Species-Specific, Strain-Specific, and CNV Assay Design Considerations

1. Integrated DNA Technologies
Elisabeth Wagner
Scientific Applications Specialist
qPCR Design Strategies for Specific Applications
Species-Specific, Strain-Specific, and CNV Assay Design Considerations

2. 1
Learning Outcomes
 You will:
 Understand the different types of design specifications for species and splice-
form specific qPCR and CNV assays.
 Identify design considerations for different experimental scenarios and adjust
the basic qPCR design parameters accordingly
 Learn how to do an alignment of sequences to discover both unique and
similar regions
 Learn how to design Copy Number Variations assays

3. 2
General Design Strategy Outline for Specific qPCR Design Parameters:
1. NCBI- sequence accession www.ncbi.nlm.nih.gov
2. Clustal O alignment www.ebi.ac.uk/Tools/msa/clustalo/
3. Identify common or unique target regions
4. PrimerQuest® Tool www.idtdna.com/scitools
5. NCBI Blast http://blast.ncbi.nlm.nih.gov/Blast.cgi
6. OligoAnalyzer® Tool—for analysis of hairpins/dimers www.idtdna.com/scitools

4. 3
General Design Considerations

5. 4
Primer and Probe Design Criteria
 Primers:
 Tm: similar Tm (+/- 2°C), 60-62°C
 Length: 18-30 bases
 GC content: 35-65% (50% ideal), avoid runs of >4 G’s
 Sequence: avoid hairpins, dimers (self and hetero)
 Avoid SNPs (a single mismatch can alter Tm up to 8°C)
 Avoid non-specific primers
 Probe:
 Tm: 4-10°C higher than primers
 Length: <30bp for DLP, longer with ZEN™ (enhanced quenching)
 GC content: 30-80%, minimize runs of G
 Sequence: avoid G base at 5’ end
 Location: sense or antisense
 Amplicon:
 ~70-200bp

6. 5
Know your Gene
 Understand your gene of interest
 Transcript variants
 Exon organization
 SNP locations
 NCBI Gene database
Your gene of interest here
Tfrc

7. 6
Obtain Sequences in FASTA Format- NCBI Nucleotide
 Go to NCBI:

8. 7
Obtain Sequences in FASTA Format- NCBI Nucleotide

9. 8
Sequence Alignment (i.e., Clustal Omega)
 http://www.ebi.ac.uk/Tools/msa/clustalo/
 ClustalO

10. 9
Analyze Alignment Output to Determine Optimal Design Regions
Export alignment and
save as a word
document for easier
manipulation

11. 10
Designing to Avoid Genomic DNA Amplification
 Design primer across exon-exon junctions
 Design primers within 2 adjacent exons spanning a large intron
 DNase treatment to eliminate gDNA amplification
Decoded 1.3

12. 11
Design Strategy 1: qPCR assay to differentiate between 2 similar genes

13. 12
Sample Design: qPCR Assay to Distinguish RCI2A vs. 2B in Arabidopsis Thaliana

14. 13
1. Clustal O Sequence Alignment: RCI2A vs. RCI2B
1.
2.

15. 14
Target Sequence Entry into PrimerQuest®

16. 15
PrimerQuest® Assay Details:
• BLAST each primer pair for target specificity
• Check for SNP’s (if applicable/annotated, not necessary here)
• OligoAnalyzer- Check primers and probes for dimers/hairpins

17. 16
RCI2B Specific Design Strategy:

18. 17
PrimerQuest®
Tool
2. Enter Region of Interest into PrimerQuest® Tool

19. 18
PrimerQuest® Assay Details:

20. 19
PrimerQuest® Assay Details:

21. 20
qPCR Assay to Distinguish RCI2A vs. 2B in Arabidopsis Thaliana
• BLAST each primer pair for target
specificity, select highly specific assay
• Check for SNP’s (if applicable/annotated,
not necessary here)
• OligoAnalyzer- Check primers and probes
for dimers/hairpins
RCI2A:
Primer F: GAGAGCGTTGGTTTGTACTTTG Tm:62°C
Primer R: TGGTTAATGGTGGTCCTGT Tm: 62°C
Probe: TGGAAATTGTGTTGCCTTGGTGGA Tm: 68°C
RCI2B
Primer F: GGTTATCTTCCCGGAATCCTTTA Tm : 62°C
Primer R: AATCAGTCCCAAAGGGAGAAG Tm : 62°C
Probe: TTTCCTCTTGCTCCTCGAAGAACAGC Tm : 68°C

