oligo-rp desalting - phenomenexphx.phenomenex.com/lib/5347_clarityminibr.pdfintroducing clarity qsp...
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Purification solutions for synthetic RNA & DNA
ClarityBioSolutions
®
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Clarity QSP™ (NEW) Oligo-RP™
Desalting
A Demanding Industry Requires a Reliable Partner The increase in nucleotide demand, due to advances in biotechnology research and their recent therapeutic applications, has fostered a pressing need for more efficient and efficacious purification platforms. To better match the need of oligo manufacturers and their customers, Phenomenex introduces Clarity BioSolutions, a portfolio of purification products designed to deliver purified synthetic oligos based on requirements and specifications set forth by the industry. In addition to providing state-of-the-art purification solutions, Phenomenex offers unparalleled technical support and customer service to companies and core labs purifying synthetic DNA and RNA.
We look forward to being your partner for efficient, economical, and efficacious synthetic oligo purification.
NEW Clarity QSP Clarity Oligo-RP Clarity Desalting
Format(s)
50 mg/ 96-well plate 50 mg/ 1 mL cartridges
150 mg/ 3 mL cartridges 5 g/ 60 mL cartridges
Analytical, semi-prep, & preparative HPLC columns
200 mg/ 3 mL tubes 500 mg/ 3 mL tubes
Equipment Required Vacuum manifold or automated liquid handling system HPLC system Vacuum manifold
Purification Time ~8 minutes per cartridge ~45 minutes per well plate 15 – 30 minutes 3 15 minutes
Oligo Length ≤100 nt ≤60 nt ≤60 nt
Synthesis Scale Load nmole – 50 µmole 1 nmole – 50 µmole ≤1 µmole
Typical Purity 90 – 95 % 1 90 – 95 % 4 ~70 % 4
Typical Recovery Yield ~90 % 2 ~70 % ~80 %
1. Purity value based on ion exchange chromatography and capillary electrophoresis 2. OD260 used for quantitation 3. Time dependent on dimension of HPLC column and flow rate 4. Purity value based on reversed phase chromatography
Clarity BioSolutions for Synthetic DNA & RNA Purification A purification strategy is developed dependent upon many factors such as purity and yield requirements, synthesis scale, oligo length and sequence, and equipment available in the lab. Clarity BioSolutions offers several strategies to suit your purification needs ranging from high-throughput, high purity technologies to simple desalting. Please see below to review the Clarity solutions and select the one to fit your needs.
Web: www.phenomenex.com
Introducing Clarity QSP™ Addressing the global aim of a purification process, Clarity QSP delivers near impurity-free, concentrated full-length oligo sequences in a stable media suitable for in-vivo applications and downstream analysis conducive for MS, NMR, CE, and HPLC. Simple in practice and in theory, the product offers speed and efficacy in formats that can be readily automated for high-throughput parallel purification and is suitable for both combinatorial-scale and large-scale purifications. Quick, simple, and pure perfectly and accurately summarizes the Clarity QSP product line.
◊ 90 % typical purities and yields for both DNA & RNA
◊ Purify oligos ranging from 10 – 100 nt
◊ Simple three-step process delivers highly purified DNA/RNA in minutes
◊ Formats easily amenable to high-throughput parallel processing
◊ Purified oligos suitable for in-vivo applications & downstream analysis
◊ Applicable for nmole to large µmole synthesis scales
Clarity Quick, Simple, Pure (QSP) Process QSP purification of trityl-on DNA oligos begins after an equal volume of loading buffer is mixed with the cleavage and deprotection solution. After brief conditioning of the sorbent with methanol and water, the solubilized crude oligo is passed through the sorbent. The unique buffer formula synergistically works with ammonia-based deprotecting solutions to selectively retain the full-length trityl-on DNA sequence, while eliminating tenaciously bound unlabeled truncated sequences and damaged fragments. The improved cleaning proficiency of the buffer when mixed directly with an alkaline solution eliminates the need for subsequent wash steps leaving only detritylation and elution to follow. For RNA TBDMS chemistry, the sequential purification steps are consistent with the above descriptions; however, the crude oligo deprotection solution is diluted with water or an appropriate buffer prior to mixing the equal volume of loading buffer.
