Increased Options for Design and Manufacture of Custom ...

Increased Options for Design and Manufacture of Custom Molecular Diagnostic Assays

Integrated DNA Technologies

The Custom Biology Company

Bob Setter, Sales Manager, Canada

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INTEGRATED DNA TECHNOLOGIES

About IDT • Founded in 1987 by Dr Joseph Walder

• Serves research and diagnostics life science markets

• Largest custom oligonucleotide manufacturer worldwide

• 82,000 active customers globally

• >830 employees in 5 locations

• 4 manufacturing sites

• Active & collaborative research group

• Average 44,000 oligos per day with 2500 shipments per day

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IDT Custom Nucleic Acid Manufacturing and QC

CHEMISTRY, PRODUCTION & DEVELOPMENT

ANALYTICAL CAPABILITIES

CustomerCare and

Tech Support

ENGINEERING

CUSTOM SYNTHETIC NUCLEIC ACIDS Core Business

Strategic Differentiators for Diagnostic Labs customers

CUSTOM SYNTHETIC NUCLEIC ACIDS *Highly consistent manufacturing

*Documented QC

RAPID GLOBAL

SHIPPING

IT

CUSTOMER INPUT USING WEB-BASED DESIGN, ANALYSIS

& ORDERING

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ISO Certification for all manufacturing • ISO 9001:2008 certification for Coralville, Belgium, San Diego and Singapore

manufacturing facilities • Automation Dependent

• Average 44,000 oligos manufactured per day (peak capacity appx 75,000 oligos per day)

• Emphasis on continuous improvement

• Mission is to provide quality research oligos in shortest TAT possible at market price

• ISO 13485:2003 certification for Coralville and Leuven • cGMP compliant under US FDA QSR 21 CFR Part 820 requirements

• Registered with the US FDA

• Services clients that use oligos in regulated/clinical applications requiring a higher level of process control

• ISO 14001 certification for Coralville • Continual improvement of environmental performance

• Enrichment of existing practices and resources managed by EHS

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Industry-Leading Quality Control and Traceability Throughout the Manufacturing Process

• Raw material QC testing on all incoming reagents

Only the highest quality products are accepted, stocked and used for synthesis.

• Manufacturing traceability

Record of reagents and equipment used in every synthesis

• Quality information

QC data available for every sequence

• Shipment traceability

Tubes are scanned individually for every shipment

• Informed CustomerCare

All manufacturing information readily available to address any customer questions

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Every Oligo Undergoes Quality Control (100% QC)

Capillary electrophoresis traces for oligos ≤60 bases

delivered with a purity guarantee The only oligo manufacturer to offer

electrospray ionization mass spectrometry for QC

Oligos that do not pass stringent QC requirements are resynthesized.

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Higher Coupling Efficiency, More Full-Length Product

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SciTools - Free Online Analysis and Design Tools

• qPCR and PCR

• Gene regulation and knockdown

• MicroRNA analysis

• Gene design

• Next Gen Sequencing

• Oligo dilution and resuspension Find these at www.idtdna.com/scitools.

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Application Support

• Team has doubled in size

• IDT will design your assays for you • qPCR

• rhPCR

• NGS

• Help develop parameters for multiplexing

• Design positive controls

• Assist with trouble shooting

• [email protected]

mailto:[email protected]

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Regular Dual-Labeled qPCR probes

ZEN and TAO Double-Quenched probes™ can be up to 45 bases

• Dyes Fam, Tet, Hex, Max, YAK or Joe • Internal Quencher ZEN • 3’quencher Iowa Black FQ

• Dyes Cy® 5

• Internal Quencher TAO • 3’quencher Iowa Black RQ

PrimeTime® qPCR ZEN & TAO double quenched probes

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Consistent reduced background from ZEN and TAO

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Fluors and Quenchers Fluors PrimeTime™ Assays FAM, HEX, TET, Cy5

*Yakima Yellow (alternate for VIC) PrimeTime™ Probes Joe, Max, Tye, Cy3, Texas Red, Tex 615 Other Dyes

*Atto dyes, (alternate for Alexa dyes)

Quenchers Zen Iowa Black FQ

*TAO Iowa Black RQ Iowa black FQ and RQ BHQ-1 and BHQ-2 Other quenchers

*MGB Eclipse Dabcyl-Molecular Beacons TAMRA- traditional quencher

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MGB Eclipse® Probe. The MGB enables the use or short (13–20 base) probes that improve allelic discrimination and targeting of AT-rich regions in qPCR assays.

