high throughput oligonucleotide analysis using the combisep cepro 9600 system ™
DESCRIPTION
High Throughput Oligonucleotide Analysis Using the CombiSep cePRO 9600 System ™. www.combisep.com. Introduction. Massive quantities of ssDNA and ssRNA oligonucleotides are produced daily to support the growth of genomic-related applications - PowerPoint PPT PresentationTRANSCRIPT
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High Throughput Oligonucleotide Analysis Using the CombiSep cePRO 9600 System™
www.combisep.com
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Introduction
• Massive quantities of ssDNA and ssRNA oligonucleotides are produced daily to support the growth of genomic-related applications
• These DNA products need to be characterized to ensure proper sizing and acceptable batch-to-batch purity
• Traditional slab gel-based methods for characterizing DNA products are time-consuming, labor intensive, not readily automated, and provide relatively poor resolution
• While useful for assessing compound identity, Mass Spectrometry cannot quantitatively assess oligonucleotide purity due to size dependent variations in ionization efficiencies
• As a result, a bottleneck exists for characterizing the purity of ssDNA oligonucleotides
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Capillary Gel Electrophoresis (CGE)
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UV
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• Size-based separation of species possessing a constant mass-to-charge ratio (e.g., denatured SDS-protein complexes, ssDNA oligonucleotides, dsDNA)
• Separation medium is a gel-based sieving matrix; smaller sized species migrate faster
• Throughput for single capillary CGE methods range from 15 min – 100 min per sample; not adequate for high throughput quality control
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Principles of MCE-UV Operation
• UV light passing through the detection window of a 96-capillary array is imaged onto a linear photodiode array detector
• Capillary inlets are arranged 8 x 12 for direct injection from 96-well sample plates• Capillary outlets are bundled to a common reservoir enabling pressure or vacuum to be
applied to the array• Samples are separated by the application of a high voltage with optional vacuum flow• 96 individual CE separations are performed in parallel with simultaneous UV detection
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• CGE-UV is the method of choice for assessing oligonucleotide purity, providing higher resolution and superior quantification vs. slab gels.
• Label-free UV detection provides a low cost, toxic free alternative and improved quantification compared to fluorescent label approaches for ssDNA which may possess size and sequence dependent fluorescent labeling efficiencies
• CGE-UV advantages for characterizing oligonucleotide purity include low sample consumption, high resolving power, direct on-line UV detection and automated operation.
• A proprietary gel sieving matrix (Oligel) has been developed for multiplexed
operation. When analyzed at low M concentrations, single nucleotide resolution can be obtained from 10mer to 80mer oligonucleotide lengths, allowing identification of low level n-1, n-2, etc. impurities.
• Multiplexed CGE-UV provides a nearly 50-fold improvement in sample throughput over single capillary CE methods with a minimal loss in separation performance. A throughput of 96 samples/h is achieved for 80mer length oligonucleotides.
ssDNA Oligonucleotide Analysis by Multiplexed CGE-UV
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cePRO 9600™ System (CombiSep, Ames, IA USA)
• Second generation 96-capillary array CE instrument• Fixed wavelength UV or visible detection• Slide-out stage accommodates four 96-well plates (1 waste, 1 buffer, 2 samples)• System can be interfaced to a robotic arm for unattended well plate exchange
Automatic Tray Handler
Sample Tray #1
Sample Tray #2
Waste Tray
Buffer Tray
Front Access Panel
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Capillary Array Cartridge
LampHousing
HV Power Supply
Syringe Pump
Optical Platform Housing
Capillary Array Detection Window
Inside View of the cePRO 9600™ Instrument
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High Pressure Pump Option for Oligonucleotide Analysis on the cePRO 9600™ Instrument
Oligel MatrixCapillary Conditioning
Solution
A/B Switching Valve
• Pressures up to 400 psi can be applied to the capillary array
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96-Capillary Array Viewed from Detector Position
Capillary Outlets(12 Bundles of 8 Capillaries)
Detection Window(Polyimide coating removed)
Capillary Inlets(Arranged in 8 x 12 Format)
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Capillary Array Inlets Viewed from Below
• Direct injection by voltage from 96-well plates• Working injection volume typically 100 l
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Image of 96-Capillary Array on PDA Detector
• Continuous measurement of UV intensity simultaneously in all 96 capillaries• Absolute light intensity does not have to be equal as the relative absorbance is
measured in each capillary
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cePRO 9600 Specifications for Oligonucleotide Analysis
Detection • Fixed wavelength UV at 254 nm using mercury lamp and narrow band pass filter
Capillary Array Dimensions • 75 m i.d., 200 m o.d.• Effective lengths 40 cm to 55 cm; ~20 cm fixed length from detector to outlet
Sample Preparation• Typical oligonucleotide working concentration of 1-5 M for a standard EK injection
Sample Injection• Electrokinetic; 100 L working injection volume; -2 to -5 kV for 10 - 20 sec