◊ Consists of 2 components: loading buffer and polymeric sorbent
◊ Provides complete discrimination of trityl-on full length sequences from trityl-off impurities
◊ Ability to load oligo direct from synthesizer
◊ No toxic ion-pairing reagents used that interfere with downstream applications
◊ Negligible depurination caused by detritylation step
Pre-treatment: Trityl-on oligo sample preparation
Mix equal volume of loading buffer with cleavage/deprotection solution
Web: www.phenomenex.com
Detritylated Failure SequencesTrityl GroupFull Length Target Oligo
Full Length Trityl - On OligoImpurityN-1 Sequence
STEP 3
Elution of Target Oligo
Organic M
ixture
Elute target oligopH buffered solutions used to
maintain safe pH for oligo;select elution buffer based on
downstream requirements
STEP 2
Detritylate
DC
A
DetritylateLess than 2%
depurination observed.A faint orange band
will appear at top halfof cartridge indicating
DMT retention
STEP 1
Loading of Crude Oligo
Load crude oligo cocktailAll trityl-off impurities flowdirectly through; no wash
required
Clarity QSP Components Clarity QSP consists of two components, a loading buffer and a polymeric sorbent. Housed in three cartridge formats and a 96-well plate, the QSP resin is pH-stable and purifies oligo sequences of lengths ranging from 10 nt to 100 nt. In addition, the QSP media has enhanced flow characteristics to ensure consistent flow rates for increased analyte contact time resulting in unfailing performance. The accompanying loading buffer is composed of biological compatible agents and is free of toxic and meddlesome ion-pairing agents. Together, the sorbent and buffer create a simple three-step process that in minutes delivers highly purified synthetic DNA with exceptional recovery yields.
◊ Ideal for high-throughput, parallel processing of purified oligos
◊ Load 0.2 µmole synthesis scale per well
◊ Purify 96 crude oligo samples in ~45 minutes
◊ Use either vacuum or positive pressure systems
◊ Easily amenable to automated liquid handling systems
Web: www.phenomenex.com
◊ 50 mg/ 1 mL ideal for purifying 0.2 µmole synthesis scale or less
◊ 150 mg/ 3 mL ideal for purifying up to 1.0 µmole scale
◊ 5 g/ 60 mL ideal for large scale purifications up to 50 µmole
◊ Purify crude oligo samples in ~8 minutes
◊ Use either vacuum or positive pressure systems
96-well plate
Cartridge
Web: www.phenomenex.com
Sequence: GTG GAT CTG CGC ACT TCA GGC TCC TGG GCA Format: 150 mg/ 1 mL cartridge
DNA Purification
DNA & RNA Purified by Clarity QSP Clarity QSP can be used for any synthetic single-stranded oligonucleotide regardless of length (up to 100 nt) or synthetic derivatization and/or adjunct. Our collaborators and we have purified several different types of DNA and RNA on each of the formats and have found that Clarity QSP is an quick and simple process to obtain highly purified oligos.
RNA PurificationSequence: GAC UCA CAU CAA CUA CGA UCG AGC ACTT Format: 50 mg/ 1 mL
min2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20
25
1.868
min2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20
25
1.957
min2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20
25
1. Crude trityl-on: OD: 43 2. Load: OD: 8.65 3. Detritylated final elution: OD: 29 [Purity: 87.6 % (total peak area)
1. Crude trityl-on: OD 28 2. Load OD 5.550 3. Detritylated final elution OD: 20.3 [Purity: 92.7 % (total peak area)
min2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20
25
1.932 2.045
min2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20
25
min2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20
25
Crude DMT-on DNA OD 28
Load Fraction OD 5.550
Detritylated Target DNA OD 20.3 Purity: 92.7 % (total peak area)
Crude DMT-on RNA OD 43
Load Fraction OD 8.65
Detritylated Target DNA OD 20.3 Purity: 87.6 % (total peak area)
Clarity Oligo-RP™ HPLC Columns Clarity Oligo-RP has been specifically designed for the reversed phase purification of oligonucleotides with balanced hydrophobicity and polar selectivity. The media is based on composite particle TWIN™ technology that is able to recognize minute differences in interaction features of two oligonucleotides with very closely resembling structures (for example N/N-1 sequences). This recognition ability enables Clarity Oligo-RP to provide better resolution between such close pairs of sequences both on the analytical and preparative scale.
