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

1.

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?