Multiplexed CGE-UV Operational Conditions• Typical total operating current 0.4 – 0.6 mA (<6 A/capillary)• Field strength ~150 V/cm• Forced air capillary cooling at room temperature
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Contains proprietary mix of polymers and 8 M urea to fully denature oligonucleotides
Low UV background at 254 nm
Low current generation (< 5 A per capillary at 150 V/cm)
Self-coating of capillaries to reduce EOF
Low viscosity to facilitate faster pumping speeds
Single base resolution from 14 – 80mers
Relatively fast separation speed (80mers in ~1 h)
Features of Oligel™ Separation Matrix for Multiplexed CE-UV
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Sample: Desalted 14,15 - 19,20 – 29,30 – 39,40 – 59,60 – 79-80mers
Concentration: 1.5 µM (14-30mers); 0.1 µM (39-60mers); 0.2 µM (79,80mers)
CGE-UV: E = -170 V/cm; Sample injection = -5 kV, 10 s; UV = 254 nm
14,15mer
19,20mer
29,30mer
39,40mer
59,60mer
79,80mer
Separation Resolution of Oligel Sieving Matrix
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96-Capillary Separation of 14-80mers Using Oligel™ Matrix
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Analysis of Unmodified (Top) and Amine-modified (Bottom) 70mers
Sample: Desalted Unmodified, Amine-modified 70mers
Concentration: 5 µM Injection: -3 kV, 15 sCE Run: -140 V/cm, 70 min
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A:B=1:9
A:B=1:1
A:B=1:3
Separation of Various Ratios of Unmodified (A) to Amine-modified (B) 70mers
Sample: Desalted Unmodified, Amine-modified 70mers
Concentration: 5 µM Injection: -3 kV, 15 sCE Run: -140 V/cm, 70 min
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Sample: Desalted, Amine-labeled 70mersConcentration: 8 µMSample Injection: -3 kV, 10 secCE Run: -150 V/cm, 70 min
Multiplexed CE-UV Analysis of Amine-modified 70mers
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Impurity Screening of an Amine-modified 70mer Sample
• Approximate % Purity of main peak is 39.1%
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CombiSep and IDT Poster Co-Winners of Best Poster at Tides 2005 Conference
The poster titled “Comparisons between Multiplexed, Absorbance-Based Capillary Electrophoresis, Capillary Electrophoresis, and Ion Exchange Chromatography for Analysis of n-1 Oligonucleotide Impurities” by Wei Wei, Ho-ming Pang, Dennis Tallman, and Jeremy Kenseth of CombiSep, Inc and Lisa Bogh of Integrated DNA Technology was recently selected as the co-winner of the best poster at Tides 2005. The Tides Conference is an industry event for manufacturing and development of oligonucleotide and peptide products. The meeting was held May 1st – 5th, 2005 at the Boston Convention & Exhibition Center.
The poster award was sponsored by BioProcess International. The selection criteria was based on novelty, applicability, and clarity of data presented.
Co-Winner Best Poster at Tides 2005
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50:50 Mixture of 29mer:30mer by multiplexed CE-UV, CE-UV, and IEC.
• IEC method was unable to provide resolution of n-1 species
Multiplexed CGE
Single Capillary CGE
Ion Exchange HPLC
70 min = 96 Samples
25 min = 1 Sample
25 min = 1 Sample
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50:50 Mixture of 39mer:40mer by multiplexed CE-UV, CE-UV, and IEC.
• IEC method was unable to provide resolution of n-1 species
Multiplexed CGE
Single Capillary CGE
Ion Exchange HPLC
70 min = 96 Samples
27 min = 1 Sample
26 min = 1 Sample
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50:50 Mixture of 49mer:50mer by multiplexed CE-UV, CE-UV, and IEC.
• All methods could provide resolution of n-1 species
Multiplexed CGE
Single Capillary CGE
Ion Exchange HPLC
70 min = 96 Samples
30 min = 1 Sample
28 min = 1 Sample
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50:50 Mixture of 59mer:60mer by multiplexed CE-UV, CE-UV, and IEC.
• IEC method was unable to provide resolution of n-1 species• IEC provided no n-1 resolution at 70mer, 80mer lengths• The CGE methods resolved n-1 at 70mer, 80mer lengths
Multiplexed CGE
Single Capillary CGE
Ion Exchange HPLC
70 min = 96 Samples
38 min = 1 Sample
30 min = 1 Sample
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Separation of 36mer and 37mer ssRNA Oligonucleotides
Sample: Two RNA oligonucleotides (36mer, 37mer) were obtained from IDT. The 36mer sequence was 3’-CAGGGACAAGCCCGCCGUGACGAUCUCUAAACAAGC-5’; the 37-mer had an additional A residue on the 3’ end. Samples were diluted to ~ 1 M in water.
Capillary Array: 75 m i.d., 150 m o.d.; 55 cm effective/80 cm total length
CGE: E = 150-170 V/cm. Sample injection: -2 kV, 15 sec
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Coming Soon: Oligo Analyzer PRO System
• Multiplexed CE-UV system designed and optimized specifically for oligonucleotide analysis
• Improved ease-of-use and automation
• Enhanced data analysis and report generation capabilities designed with feedback from scientists directly engaged in oligo production
• Scheduled for release in Q2 of 2006
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Summary
• Multiplexed CGE-UV provides a powerful method for oligonucleotide purity analysis, providing superior resolution, throughput and automation compared to slab gel methods
• Only minimal sample preparation is required for analysis
• The developed Oligel™ matrix is capable of achieving single base resolution from 14-80mers in about 1 h
• Multiplexed CGE-UV provides much higher sample throughput and superior resolution to HPLC methods. The standard IEC method could not resolve n-1 species at 30mer, 40mer, or above 60 mer lengths
• RNA oligonucleotides as well as dsRNA duplexes can be analyzed for purity in addition to DNA oligonucleotides