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Real-Time PCR >
Design Guidelines
Guidelines for designing primers and
probes using Primer Express
Primer Express designs probes and primers
suitable for the universal thermal cycling conditions specified by ABI.
Notes
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Blast the sequence of the entire gene and
identify a region which is specific for that gene. Use this sequence for
designing primers and probes using primer express.
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Following guidelines are used automatically (by
default) by Primer Express
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Select the Probe first ant then design primers.
Target Sequence and Amplicon Size
Guidelines
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Primers and probes should be selected in a
region with G/C contents of 20-80%. Lower G/C content is preferred.
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Design primers to amplify short segments of DNA
within the target sequence. The recommended amplicon size is 50 to 150 bp.
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Design at least one primer and/or probe which
crosses one exon junction. The primers thus created would amplify mRNA (or cDNA
made from it), but not genomic DNA. Use of Junction Annotation tool from Primer
express which allows the user to mark exon junctions and selects at least one
primer crossing at least on exon junction.
TaqMan Probe Design guidelines
0
C.
Keep G-C contents in the 30-80% range.
Avoid runs of an identical nucleotide. This is
true especially for G, where runs of four or more Gs should be avoided.
Do not put Gs on the 5’ end.
Select the strand that gives the probe more Cs
than Gs. Primer express does not automatically screen
for this feature. It needs to be done manually.
If the TaqMan probe is designed for allelic
discrimination, the position of the polymorphic site (mismatch) should be
approximately in the middle third of the sequence.
Primer Design Guidelines
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Design primers as close to probe as possible
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Keep G-C contents in the 30-80% range.
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Avoid runs of an identical nucleotide. This is
true especially for G, where runs of four or more Gs should be avoided.
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The melting temperature (Tm) of the primer
should be 58-60
0
C.
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The total number of Gs and Cs in the last five nucleotides
at the 3’ end of the primer should not exceed two. The software does this
automatically if the option is selected.
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Primers should scan exon-exon junction. Contaminating
genomic DNA will not be amplified by these primers.
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Use primers that contain dA nucleotides near the 3’ ends so
that any primer-dimer generated is efficiently degraded by AmpErase UNG. If
primers cannot be selected with dA nucleotides near the ends, the use of primers
with 3’ terminal dU nucleotide should be considered.
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