AMX DESIGN XPRESS V 1.5 - PROGRAMMER GUIDE Manuel d'utilisateur télécharger pdf (Page 44)

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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

Primer Express Software 3.0 Getting Started Guide 37

Notes

3. Paste (Ctrl+V) the sequence into the Probe 2 field. The Primer Probe Test Tool

displays the Tm, %GC, and sequence length to the right of the field. Note that the

original Allele 2 variant base will appear in lower case on the Primer Probe Test

Tool.

4. If the Tm is not between 65 °C to 67 °C, highlight a section of the sequence to view

the corresponding Tm, %GC, and oligonucleotide length of the highlighted region.

Once the highlighted region results in the desired Tm, click on Trim to delete the

non-highlighted bases. Keep in mind the general design guidelines previously listed

on page 35.

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.

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 as if those highlighted

bases were deleted. Once the highlighted region results in the desired Tm, click on

Trim to delete the non-highlighted bases.

Ensure the following guidelines are met (for more information on design

guidelines, refer to Primer Express Software Version 3.0 Online Help):

• Amplicon Length – 50 to 150 bases for optimum PCR efficiency.

• Optimal Primer Length – 20 bases. Do not overlap primer and probe

sequences.

• Tm – 58 °C to 60 °C (Optimal Tm – 59 °C).

• % GC – 30% to 80%.

• 3′ end – Make sure the last five nucleotides at the 3′ end contain no more

than two G + C residues.

Avoid the following motifs:

• Repeating oligonucleotides – Avoid runs of identical nucleotides. If

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

For secondary structure design considerations, see Primer Express Software Version

3.0 Online Help.

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