MGB Eclipse® Probes and Primers

IDT is an alternate source for qPCR (5’ nuclease assay) probes incorporating a minor groove binder

Manufactured under GMP conditions (ISO 13485) for use in Human in vitro diagnostics only

• FAM, HEXTM, TETTM or Yakima Yellow®

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Super T® & Super G® bases

• Super T (5-hydroxybutynl-2’-deoxyuridine)

• duplex-stabilizing modified base that increases oligonucleotide Tm.

• useful modified base for designing short primers or probes for low-complexity, A-T

rich sequences

• Super G (8-aza-7-deazaguanosine)

• eliminates naturally occurring, non-Watson-and-Crick secondary structures

associated with guanine-rich sequences.

• Super G does not quench fluorophores, potentially improving probe performance

• Oligonucleotides containing Super G and T can be extended normally by

polymerases, including Taq polymerase,

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RNase H2-dependent PCR (rhPCR)

• Method for increasing PCR specificity

• RNase H2 from Pyrococcus abyssi (P.a.)

• Blocked primers that contain a single ribonucleotide residue

• Eliminates primer dimers

• Allows discrimination of highly similar targets

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RNase H2-dependent PCR (rhPCR)

• Primer deblocking is required for PCR, which in turn requires that primers be annealed to the target DNA sequence.

• The enzymatic deblocking cleavage event is highly sensitive to base mismatch and confers added specificity to the ensuing PCR reaction.

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LNA PrimeTime® Probes • Synthesis of the custom probes (10 - 25 nt) • Includes up to 6 LNA base insertions, reporter, quencher, and HPLC

• Mini LNA PrimeTime® Probes are available with a low normalized yield (0.5 nmole)

• FAM, HEX, and YAK fluors

• LNA and Mini LNA PrimeTime Probes are shipped in 4–6 business days

• *Fantastic performance on ddPCR platforms*

• contact IDT Technical Support for design assistance with SNP genotyping assays



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Synthetic gBlocks™ Template For Standard Curves

ACVR2B-LIMK1-ACVR1B-CDK7 wt

TCATACCTGCATGAGGATGTGCCCTGGTGCCGTGGCGAGGGCCACAAGCCGTCTATTGCCCA

CAGGGACTTTAAAAGTAAGAATGTATTGCTGAAGAGCGACCTCACAGCCGTGCTGGCTGACT

TTGGCTTGGtttttGAACATCATCCACCGAGACCTCAACTCCCACAACTGCCTGGTCCGCGA

GAACAAGAATGTGGTGGTGGCTGACTTCGGGCTGGCGCGTCTCATGGTGGACGAGAAGACTt

ttttGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCAC

TGCGGGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGtttttgGATGTATGGTGTAGGTG

TGGACATGTGGGCTGTTGGCTGTATATTAGCAGAGTTACTTCTAAGGGTTCCTTTTTTGCCA

GGAGATTCAGACCTTGATCAGCTAACAgcggccgc

• Separate sequences by a series of ttttt bases

• If cloning add in a restriction site for linearization of plasmid if necessary

A single gBlock used for 4 different standard curves

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gBlocks™ Gene Fragments as Quadruplex Standards

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

2.00E+06 2.00E+04 2.00E+02

Cq

Valu

es

Copies

G Block Standards

Hs LIMK1

Hs CDK7

Hs ACVR1B

Hs ACVR2B

Fourplex Reaction Conditions

Reagent Final Conc.

10X buffer 1X

100 mM dNTPs 800 nM

50 mM MgCl2 3 mM

25 µM Forward Primer 1 500 nM

25 µM Reverse Primer 1 500 nM

12.5 µM Probe 250 nM










Immolase polymerase 0.8 U

H2O ----

Template

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Using gBlocks™ Gene Fragments As Modified Standards

While both template sequences contain the primers and probe binding sites, by altering the length of one, the modified amplicon can be distinguished from the endogenous one.

Hs.PT.51.4056836 LIMK1

Hs LIMK1 Forward GAACATCATCCACCGAGACC

Hs LIMK1 Reverse AGTCTTCTCGTCCACCATGA

HS LIMK1 Probe CCAGCCCGAAGTCAGCCACC

Hs LIMK1 endogenous amplicon sequence

GAACATCATCCACCGAGACCTCAACTCCCACAACTGCCTGGTCCGCGAGAACAAGAATGTGGTGGTGGCTGACTTCGGGCTGGCGCGTCTCATGGTGGACGAGAAGACT

Hs LIMK1 endogenous amplicon sequence – 10 bases

GAACATCATCCACCGAGACCTCAACTCCCACAACTGCCTAACAAGAATGTGGTGGTGGCTGACTTCGGGCTGGCGCGTCTCATGGTGGACGAGAAGACT

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SYBR Green Dissociation Curve

By deleting or adding bases, a unique

standard can be used that is

distinguishable from the endogenous

sequence.

