Yong You MS, Associate Product Manager Integrated DNA...

Integrated DNA Technologies

PrimeTime and qPCR

Yong You MS, Associate Product Manager

IDT Confidential: This document may contain confidential and proprietary information. Any unauthorized review, use, disclosure or distribution is prohibited

Webinar for Hong Kong University, March 29th, 2011

Overview

Basics of qPCR

SYBR vs Hydrolysis Probes

PrimeTime vs MGB TaqMan Assays

IDT Pre-designed PrimeTime Assays

IDT ZEN Double Quenched Probes

IDT SciTools

BASICS OF qPCR

Real Time quantitative PCR

• The Polymerase Chain Reaction (PCR) technique was invented in 1985 by Dr. Kary B. Mullis

• Real Time PCR was Developed by Perkin Elmer in 1995.

• Mechanism: Fluorescent signal increase proportionally to the amplicon increase.

Difference between traditional PCR and qPCR

PCR - Gel based analysis at the end of amplification in the plateau phase

qPCR - Fluorescent measurements during the entire amplification cycle

qPCR is THE method of choice for quantitative gene expression analysis

Real TimeNo. of DNA molecules 2500 500 100 20

4

Traditional PCR detection

RT-PCR

Threshold

Cycle No. 15 20 25 30 35

Plateau

Results of a qPCR assay

• Cycle number (Cq) reflects number of copies of starting template DNA

Early cycle number -----more DNA

Late cycle number -----less DNA

*

• Fluorescence is a property of a class of molecules

• Absorb light energy at a short wavelength

• Release this light energy at a longer wavelength

• Length of time between absorption and emission is 10-8 to 10-9s

SO

S1’

S1

hνex hνem

1

2

3

Brought to you by the phenomenon of

Fluorescence!

Release of energy as fluorescence

Fluorescence （熒光現象）

Types of qPCR

1 2

3 4

1 2

1 2 31 2

Intercalators

(Sybr Green)

Hybridization FRET

Molecular Beacon5’ Nuclease assay,

Prime Time Assay,

Taqman assay

cgctacc

Fam

494/518 IablkFqZ

SYBR is a fluorescent dye not linked to a sequence,all other probes have the fluorescent dye attached to certain nucleic acid

熒光染料 : SYBR Green, EvaGreen

熒光探針 : FRET（熒光共振能量轉移）Molecular Beacon（分子信標）

5’ Nuclease Assay

（ 5’ 水解酶分析技術）

5’ nuclease qPCR assay

5’ 3’3’ 5’

a c g a c g a

3’ 5’Primer/oligo

t c g a t c g a t c g a t a g g c c a t c g c g a c t c c t t g c t g c t

gctagct


gcta ccc

R Q

aa ta gct

a g c t a g c t a g c t a t c c g g t a g c g c t g

3’ 5’

a g g a a c g a g ac

t tt tt tt t t

12

3

95C

65C

During amplification, the 5‘ to 3’ exonuclease activity of polymerase will hydrolyze the probe, separating the fluorophorfrom the quencher, the detection system will detect fluorescence signal. Each cycle, more DNA is made, more signal is generated. This is the basis of qPCR quantification.

PCR amplification and copy number

Real-time qPCR general design rules

• Good primer and probe design

• Check qPCR instruments compatibility with dyes

• Use a hot-start, high-fidelity enzyme

• Additional considerations for multiplex experiments• Especially in assay design and choices of dyes

• Determine controls (both positive and negative)

Instrument Compatibility with Reporter

Dyes

Multiplex qPCR

Primer and Probe design criteria for PrimeTime

Primers

equal Tm (60-64 C)

18-30 bases in length

no runs of consecutive Gs

Probes

Tm value 6-10C higher than primers

no longer than 30-35 bases

no runs of consecutive Gs

No Gs on the 5’ end

Designed to limit genomic DNA amplification—How?

Exon Exon Exon Exon1 2 3 4

intron intron(1000bp) intron

DNA

1 2 3 4RNA(immature)

Transcription

Splicing

(amplicon is large)

• Assays are designed to span exons

• Exon – Exon junctions maybe targeted

• Primers could span an intron greater than 1 kb

(amplicon is small and specific)

Mature RNA1 2 43

IDT SciTools

IDT offers a variety of design and analysis tools. The SciTools suite includes tools for designing assay components as well as determining the properties of any sequence entered.

OligoAnalyzerProvides accurate analysis of melting temperature

PrimerQuestCustom design of primers and probes for PCR and qPCR reactions

UNAFoldProvides analysis of secondary structures

RealTime PCR DesignqPCR assay design based on exon boundaries

IDT SciTools

SciTools – RealTime PCR

IDT recommends using the RealTime PCR design tool located on the IDT website to ensure all of the important parameters will be included in the assaydesign.

