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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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
Higher Coupling Efficiency, More Full-Length Product
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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
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INTEGRATED DNA TECHNOLOGIES
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
Consistent reduced background from ZEN and TAO
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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
25 µM Forward Primer 2 500 nM
25 µM Reverse Primer 2 500 nM
12.5 µM Probe 250 nM
25 µM Forward Primer 3 500 nM
25 µM Reverse Primer 3 500 nM
12.5 µM Probe 250 nM
25 µM Forward Primer 4 500 nM
25 µM Reverse Primer 4 500 nM
12.5 µM Probe 250 nM
Immolase polymerase 0.8 U
H2O ----
Template
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
FREE 1ml Samples available!!
PrimeTime® Gene Expression Master Mix
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
Exceptional Stability Reproducible amplification after heating the Master Mix at 55°C (4 or 8h)
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
• 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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
Custom xGen® Lockdown® Probes are shipped within 2 weeks
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IDT turnaround time
Competitors’ turnaround time
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INTEGRATED DNA TECHNOLOGIES
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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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INTEGRATED DNA TECHNOLOGIES
Target capture can be completed in 1 day with xGen® Lockdown®
Probes and Reagents
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INTEGRATED DNA TECHNOLOGIES
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
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INTEGRATED DNA TECHNOLOGIES
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INTEGRATED DNA TECHNOLOGIES
IDT: Your commercialization partner
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GMP Oligo and Contract Manufacturing Services
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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