azco biotech, inc. synthesis training july, 2011
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Azco Biotech, Inc.
Synthesis Training
July, 2011
2
Synthesis Training
Applications
Introduction to DNA/RNA Synthesis Review of Synthesis Chemistry Instrument Review
Instruments
Reagents
FAQs
Conclusion
Applications: Oligo Synthesis
Uses for Oligonucleotides Great!
Gene Therapy- Cystic Fibrosis- Diabetes- Cancer
Microarray- Diagnostics - DNA Forensics- Predisposition to Disease- Detection of Infectious Agents
Antisense Research- Therapeutics- Viral Infections- Immune Disorders- Cancer
Main Applications- Primers & Probes for PCR/RT-
PCR- Synthetic Genes- 2nd Gen Sequencing
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Oligonucleotide Synthesis Chemistry
Nucleic Acid Synthesis
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Developed by Marvin Carruthers, U. Colorado in 1982
Oligonucleotide Synthesis
Basic Synthetic Cycle Steps (NOTE: Very little has changed since original Carruthers paper in 1982)
The Detritylation StepThe Coupling (Activation) StepThe Capping StepThe Oxidation StepThe Deprotection and Cleavage Step
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The Detritylation Step 5’ protecting group is removed with TCA
or DCA in DCM (3% w/v). There is now a hydroxyl at the 5’
carbon, which will react with the next base to be added.
O
O CPG
DMTO Base 1O
O CPG
HO Base 1
3% TCA in DCM5% DCA in DCM
Detritylation
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The Coupling (Activation) Step
To react with the free hydroxyl of the first base, phosphoramidites are reacted with a weak acid,generally 1-H-tetrazole, ETT or DCI.
The acidic conditions protonate the dialkylamino group and then tetrazole attacks as a nucleophile generating the reactive tetrazolophosphane intermediate.
The result is a phosphite triester bond formed between the first and second bases.
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The Coupling (Activation) Step
O
O CPG
HO Base 1
O
O
DMTO Base 2
PNCCH2CH2O
N
O
O
DMTO Base 2
PNCCH2CH2O
O
O CPG
O Base 1
ACTIVATION
0.25M ETT
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Water (H-O-H) Kills Coupling!
The Capping Step The coupling step is not 100% efficient. Need to stop the elongation of sequences that
are missing bases, e.g. failure sequences. This is done by capping the free hydroxyls
through acetylation, making these sequences terminally unreactive.
Capping is in two steps: acetic anhydride and lutidine to form the cap and N-methylimidazole to form the reactive intermediate.
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The Capping Step
O
O CPG
HO Base 1 Acetic AnhydrideLutidine (1:1)(10%ea. w/v THF)
N-methylimidazole(10% w/v THF)
O
O CPG
O Base 1H3CC
O
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The Oxidation Step
After the capping step is done, the unstable phosphite triester is oxidized to the stable phosphate triester.
Iodine acts as the oxidant with water providing a hydroxyl.
This step must occur after capping because acetic anhydride will react with trace amounts of water to form acetic acid.
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The Oxidation Step
O
O
DMTO Base 2
P
O
O CPG
O Base 1
OH2CH2CCNO
O
O
DMTO Base 2
P
O
O CPG
O Base 1
OH2CH2CCN
Iodine/ pyridine
H2O/ THF
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Post Synthesis
Deprotection and CleavageColumn Oligo
The N-benzoyl and isobutyryl protecting groups are removed completely by treatment with 9N ammonia in 8 hours at 55° C or 90 minutes at 70° C.
Fast Deprotect can occur in 15 minutes at 50° C temperature…
Simultaneously, the first base is cleaved from the solid support.
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Deprotection & CleavageChip Synthesis
Deprotection and Cleave• Deprotection– same as normal, aminolysis with either
ammonium hydroxide or AMA, regulated by amidites for Oligo Pools. EDA/EtOH for Microarray
• For Oligo Pools Cleave from Chip – If the oligos are for pooled applications they are cleaved during aminolysis. The chip contains a cleavable linker (same as with column based synthesis).
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Instruments
Instruments
The instrument just automates the above steps
The instrument range is based on throughput and quantity
Select the instrument that meets your throughput requirements
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New Synthesizers• Oligo 800 • Oligo 96/192• OM LS2• OligoArray
Refurbished Synthesizers• ABI 391-4• ABI Expedites• BLP 96/192• Dr. Oligo 96/192• Polyplex
Azco Synthesizers
Azco Synthesizer: Oligo-800
Automated 8 column synthesizer
8 amidite/10 reagent ports
Highly accurate reagent delivery
via syringe pump
Wide range of synthesis scales
Ideal for long oligos or modified
oligos including FRET probes
Very Economically Priced!