SYBR GREEN VS HYDROLYSIS PROBES

Real Time qPCR Method of Detection

cDNA+

Water, Buffer, Primers, dNTPs, Enzyme, MgCl2 (MASTERMIX)+

Method of detection

cgctacc

R Q

Probe (PrimeTime Assays)

R= reporter/fluorophore,Q=quencher

PrimeTime assays have adual labeled probeand is a superior method of detection

SYBR Green OR(fluorescentIntercalating dye)

SYBR – non-sequence specific

550CPrimers anneal, dsDNASYBR Green intercalates and fluoresces

720CAs more dsDNA is synthesizedIncreased fluorescence is observed

940CDNA is denatured to ssDNA formNo SYBR green binding

(SYBR Green only intercalates with dsDNA)

Method of detection: SYBR Green (494nm/520nm)

SYBR Green Summary

Advantages: Inexpensive (as do not require a dye-labeled probe)

General screening prior to moving to probe based assays

OK when the target is abundant

Disadvantages: Not sequence specific: bind to primer-dimers and incorrect amplicon

Requires melting curve analysis

Cannot perform multiplex reaction

Cannot be used for Allelic Discrimination

PRIMETIME VS TAQMAN MGB ASSAYS

IDT PrimeTime qPCR Products

PrimeTimeTM qPCR Assays Gene expression PrimeTime Pre-designed Assays PrimeTime qPCR Assays

PrimeTimeTM qPCR Probes Gene Expression Copy Number Estimation

PrimeTimeTM LNA Probes Genotyping

Components of a PrimeTimeTM Assay

Each PrimeTimeTM Assay consists of two PCR primers and a dual labeled PrimeTimeTM probePre-mixed in a tube primer probe ratio = 2:1(Mini), 1:1 (Std) and 4:1 (XL)

gctagct


a c g a c g a


cgctacc

R Q

Probe

R= reporterQ=quencher

+Z

PrimeTime Assay include IDT’s proprietary ZEN Double Quenched Probes (DQP).The DQPs are linear probes, in addition to the 5’ fluorophor and 3’ quencher, they also containan internal ZEN quencher.

PrimeTime Assays

5' Dye 3' Quencher Mini Standard XL

FAM ZEN/Iowa Black FQ* • • •

FAM TAMRA • •

HEX Iowa Black FQ • •

TET Iowa Black FQ • •

Cy5 Iowa Black RQ • •

Available Dye and Quencher Combinations for PrimeTime qPCR AssaysPrimer/probe ratios from 1:1 to 4:1(primer concentration varies, probe is fixed)

PrimeTime Assay Performance = Gold Standard Performance

Efficiency 101.1%Std Curve R2 0.9996

Gene CCL5Copies shown 10 to 1E7Master Mix ABI MMxInstrument ABI7900

NTC

10 copies

First 4 assays run on ABI machine, the Cy5 assay is run on theRoche LC480

Performance of Assays with different dye /quencher

0 Freeze - Thaws 15 Freeze - Thaws 30 Freeze - Thaws

Stability Test for PrimeTime Assays

PrimeTimeTM for gene expression

PrimeTime qPCR probes and assays can be used to study gene expression.

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product.

Protein-coding genes

Non-protein coding genes:ribosomal RNA (rRNA) genes transfer RNA (tRNA) genes

proteins

functional RNA

PrimeTime qPCR for Copy Number Estimation

Copy Number Estimation – using techniques such as real time qPCR to estimate the copy number of certain genes of interest, this will require an internal standard

Copy-number variations (CNVs): Alterations of genomic DNA that have been deleted or amplified on certain chromosomes

PrimeTime qPCR probes can be used to study Copy Number Variations.

PrimeTimeTM LNA probes for Genotyping

Genotyping (Allelic discrimination): determining the genotype of an individual by examining its DNA sequence using molecular techniques

The Allelic Discrimination assay detects:• Homozygotes

(samples having only allele 1 or allele 2)• Heterozygotes

(samples having both allele 1 and allele 2)

PrimeTime qPCR LNA probes can be used to study allelic discrimination.

Allelic discrimination assay

• Multiplexed assay to detect variants of a single nucleic polymorphism (SNP)

For each sample in an AD assay, a unique pair of probes is used, for example, two PrimeTime LNA probes that target the same SNP site. • 1 probe is a perfect match to the wild type (allele 1) • 1 probe is a perfect match to the mutation (allele 2)

TaqMan MGB probes by ABI (MGB = Minor groove binder)

These probes are sold exclusively by ABI because they have the license for MGB

• MGB clamps the probe and stabilizes the probe onto the target

• MGB raises the Tm by as much as 10oC

• Probes can be shorter than traditional dual labeled probes, but still maintain Tm.

