Primer Design using IDT Quest
Primer Quest is part of the IDT suite of genomic tools for identifying and
mapping qPCR primers to target sequences.
In essence, target sequences are imported from NCBI and then primers are
designed and mapped to this sequence based on specified parameters
This primer design tool is a useful facility for making pan specific transcript
primers to cDNA but is less effective than primer BLAST at making exon
junction splice form specific primers
Identifying NCBI transcript & associated primers
1. Go to the Entrez gene (‘Gene’) database (formely ‘Locus Link’):
http://www.ncbi.nlm.nih.gov/gene
2. Click on the ‘RefSeqGene link’: ‘Resources_DNA & RNA_RefSeqGene’
3. This will take you to the portal for NCBI Reference sequences
4. Type the generic name of your gene into the search box_Search
5. Now select the link to your gene
6. This should provide a summary page of your gene:
7. On the right hand side you should see ‘NCBI Reference sequences’
8. Click on this and you will come to a page giving details of transcripts
9. One of the headings is ‘mRNA & Proteins’ & under that links are provided to
individual transcripts, denoted by accession numbers ‘NM*****’
10. Now click on the ‘NM******’ link: This will take you to a detailed description
of the transcript
11. On the right hand side is the heading ‘RefSeq alternative splicing’:
12. Click on this link and you are taken to links of respective transcript variants
Entering & submitting NCBI Ref Sequences using IDT Primer Quest
Open IDT Primer Quest:
http://www.idtdna.com/primerquest/home/index
13. Under Sequence Entry Enter NCBI ID #
14. Hit get Sequence
15.This should upload sequence
16.Now within ‘Custom design parameters’ select qPCR intercalating dyes (for
probe type) and then adjust parameters like amplicon size etc (as before):
‘Select parameters for’ ‘qPCR intercalating dyes (primers only)’
Reaction Conditions:
Sodium = 50mM
Primer DNA concentration = 200nM
Divalent salt (mg2+) = 2mM
Primer details:
Tm Min =’59’ + Opt ‘65’ Max 70
3’ GC clamp(nt) = ‘2’
Amplicon Details:
Size =100 min + 150 opt +300 max
17.‘Get assays’:
18. Returns a series of primers optimised for qPCR mapped to transcript
sequence
19. Click on green icon corresponding to most 3’ primers initially: Your primers
should be within 2kb of 3’ end and preferably 1kb from 3’ Poly A site
20. Now click on ‘view assay Details’
21. Examine primers:
1. On average, make primers 18-22 mers and incorporate 50 – 60% GC. This equates to an
average Tm of ~ 60oC
2. Avoid runs of 3 or more of the same bases, particularly G’s & C’s or consecutive G’s
3. Keep primer Tm’s in an optimal window of 55 oC_65oC
4. Ensure that primer pairs have Tm’s within 2oC of one another
5. Preferably match primers for Δ G values as well: No more than -10Kcal/mol between primer
pairs
6. Pay attention to the last 5 bases:
Termination with either a ‘G’ or ‘C’ residue preceded by a pyrimidine base is acceptable
Avoid terminating with ‘T’ residues
If primer must end in 3 purine (GC) residues (by virtue of restricted placement) terminate
with an ‘A’ residue
Termination with either a GG/GC or CC residue is satisfactory
Indeed some commentators think that this so called ‘GC clamp’ increases priming
efficiency and so is preferable for qPCR primers
22. If suitable click on ‘hairpin’
23. If > -5Kcal/Mol ignore structure as thermodynamically it is unstable
24. Go back to primer & click ‘BLAST’
25.This takes you to the NCBI BLASTn Suite
26. Select ‘Reference RNA Sequences’ under data base & ‘Human’
27. Also Select ‘Exclude models’ = XM/XP
28. ‘Show results in a separate window’_BLAST
29. Click on blue lines in schematic
30. This takes you to aligned sequence(s)
31. Make sure 100% coverage and 100% query just correspond to your
transcript of interest
32. Above the aligned primer sequence select ‘Down load’_’FASTA Sequence (full
sequence)
33. This opens a FASTA file with your sequence in
34. Use the find facility to identify primers primers
Mapping & archiving primer sequences to target RefSeq
35. Go back to NCBI BLAST result(s) window
36. Select ‘GenBank’ for aligned sequence
37. This will bring up the transcript sequence
38. Select ‘FASTA’
39. Now select ‘find in this sequence’
40. Identify primer sequence then copy and paste into word in order to
format/highlight primer sequences
