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Species-Specific, Strain-Specific, and CNV Assay Design Considerations
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Integrated DNA Technologies
Elisabeth WagnerScientific Applications Specialist
qPCR Design Strategies for Specific ApplicationsSpecies-Specific, Strain-Specific, and CNV Assay Design Considerations
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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
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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
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General Design Considerations
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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
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Know your Gene
Understand your gene of interest Transcript variants Exon organization SNP locations
NCBI Gene database
Your gene of interest here
Tfrc
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Obtain Sequences in FASTA Format- NCBI Nucleotide
Go to NCBI:
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Obtain Sequences in FASTA Format- NCBI Nucleotide
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Sequence Alignment (i.e., Clustal Omega) http://www.ebi.ac.uk/Tools/msa/clustalo/ ClustalO
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Analyze Alignment Output to Determine Optimal Design Regions
Export alignment and save as a word document for easier manipulation
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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
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Design Strategy 1: qPCR assay to differentiate between 2 similar genes
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Sample Design: qPCR Assay to Distinguish RCI2A vs. 2B in Arabidopsis Thaliana
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1. Clustal O Sequence Alignment: RCI2A vs. RCI2B
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2.
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Target Sequence Entry into PrimerQuest®
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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
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RCI2B Specific Design Strategy:
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PrimerQuest®Tool
2. Enter Region of Interest into PrimerQuest® Tool
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PrimerQuest® Assay Details:
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PrimerQuest® Assay Details:
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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
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Design Strategy 2: qPCR Assay to Distinguish Between 2 Homologous Microbial Sequences
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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
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qPCR Assay to Distinguish Related Viral Strains
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Input the Targeted Design Area into PrimerQuest® Tool
PrimerQuest®Tool
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Adjust Parameters for qPCR (Probe Assay)
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Use the Custom Design Parameters to Target Probe Area
Target the probe region using the Excluded Region List
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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
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Probe Specificty- Amigera Strain Specific Design
TGGCGTGATTTTAAATCTATTTAA |||| | ||| |||||| TGGCGTTTAATCAATCTTTGGCGT
Probe mismatch:Probe won’t be able to bind
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Repeat Process to Obtain Zea Strain Specific DesignZea (top sequence)
Identify unique target region for design
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Analyze Potential Assays
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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
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Copy Number Variation (CNV) Assays
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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.
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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
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• 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
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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
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Questions?