The MGB is attached in addition to the standard fluorophore reporter such as FAM and quencher

http://nar.oxfordjournals.org/content/vol28/issue2/images/large/gkd15604.jpeg

If MGB probe sequences were used for PrimeTime…

Probe Tm has to be 6 to 10oC higher than primers

Please lengthen the probe flanking sequence to increase Tm

Length of MGB probes likely less than 18 bpWithout MGB Tm is going to be low at 45-54oC(Please check with IDT OligoAnalyzer, Mg2+ 3 mM, dNTPs 0.8mM)

How to find PrimeTime products on IDT website?

IDT PRE-DESIGNED PRIMETIME ASSAYS

Introducing PrimeTime Pre-designed qPCR Assays

Pre-designed assays are available for human, mouse and rat Designed to span every exon boundary in the transcriptome

Current coverage rate for human is 97.4% of exon junctions

Assays are designed to Avoid SNPs locations

Eliminate non-specific amplification

Reduce impact from secondary structure formation

Simple ordering tool Filter to quickly view common or transcript specific assays

Sort by recommended assays

Select reaction size, dye-quencher, or primer to probe ratio

Group by species, gene symbol or refseq number for quick assay selection

Designing successful qPCR assays

1. Up-to-date transcript sequence information, including: exon boundaries

splice variants

SNP locations

2. Design of primers and probes, including: accurate melting temperature

elimination of non-specific amplicon generation

reduction of secondary structure formation

3. High quality oligos that reduce impurities and provide reproducible results over time

Transcript information is changing rapidly

Assay performance heavily relies on the quality of sequence information you are trying to target.

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# o

f SN

Ps

Number of SNPs in dbSNP

Identified

Validated

Next Generation Sequencing has significantly increased the number of SNPs and splice variants identified. Having up to date sequence information is critical to qPCR assay performance

April 200815M SNPs

Sept 201030M SNPs

IDT has expertise in oligo thermodynamics

For over ten years, IDT has dedicated a team to understanding oligo thermodynamics. With thousands of melt curves performed, IDT oligo melting temperature calculations are estimated to be the most accurate in the industry.

IDT continues innovative DNA thermodynamic research and integrates those improvements into its calculation and oligonucleotide design tools

Publications:Owczarzy R, Tataurov AV, Wu Y, Manthey JA, McQuisten KA, Almabrazi HG, Pedersen KF, Lin Y, Garretson J, McEntaggart NO, Sailor CA, Dawson RB and Peek AS

(2008) IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res., 36:163-169.

Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA (2008) Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry, 47: 5336-5353. Additional data

Tataurov AV, You Y, Owczarzy R (2008) Predicting ultraviolet spectrum of single stranded and double stranded deoxyribonucleic acids. Biophys. Chem., 133: 66-70.

You Y, Moreira BG, Behlke MA, Owczarzy R (2006) Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res., 8: 60.

Owczarzy R (2005) Melting temperatures of nucleic acids: Discrepancies in analysis. Biophys. Chem., 117: 207-215.

Moreira BG, You Y, Behlke MA, and Owczarzy R (2005) Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability. Biochem. Biophys. Res. Comm., 327: 473-484

Owczarzy R, You Y, Moreira B, Manthey JA, Huang L, Behlke MA, and Walder JA (2004) Effects of sodium ions on DNA duplex oligomers: Improved predictions of melting temperatures. Biochemistry, 43: 3537-3554

Owczarzy R, Dunietz I, Behlke MA, Klotz IM, and Walder JA (2003) Thermodynamic treatment of oligonucleotide duplex-simplex equilibria. Proc. Natl. Acad. Sci. USA, 100: 14840-14845

Pre-designed Validation Results

Validation Highlights

More than 99% of assays exhibited >90% efficiency

100% of the assays had R2 values >0.99

Mean efficiency across all 150 assays was 94.5%

Over 2/3rds of the assays had efficiencies >94%

Example Dilution Series Results

Comparison to competitor pre-designed assays

25 assays from Competitor A were compared to an equal number of IDT’s PrimeTime Pre-designed qPCR Assays.

The Competitor A assays consisted of 15 inventoried pre-designed assays and 10 made-to-order pre-designed assays.

To ensure an accurate comparison was made, the PrimeTime Assays and Competitor A assays were selected to span the same exon boundary of each gene.

The reactions were run with the Applied Biosystems Gene Expression Master Mix and identical thresholds were set for all runs

qPCR Efficiency

PrimeTime Pre-designed qPCR Assays Have Higher qPCR Efficiency and a Smaller Distribution Range than Competitor A Assays.

