for Allele 2:

Note: Keep the Allele 1 and Allele 2 probe Tms within one degree of each other.

1. In the Sequence tab, select the sequence for the probe (13 to 25 bases and includes

the SNP site) then select Edit > Copy with Allele 2.

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.

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

NNNNNNNNNN N NNNNNNNNNN

SNP

site

If possible, position the SNP site

in the middle third of the probe.

If necessary, position the

SNP site toward the 3’ end.

5’ 3’

Do not position the SNP

site in the last two bases.

1 2 ... 38 39 40 41 42 43 44 45 46 47 48 ... 51 52

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