22. 21
Design Strategy 2: qPCR Assay to Distinguish Between 2 Homologous
Microbial Sequences

23. 22
Strain Specific qPCR Design for 2 Helicoverpa NPV Strains
Helicoverpa zea single nucleopolyhedrovirus strain—virus that infects earworm, which
feeds on plants/crops
Obtain sequences of interest from NCBI
 >Helicoverpa_zea
 CGCCCAAAAATAACGTACTTTTAAACTGGTCTTGGATCATTTCGTTCGAAACGGGCCGTGATCTTTTGTTTCGCTTCGTGACCCAAAAAAAACAAATTACGTCATCGACCAAA
GTAAAAATTCTTGCGCATGTTTAAACTAGTCTTGGATATTTTCGTTCGAAACGGGCCGTGATCTTTTGTTTCGCTTCGTGACCCAAAAAAACAAATTACGTCATTCGTTTAAAA
TATTGCATCATCTTTAAATTCGAAACCCGCCCGCGCTTTCATATGAAACCGTCGGCGAAGATCGATAAATTTTGTTCTAGAACGTTCGATGGTTTGACCCAAAAAACAAATGA
CGTCATATAGCGTGCGTCCAATCACAACACGAATCACGCCTTGTCTAAAGATAACATTTCCCGCGCATGTTTAAACTAATCTTGGATCTTTTCGTTCGAAACGGGCCGTGATC
TTTTGTTTCAATTCATGATTTAGAAAAAAACGAACATAAAATTTTACCGCGCATTTTTAAACTAGTGTTGGATTTTTTTTGTTTGAAACGAGCCGTGATCTTTTCGTTCGAAAC
GGGCCGTGATCTTTTCGTTCGAAACGGGCCGTGATCTTTTGTTTCGCTGACTCGTGACCCAAAAAAACAAATCACGTCATTCGTTTAGAATATTGCATCATCTTTAAATTCGA
AACTCGCCCGCGCTTTCATACGAAACCGCCGGCAAAGATCGGTAAAATTTGTTCTAGAACTTTCCACGGCTTGACCCAAAAAAACAAATGACGTCATATGGCGTGATTTTAA
ATCTATTTAATCGTCTCTGGCGTACAAAAGTAAATTACACACGAAACGTGCCATGTTAAGTTTGTTTACAATGAAACTGATTGTGTCGATTTTAATATGGACATAAGATTTTT
GCAAAAAAATTCCATTAATCGAACGAATGCGACAATAAACAGTTCGTTTGTTATACCAAATCGAAATGCGTTTGTATATTATTCACAATCCATCAATTCAAAACATGCCTCGT
CGACGTCGTTCGCGTACGCATAATTATAATGATCGAACAATTGTTTCAATGAAGTGAAACCGGTT
 >Helicoverpa_armigera
 AACTGTCTGATCTTTGTTGAAACGGGCCGTGATCTTGTTCGACTCGTGACCAAAAAACAAATGACATCATCGACCAAAAATCCCGCGCATGTTTAAACTAGTCTTGGATCTTT
CGTTCAAAACATGACGTAATCTTTCGTTCTACTCGTGACCCAAAAAAACAAATTACGTCATTTGTTTAAATTATTGCATCATCTTTAAATTCAAAACTCGCCCGCGCTTTCATAT
AAAACCGTCGGCGAAGATCGATAAAATTTGTTTTAGAACATTCCACGGCTTGACCCAAAAAAACAAATGACGTCATATAGCGTGATTTGAAAATCGTCCAATCACAACACGA
ATCACGCCTTGTCTAAAGATAACATTTCCCGCGCATGTTTAAAATAGTCTTGGATCTTTTCGTTCGAAACGGGCCGTGATCTTTTGTTTCGACTTATGATTTAGAAAAAAACG
AACATAAAATTTTACCGCGCATTTTTAAACTAGTCTAGGATCTTTTCGTTCAAAACGGGCCGTAATCTTTTGTTCAAAACGGGCCGTAATCTTTTCGTTCGAAACGGGCCGTG
ATCTTTTGTTTCGCTGACTCGTGACCCAAAAAAACAAATCACGTCATCCGTTTAGGATATTGCATCATCTTTAAATTCAAAACCCGCCCGCGCTTTCATATGAAACCGTCGGC
AAAGATCGGTAAAATTTGTTCTAGAACGTTCCACGGCTTGACCCAAAAAACAAATGACGTCATATGGCGTTTAATCAATCTTTGGCGTACAAAAGTAAATTACACACGAAAC
GTGCCATGTTAAGTTTGTTTACAATGAAACTGATTGTGTCGATTTTAATATGGACATAAGATTTTTGCAAAAAAATTCCATTAATCGAACGAAAGCGACAATAAACAGTTCGT
TTGTTATACCAAATCGAAATACGTTTGTATATTATTCACAATCCATCAATTCAAAACATGCCTCGTCGACGTCGTTCGCGTACGCATAATTATAATGATCGAACAATTGTTTCA
ATGAAGTGAAACCGGTT

