qpcr design strategies for specific applications
DESCRIPTION
Species-Specific, Strain-Specific, and CNV Assay Design ConsiderationsTRANSCRIPT
![Page 1: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/1.jpg)
Integrated DNA Technologies
Elisabeth WagnerScientific Applications Specialist
qPCR Design Strategies for Specific ApplicationsSpecies-Specific, Strain-Specific, and CNV Assay Design Considerations
![Page 2: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/2.jpg)
2
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
![Page 3: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/3.jpg)
3
General Design Strategy Outline for Specific qPCR Design Parameters:
1. NCBI- sequence accession www.ncbi.nlm.nih.gov2. Clustal O alignment www.ebi.ac.uk/Tools/msa/clustalo/3. Identify common or unique target regions4. PrimerQuest® Tool www.idtdna.com/scitools5. NCBI Blast http://blast.ncbi.nlm.nih.gov/Blast.cgi 6. OligoAnalyzer® Tool—for analysis of hairpins/dimers www.idtdna.com/scitools
![Page 4: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/4.jpg)
4
General Design Considerations
![Page 5: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/5.jpg)
5
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
![Page 6: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/6.jpg)
6
Know your Gene
Understand your gene of interest Transcript variants Exon organization SNP locations
NCBI Gene database
Your gene of interest here
Tfrc
![Page 7: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/7.jpg)
7
Obtain Sequences in FASTA Format- NCBI Nucleotide
Go to NCBI:
![Page 8: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/8.jpg)
8
Obtain Sequences in FASTA Format- NCBI Nucleotide
![Page 9: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/9.jpg)
9
Sequence Alignment (i.e., Clustal Omega) http://www.ebi.ac.uk/Tools/msa/clustalo/ ClustalO
![Page 10: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/10.jpg)
10
Analyze Alignment Output to Determine Optimal Design Regions
Export alignment and save as a word document for easier manipulation
![Page 11: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/11.jpg)
11
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
![Page 12: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/12.jpg)
12
Design Strategy 1: qPCR assay to differentiate between 2 similar genes
![Page 13: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/13.jpg)
13
Sample Design: qPCR Assay to Distinguish RCI2A vs. 2B in Arabidopsis Thaliana
![Page 14: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/14.jpg)
14
1. Clustal O Sequence Alignment: RCI2A vs. RCI2B
1.
2.
![Page 15: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/15.jpg)
15
Target Sequence Entry into PrimerQuest®
![Page 16: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/16.jpg)
16
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
![Page 17: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/17.jpg)
17
RCI2B Specific Design Strategy:
![Page 18: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/18.jpg)
18
PrimerQuest®Tool
2. Enter Region of Interest into PrimerQuest® Tool
![Page 19: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/19.jpg)
19
PrimerQuest® Assay Details:
![Page 20: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/20.jpg)
20
PrimerQuest® Assay Details:
![Page 21: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/21.jpg)
21
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
RCI2BPrimer F: GGTTATCTTCCCGGAATCCTTTA Tm : 62°C
Primer R: AATCAGTCCCAAAGGGAGAAG Tm : 62°C
Probe: TTTCCTCTTGCTCCTCGAAGAACAGC Tm : 68°C
![Page 22: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/22.jpg)
22
Design Strategy 2: qPCR Assay to Distinguish Between 2 Homologous Microbial Sequences
![Page 23: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/23.jpg)
23
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 CGCCCAAAAATAACGTACTTTTAAACTGGTCTTGGATCATTTCGTTCGAAACGGGCCGTGATCTTTTGTTTCGCTTCGTGACCCAAAAAAAACAAATTACGTCATCGACCAA