PrimeTime qPCR Assays were compared to matched Competitor A assays using 5-fold dilutions of cDNA and the Applied Biosystems TaqMan™ Gene Expression Master Mix. The reactions were run on the ABI 7900HT Fast Real-Time PCR System with the following PCR cycling conditions: 2 min. 50°C;

10 min. 95°C; 45 x (15 sec. 95°C, 1 min. 60°C). Identical thresholds were set for all runs for comparison across assays.

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88 - 90 90 - 92 92 - 94 94 - 96 96 - 98 98 - 100

Fre

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en

cy

qPCR Efficency (%)

Comparison of qPCR Efficiency

PrimeTime

Competitor A

Example qPCR curves

PrimeTime Competitor A

Example curves pre-baselined

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Rn

(F

AM

/RO

X)

Cycle Number

Comparison of Assay Performance with WDR3 (NM_006784)

■ PrimeTime Assays

■ Competitor A

PrimeTime Cq’s are nearly 1 full Cq lower than Comp. A

0

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Me

an

Cq

Va

lue

Gene RefSeq Accession Number

Comparison of Mean Cq Value

PrimeTime

Competitor A

PrimeTime Assays excel with fast-cycling, Brilliant III

Locating the pre-Designed qPCR page

Search PrimeTime Pre-designed for RefSeq Number

Enter the RefSeq Number

Click “Submit”

www.idtdna.com/order/predesignedassay.aspx

Step 5: Select the Same Exon Boundary

Select Exon Boundary

IDT ZEN DOUBLE QUENCHED PROBES

Requires overlap of donor emission spectra and acceptor absorption spectra andphysical proximity

Donor Acceptor Ro (Å)

Fluorescein Tamra 58

Fluorescein BHQ-1 60

Fluorescein BHQ-2 61

Fluorescein IBRQ 45

Fluorescein IBFQ 60

Fluorescein Dabcyl 46

Fluorescein Eclipse 60

Dual labeled probes: To capture fluorescence emitted by reporter dye

Proximity: Förster Radii

5’-Fluorophore --------- 3’-Quencher

• Force reporter and quencher into proximity (Molecular Beacons, etc)

• Make probes shorter (even with LNA or MGB, it is hard to get probes much shorter than 14-18 bases)

5’-Fluorophore --------- Internal Quencher

• Place quencher internally on dT base (need a “T” nearby)

• Place quencher between bases (destabilizing Tm effect?)

Internal Quenching has many benefits, if the problem of destabilization can be solved

Methods to improve Quenching

Modification DTm

ZEN + 3.8oC

BHQ2 - 0.6oC

Eclipse - 7.6oC

IBRQ - 8.6oC

C3 spacer - 8.7oC

S9 spacer - 19.4oC

All quenchers tested were destabilizing (lowered Tm) except ZEN

Insertion

Q

5’-ATCGTTGCTA-3’

3’-TAGCAACGAT-5’

^

Tm studies on Internal quenchers - insertion

Actin qPCR Assay FAM-AGCTCACCATGGATGATGATATCGCC-Q

Lowest background was seen with the ZEN-DQ probe

Note the difference of Cq values

Zen-FQ DQP Probes vs. Standard design

RAW Baseline Adjusted

RAW

Quencher placement makes a big difference

i8 – i10 best

i6 probably not being fully cleaved by Taq

Baseline Adjusted

Position Sequence HPRT qPCR assay

i12 FAM-ATGGTCAAGGT/ZEN/CGCAAGCTTGCTGGT-SpC3

i10 FAM-ATGGTCAAG/ZEN/GTCGCAAGCTTGCTGGT-SpC3

i8 FAM-ATGGTCA/ZEN/AGGTCGCAAGCTTGCTGGT-SpC3

i6 FAM-ATGGT/ZEN/CAAGGTCGCAAGCTTGCTGGT-SpC3

Optimization of Zen Assays

5’-dye i10-Quencher 3’-Quencher

New DQP design gives lower baseline fluorescence while maintaining good signal strength

BHQ does not tolerate internal placement as well this way

RAW Baseline Adjusted

New Double Quencher Probe (DQP) design

Using internal ZEN, the efficiency of quenching does not change as probes length increases

Zen-FQ design allows use of longer probes

Summary for Double Quencher Probes

1. Proximity of Fluorophore and Quencher are crucial for efficient quenching in linear DLPs.

2. Most approaches to place a quencher internally within a sequence destabilize duplex formation (decrease Tm).

3. New ZEN Quencher stabilizes the duplex and increases Tm.

4. Novel internal ZEN + 3’-FQ (Double-Quencher format) gives excellent balance of low background combined with high signal generation and leads to increased sensitivity.

IDT SciTools

IDT SciTools – Where are they located?

SciTools - OligoAnalyzer

SciTool - PrimerQuest

SciTools – RealTime PCR

QUESTIONS?

Yong You MS, Associate Product Manager Integrated DNA...

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