◊ Ideal for trityl-off purification of DNA, RNA, thioates, and modified/labeled oligos
◊ > 90 % typical purity
◊ 50 nmole - 50 µmole loading capacity available with standard dimensions
◊ Full length oligonucleotides easily separated from N-1 and other failure sequences
Clarity Desalting Tubes Clarity QSP purification cartridges and Oligo-RP can be used to yield highly purified target oligonucleotides (~90 % purity) from a synthesis mixture. However, some applications do not require a high degree of purity. For simple desalting of a synthetic oligonucleotide, Clarity desalting tubes can be used.
◊ 70 % typical purity by removing salt and excess reagent
◊ 80 % recovery of target oligonucleotide
App
ID 1
5935
min0 5 10 15 20 25 30 35 40
mAU
0
10
20
30
40
50 21 nt20 nt
15935
App
ID 1
5935 Column: Clarity 3 μm Oligo-RP C18
Dimensions: 50 x 4.6 mmPart No.: 00B-4441-E0Mobile Phase: A: 10 mM TPAC B: MethanolGradient: A/B (75:25) to A/B (55:45) in 80 min (50 min run time)Flow Rate: 1 mL/min Detection: UV @ 260 nmSample: 1. 20 nt RNA with sequence CUGUAAUCUCUUGUCUATT (2.5 μg)
2. 21 nt RNA with sequence UCUGUAAUCUCUUGUCUATT (2.5 μg)
• RNA Purification of Failure N-1 sequence from target N sequence
Excellent selectivity characteristics of Clarity Oligo-RP allow baseline separation of failure N-1 sequences from target N sequences.
Web: www.phenomenex.com
◊ For the moderate purification of DMT-off oligonucleotides
◊ Removes salt prior to MS analysis
◊ Economical, disposable tubes
A quencher-labeled sample of DNA (25 nt) with the sequence FAM - TTTGACTTAGACTTAGACTTAGTTT was desalted using Clarity Desalting Tubes in the 200 mg/3 mL format. Collection fractions were then analyzed for purity and recovery using the above protocol.
Final Purity: 71 %
Final Recovery: 94 %
App
ID 1
5991
mAU
0
20
40
60
80
mAU
0
20
40
60
80
mAU
0
20
40
60
80
min0 2.5 5 7.5 10 12.5 15 17.5
mAU
0
20
40
60
80
15991
Crude DNAPurity: 58 %
Load
Final Elution
Wash
App
ID 1
5991 Column: Clarity 3 μm Oligo-RP C18
Dimensions: 50 x 4.6 mmPart No.: 00B-4441-E0Mobile Phase: A: 50 mM TEAA, pH 7.5 / 5 % Acetonitrile B: MethanolGradient: A/B (90:10) to A/B (40:60) in 20 minFlow Rate: 1 mL/min Detection: UV @ 260 nmSample: 25 nt DNA oligonucleotide
• Crude DNA Desalting
App
ID 1
5935
min0 5 10 15 20 25 30 35 40
mAU
0
10
20
30
40
50 21 nt20 nt
15935
App
ID 1
5935 Column: Clarity 3 μm Oligo-RP C18
Dimensions: 50 x 4.6 mmPart No.: 00B-4441-E0Mobile Phase: A: 10 mM TPAC B: MethanolGradient: A/B (75:25) to A/B (55:45) in 80 min (50 min run time)Flow Rate: 1 mL/min Detection: UV @ 260 nmSample: 1. 20 nt RNA with sequence CUGUAAUCUCUUGUCUATT (2.5 μg)
2. 21 nt RNA with sequence UCUGUAAUCUCUUGUCUATT (2.5 μg)
• RNA Purification of Failure N-1 sequence from target N sequence
Excellent selectivity characteristics of Clarity Oligo-RP allow baseline separation of failure N-1 sequences from target N sequences.