AGTAAAAATTCTTGCGCATGTTTAAACTAGTCTTGGATATTTTCGTTCGAAACGGGCCGTGATCTTTTGTTTCGCTTCGTGACCCAAAAAAACAAATTACGTCATTCGTTTAAAATATTGCATCATCTTTAAATTCGAAACCCGCCCGCGCTTTCATATGAAACCGTCGGCGAAGATCGATAAATTTTGTTCTAGAACGTTCGATGGTTTGACCCAAAAAACAAATGACGTCATATAGCGTGCGTCCAATCACAACACGAATCACGCCTTGTCTAAAGATAACATTTCCCGCGCATGTTTAAACTAATCTTGGATCTTTTCGTTCGAAACGGGCCGTGATCTTTTGTTTCAATTCATGATTTAGAAAAAAACGAACATAAAATTTTACCGCGCATTTTTAAACTAGTGTTGGATTTTTTTTGTTTGAAACGAGCCGTGATCTTTTCGTTCGAAACGGGCCGTGATCTTTTCGTTCGAAACGGGCCGTGATCTTTTGTTTCGCTGACTCGTGACCCAAAAAAACAAATCACGTCATTCGTTTAGAATATTGCATCATCTTTAAATTCGAAACTCGCCCGCGCTTTCATACGAAACCGCCGGCAAAGATCGGTAAAATTTGTTCTAGAACTTTCCACGGCTTGACCCAAAAAAACAAATGACGTCATATGGCGTGATTTTAAATCTATTTAATCGTCTCTGGCGTACAAAAGTAAATTACACACGAAACGTGCCATGTTAAGTTTGTTTACAATGAAACTGATTGTGTCGATTTTAATATGGACATAAGATTTTTGCAAAAAAATTCCATTAATCGAACGAATGCGACAATAAACAGTTCGTTTGTTATACCAAATCGAAATGCGTTTGTATATTATTCACAATCCATCAATTCAAAACATGCCTCGTCGACGTCGTTCGCGTACGCATAATTATAATGATCGAACAATTGTTTCAATGAAGTGAAACCGGTT
>Helicoverpa_armigera AACTGTCTGATCTTTGTTGAAACGGGCCGTGATCTTGTTCGACTCGTGACCAAAAAACAAATGACATCATCGACCAAAAATCCCGCGCATGTTTAAACTAGTCTTGGATCTT
TCGTTCAAAACATGACGTAATCTTTCGTTCTACTCGTGACCCAAAAAAACAAATTACGTCATTTGTTTAAATTATTGCATCATCTTTAAATTCAAAACTCGCCCGCGCTTTCATATAAAACCGTCGGCGAAGATCGATAAAATTTGTTTTAGAACATTCCACGGCTTGACCCAAAAAAACAAATGACGTCATATAGCGTGATTTGAAAATCGTCCAATCACAACACGAATCACGCCTTGTCTAAAGATAACATTTCCCGCGCATGTTTAAAATAGTCTTGGATCTTTTCGTTCGAAACGGGCCGTGATCTTTTGTTTCGACTTATGATTTAGAAAAAAACGAACATAAAATTTTACCGCGCATTTTTAAACTAGTCTAGGATCTTTTCGTTCAAAACGGGCCGTAATCTTTTGTTCAAAACGGGCCGTAATCTTTTCGTTCGAAACGGGCCGTGATCTTTTGTTTCGCTGACTCGTGACCCAAAAAAACAAATCACGTCATCCGTTTAGGATATTGCATCATCTTTAAATTCAAAACCCGCCCGCGCTTTCATATGAAACCGTCGGCAAAGATCGGTAAAATTTGTTCTAGAACGTTCCACGGCTTGACCCAAAAAACAAATGACGTCATATGGCGTTTAATCAATCTTTGGCGTACAAAAGTAAATTACACACGAAACGTGCCATGTTAAGTTTGTTTACAATGAAACTGATTGTGTCGATTTTAATATGGACATAAGATTTTTGCAAAAAAATTCCATTAATCGAACGAAAGCGACAATAAACAGTTCGTTTGTTATACCAAATCGAAATACGTTTGTATATTATTCACAATCCATCAATTCAAAACATGCCTCGTCGACGTCGTTCGCGTACGCATAATTATAATGATCGAACAATTGTTTCAATGAAGTGAAACCGGTT
![Page 24: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/24.jpg)
24
qPCR Assay to Distinguish Related Viral Strains
11
![Page 25: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/25.jpg)
25
Input the Targeted Design Area into PrimerQuest® Tool
PrimerQuest®Tool
![Page 26: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/26.jpg)
26
Adjust Parameters for qPCR (Probe Assay)
![Page 27: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/27.jpg)
27
Use the Custom Design Parameters to Target Probe Area
Target the probe region using the Excluded Region List
![Page 28: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/28.jpg)
28
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
![Page 29: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/29.jpg)
29
Probe Specificty- Amigera Strain Specific Design
TGGCGTGATTTTAAATCTATTTAA |||| | ||| |||||| TGGCGTTTAATCAATCTTTGGCGT
Probe mismatch:Probe won’t be able to bind
![Page 30: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/30.jpg)
30
Repeat Process to Obtain Zea Strain Specific DesignZea (top sequence)
Identify unique target region for design
![Page 31: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/31.jpg)
31
Analyze Potential Assays
![Page 32: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/32.jpg)
32
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
![Page 33: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/33.jpg)
33
Copy Number Variation (CNV) Assays
![Page 34: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/34.jpg)
34
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.
![Page 35: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/35.jpg)
35
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
![Page 36: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/36.jpg)
36
• 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
![Page 37: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/37.jpg)
37
Single Copy Reference- Important for CNV Assays
Commonly used examples: Human- RNasePPrimer F: AGATTTGGACCTGCGAGCGPrimer R: GAGCGGCTGTCTCCACAAGTProbe: 5’Hex/TTCTGACCT/ZEN/GAAGGCTCTGCGCG/3IABkFQ/
Mouse- TFRC (or TERT)Primer F: CTAAGTCTACAGTGGCTGTATTCCPrimer R: GATCATTGATTTCCCTCATGACAAAProbe: /5HEX/TCGTGGAGA/ZEN/CTACTTCCGTGCTACT/3IABkFQ
![Page 38: qPCR Design Strategies for Specific Applications](https://reader033.vdocument.in/reader033/viewer/2022061103/5409edd58d7f72e54b8b45c3/html5/thumbnails/38.jpg)
38
Questions?