24. 23
qPCR Assay to Distinguish Related Viral Strains
11

25. 24
Input the Targeted Design Area into PrimerQuest® Tool
PrimerQuest®
Tool

26. 25
Adjust Parameters for qPCR (Probe Assay)

27. 26
Use the Custom Design Parameters to Target Probe Area
Target the
probe region
using the
Excluded
Region List

28. 27
Analyze Potential Assays:
• BLAST each primer pair for target
specificity, select highly specific
assay
• Check for SNP’s
• OligoAnalyzer- Check primers and
probes for dimers/hairpins

29. 28
Probe Specificty- Amigera Strain Specific Design
TGGCGTGATTTTAAATCTATTTAA
|||| | ||| ||||||
TGGCGTTTAATCAATCTTTGGCGT
Probe mismatch:
Probe won’t be able to bind

30. 29
Repeat Process to Obtain Zea Strain Specific Design
Zea (top sequence)
Identify unique target region for design

31. 30
Analyze Potential Assays

32. 31
Zea Strain Specific Design
In this example, the probe again won’t bind, but also the forward primer has multiple mismatches
• BLAST each primer pair for target
specificity, select highly specific
assay
• Check for SNP’s
• OligoAnalyzer- Check primers and
probes for dimers/hairpins

33. 32
Copy Number Variation (CNV) Assays

34. 33
Designing Assays for Copy Number Variation
Estivill and Armengol, (2007) PLOS Genetics
 Copy Number Variations (CNVs) are important polymorphisms that can influence
the expression of genes within and close to a rearranged region.
 This allows for transcription levels to be higher or lower than those that can be
achieved by control of transcription of a single gene copy.
 CNVs are being associated more and more with genetic diseases such as cancer,
neurological disorders, and immune diseases.
 PrimeTime® qPCR Assays can be designed to specifically evaluate the copy number
of genomic DNA targets.

35. 34
Important Considerations for CNV Designs
 1. Design an assay that is within a single exon of the gene of interest
 Obtain sequence information for a single exon in NCBI Nucleotide
 By accession number or BLAST
 Exon information also available in NCBI Gene

36. 35
• BLAST each primer pair for
target specificity, select
highly specific assay
• Use OligoAnalyzer® Tool to
check primers and probes
for dimers/hairpins
• Check for SNPs
Input Sequence for a Single Exon Using PrimerQuest® Tool

37. 36
Single Copy Reference- Important for CNV Assays
Commonly used examples:
 Human- RNaseP
Primer F: AGATTTGGACCTGCGAGCG
Primer R: GAGCGGCTGTCTCCACAAGT
Probe: 5’Hex/TTCTGACCT/ZEN/GAAGGCTCTGCGCG/3IABkFQ/
 Mouse- TFRC (or TERT)
Primer F: CTAAGTCTACAGTGGCTGTATTCC
Primer R: GATCATTGATTTCCCTCATGACAAA
Probe: /5HEX/TCGTGGAGA/ZEN/CTACTTCCGTGCTACT/3IABkFQ

38. 37
 Questions?