Web: www.phenomenex.com
Ordering Information
Clarity Oligo-RP HPLC Columns
Clarity QSP Well Plates & CartridgesFormats
Part No. Description Unit
8E-S102-DGB Clarity QSP Well Plate 50 mg/well 1/Box
8B-S102-DAK Clarity QSP Cartridge 50 mg/1 mL 50/Box
8B-S102-SBJ Clarity QSP Cartridge 150 mg/3 mL 50/Box
8B-S042-LFF Clarity QSP Cartridge 5 g/60 mL 20/Box
BuffersPart No. Description Unit
AL0-8279 Clarity QSP DNA Loading Buffer 100 mL Ea
AL0-8280 Clarity QSP DNA Loading Buffer 1 L Ea
AL0-8281 Clarity QSP RNA Loading Buffer 200 mL Ea
AL0-8282 Clarity QSP RNA Loading Buffer 1 L Ea
AH0-7858 Clarity Nuclease Free Water 1 L EaAccessories
Part No. Description Unit
AH0-7284 96-Well Plate Manifold Acrylic Ea
AH0-6024 24-Position Vacuum Manifold Complete Set Ea
AH0-7194 96 Square Well Collection Plate2 mL/well
(Polypropylene)50/pk
AH0-7408 Solvent Waste Reservoir Trayfor Well Plate
Manifolds25/pk
AH0-7195 96-Well Pierceable Sealing Mat Square Well 50/pk
*SecurityGuard™ Analytical Cartridges require universal holder Part No.: KJO-4282
3 µm Minibore & Analytical Columns (mm) SecurityGuardTM Cartridges
50 x 2.0 100 x 2.0 50 x 4.6 100 x 4.6 4 x 2.0* mm 4 x 3.0* mm
Phase /10pk /10pk
C18 00B-4441-B0 00D-4441-B0 00B-4441-E0 00D-4441-E0 AJ0-8134 AJ0-8135
for ID: 2.0-3.0 mm 3.2-8.0 mm
3 µm Semi-Prep Columns (mm) SecurityGuardTM Cartridges
50 x 10.0 10 x 10 mm‡
Phase /3pk
C18 00B-4441-N0 AJ0-8136
for ID: 9-16 mm
‡Semi-prep SecurityGuard™ Cartridges require holder, Part No.: AJ0-7220**PREP SecurityGuard™ Cartridges require holder, Part No.: AJ0-8223
for ID: 9-16 mm 18-30 mm
*SecurityGuard™ Analytical Cartridges require universal holder Part No.: KJO-4282
for ID: 3.2-8.0 mm
Clarity Desalting Tubes.
Clarity Desalting Tubes
200 mg/3 mL* 500 mg/3 mL**
Phase 50/box 50/box
C18 8B-SO41-FBJ 8B-SO41-HBJ
* For 200 µmole synthesis ** For 1 µmole synthesis
Clarity is a registered trademark of Phenomenex, Inc. SecurityGuard, Oligo-RP, QSP and TWIN Technology are trademarks of Phenomenex, Inc. © 2007 Phenomenex, Inc. All rights reserved.
5 µm Semi-Prep and PREP Columns (mm) SecurityGuardTM Cartridges
50 x 10.0 100 x 10.0 250 x 21.2 10 x 10 mm‡ 15 x 21 mm**
Phase /3pk ea
C18 00B-4442-N0 00D-4442-N0 00G-4442-P0 AJ0-8136 AJ0-8210
5 µm Analytical Columns (mm) SecurityGuardTM Cartridges
50 x 4.6 150 x 4.6 4 x 3.0 mm*
Phase /10pk
C18 00B-4442-E0 O0F-4442-EO AJ0-8135
Evaluate Clarity™ Biosolutions in your lab for 45 days, if you are not completely satisfied return it for a full refund.
ClarityBioSolutions
®
pate
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ng
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www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department by telephone, fax or email: [email protected].
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