If you have trouble with contamination

you will always be able to tell the

standard from the endogenous

amplicon.

G Block (-10 bases)

G Block (endogenous )

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FREE 1ml Samples available!!

PrimeTime® Gene Expression Master Mix

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Probe-Based qPCR Master Mix

• 2X solution designed for use in two-step RT-qPCR

• Provides high efficiency qPCR under Fast or Standard cycling conditions

• Multiplex without loss of sensitivity

• Obtain consistent results from overnight experiments by capitalizing on exceptional

benchtop stability

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Exceptional Stability Reproducible amplification after heating the Master Mix at 55°C (4 or 8h)

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Mix designed for 2-step RT-PCR

For 2 step RT-PCR

• if analyzing gene expression RNA will need to be converted to cDNA prior to qPCR

• 2 step is beneficial to interrogate multiple genes

• Can also be used for direct analysis of DNA

• *Works well in presence of PCR inhibitors*

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cDNA (ng) DCq gBlocks DCq

50 -2.022 1.00E+07 -2.463

10 -2.169 1.00E+06 -2.675

2 -2.679 1.00E+05 -3.098

0.4 -2.386 1.00E+04 -3.096

0.08 -2.400 1.00E+03 -3.169

1.00E+02 -3.784

1.00E+01 -3.862

IDT MM - Life Tech MM

IDT MM vs. AB Gene Expression MM (HPRT)

Same threshold and same y-axis

IDT MM AB MM

IDT MM AB MM

cDN

A

gBlo

cks

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xGen® Lockdown® Probes

Target capture probe pools for NGS

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•5' biotin modified probes from 60-120 bases manufactured using our proprietary synthesis method with the highest coupling efficiency •Individually assessed by mass spectrometry for QC •Up to 2000 probes per pool; larger probe pools by request •Also available in individual wells (96 well plate) for you to mix and match

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• High uniformity allows capture of regions

of high or low GC content

• Uniformity is a product of:

• Individual probe synthesis using IDT

proprietary platform

• Equimolar pooling of individual probes

• Individual mass spec measurement

• Individual OD measurement

xGen® Lockdown® Probes provide more uniform coverage

than array-synthesized probes

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[Data courtesy of Genoptix, Inc.]

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xGen® Lockdown® Probes and Panels allow complete

flexibility in panel composition

• Supplement panels with additional xGen Lockdown Probes to capture more targets

• Combine panels to meet changing project needs

• Spike in probes to rescue areas that were poorly covered by array based capture methods

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Custom xGen® Lockdown® Probes are shipped within 2 weeks

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IDT turnaround time

Competitors’ turnaround time

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Hybridization and Wash Kit—xGen® Lockdown® Reagents •Achieve uniform coverage with hybridization and wash buffers optimized for use with xGen Lockdown Probes •Generate results quickly with a short, 4-hour hybridization protocol

Blocking Oligos—xGen® Universal Blocking Oligos •Effectively block multiple barcoded adapters using just one pair of blocking oligos •Improve on-target performance by reducing adapter-to-adapter hybridization •Illumina® dual- or single-index adapters •Ion Torrent® adapters •Increase flexibility in custom barcode design

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Target capture can be completed in 1 day with xGen® Lockdown®

Probes and Reagents

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RESEARCH ARTICLE F1000Research 2015, 4:1062 Last updated: 26 OCT 2015

ve-SEQ: Robust, unbiased enrichment for streamlined detection and whole-genome sequencing of HCV and other highly diverse pathogens [version 1; referees: 1 approved]

David Bonsall1*,

M. Azim Ansari1,2*, Camilla Ip3*, Amy Trebes3, Anthony Brown1, Paul Klenerman1,4, David Buck3, STOP-HCV Consortium, Paolo Piazza3, Eleanor Barnes1,4, Rory Bowden3

1Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, OX1 4BH, UK

2Oxford Martin School, University of Oxford, Oxford, OX1 4BH, UK

3Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX1 4BH, UK

4National Institute for Health Research Biomedical Research Centre, John Radcliffe Hospital, Oxford, UK

http://f1000research.com/articles/4-1062/v1




















































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IDT: Your commercialization partner

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GMP Oligo and Contract Manufacturing Services

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Meeting your specifications

• IDT offers a full range of final-fill, labeling, and

OEM solutions with services including:

• Customer-specified formulation

• Custom reagent formulations including buffers,

beads, master mixes and washes

• Custom packaging

• Third party labeling

• OEM and kitting solutions

• Plate packaging

• Stability programs

• Functional QC 40

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