Oligo-800 Plumbing Diagram
Azco Synthesizer: Oligo 96/192
96, 192 column/well format
Up to 36 Amidite Ports
Low volume “specials” upgradeable
10 Reagent Ports
Fully controlled via Oligo v 4.0
Workstation
Full process monitoring with audit
tracking and error reporting
Trityl monitoring capability
Ideal for high throughput applications
OligoArray In-situ Synthesizer
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OligoArray Plumbing Diagram
Reagents
Synthesis Reagents
Phosphoramidites
Liquid Reagents
Solid Supports and Columns or Chips
The key to quality synthesis is the reagents you select
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Standard Phosphoramidites • dA, dG, dC and T
Fast Deprotect Phosphoramidites • Acetyl - dC
• dmf- dG
High Quality
DNA Phosphoramidites
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T-CE Phosphoramidite
Adds a “T” to an oligo Catalog Numbers:
20-8100-xx
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dG-CE Phosphoramidite
Adds a “G” to an oligo
Catalog Numbers:20-8110-xx
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dA-CE Phosphoramidite
Adds an “A” to an oligo
Catalog Numbers:20-8120-xx
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dC-CE Phosphoramidite
Adds a “C” to Catalog Numbers:
20-8130-xx
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Acetyl-dC-CE Phosphoramidite
Fast Deprotecting “C” Catalog Numbers:
20-8230-xx
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dmf-dG-CE Phosphoramidite
Fast Deprotecting “G”
Catalog Numbers:20-8210-xx
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Fast Deprotect RNA Phosphoramidites • 2’ TBDMS (n Acetyl) – rA, rG, rC, rU
• Requires 2nd deprotection to remove TBDMS
RNAse resistant Phosphoramidites • 2’ Fluoro (nAcetyl) rC, rU, rA and rG
• 2’ OMe (nAcetyl) rC, rU, rA, rG
ONLY USE MILD DEPROTED RNA AMIDITES!
RNA Phosphoramidites
Silyl RNA Amidites
Popular for small RNA pH stability Ease of use Catalog Numbers:
28-8800-xx rU
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rU-CE Phosphoramidite Adds a “U” to an
oligo Catalog Numbers:
20-8800-xx
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(Acetyl) rG-CE Phosphoramidite
Fast deprotect rG Adds a “rG” to an
oligo Catalog Numbers:
20-8810-xxO-TBDMS
Ac
rA-CE Phosphoramidite
Adds an “rA” to an oligo
Catalog Numbers:20-8820-xx
O-TBDMS
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(Acetyl) rC-CE Phosphoramidite
Fast Deprotecting “C” Catalog Numbers:
20-8810-xx
O-TBDMS
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2’ OMe rU-CE Phosphoramidite
2’ OMe rU RNAse Resistant Product Numbers:
20-5200-xx
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2’ OMe (N-Ac) rG-CEPA
2’ OMe rG Fast Deprotect RNAse Resistant Product Numbers:
20-5310-xx
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2’ OMe (N-Bz) rA-CEPA
2’ OMe rA RNAse Resistant Product Numbers:
20-5220-xx
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2’ OMe (N-Ac) rC-CE PA
2’ OMe rC Fast Deprotect RNAse Resistant Product Numbers:
20-5330-xx
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2’ F rU-CE Phosphoramidite
2’ F rU RNAse Resistant Product Numbers:
20-9000-xx
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2’ F (N-Ac) rC-CE PA
2’ F (N-Ac) rC RNAse Resistant Product Numbers:
20-9030-xx
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Specialty Phosphoramidites
Fluorescent Phosphoramidites
TF and TQ dyes and quenchers
Biotin
Linkers and Spacers
Modified and non-natural Bases
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Specialty Phosphoramidites
Phosphorylation Reagent
Amino Modifiers
Thiolation Reagent
Cholesterol
Custom Phosphoramidites
Dyes and Quenchers
We offer a full line of dyes and quenchers FAM, TET, HEX, Cy3, Cy5, TF Phosphoramidites (TF1, 2, 3, 4, 5, 6) TQ Quenchers (wavelength matched to TF) JOE, ROX, All other dyes as free dye and NHS esters
Used in Dual Labeled Probes TeHP Probes TaqMan (FQ) Probes Molecular Beacons Other
Deprotection Depends on Amidite
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Amidtite Deprotect Soln Time
DNA All Standard Ammonium Hydroxide 10 hours at 60C14 hours at 50 C
DNA All Fast AMA (1:1 MeNH4/NH4OH)
15 minutes at 55C
DNA w/ dmf-dG Ammonium Hydroxide 4 hours at 55C
RNA (only use fast deprotect)
AMA 15 minutes at 50C
Std for Microarray EDA/EtOH (1:1) 1 hour at 55C
Std for Oligo Pool Ammonium Hydroxide 7 hours at 65C14 hours at 55C
Fast for Oligo Pool Same as above…
Liquid Reagents
Acetonitrile/ Diluent
Used to re-suspend amidites and wash. Supply one grade of Acetonitrile: <10ppm.
• Need to use only 10ppm or less.
Low water content ensures quality synthesis, should also use MolSieves.
Water will cause precipitation of dG.
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ETT Activator
Turn key solution. Optimal Concentration 0.25M Produced in US & UK Preferred by Chip Co.’s Great for RNA (0.3M) Catalog Numbers:
17-1124-xx 17-1224-xx
N N
N
N SCH2CH3
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Other Activators
DCI, 5-BMT• Slight acidic conditions excellent for DNA
and RNA synthesis.
N
NCN
CN
N
NN
N
S
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Cap A Solution
One of Two reagents used to stop the elongation of failure sequences.
Components (17-1130-xx):TetrahydrofuranAcetic AnhydrideLutidine
Other formulations: (17-1131-xx)
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Cap B Solution
The second solution used to form the chemical cap.
Components (17-1137-xx):TetrahydrofuranN-MethylimidazolePyridine
Other Formulations: (17-1138-xx)
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Oxidizer Solution
Used oxidize the unstable phosphite triester to the stable phosphate triester.
Three formulations: 0.1M & 0.02M & 0.05M Iodine acts as the oxidant with water. Components
Tetrahydrofuran Iodine Pyridine Water
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Deblock Solutions
Removal of DMT group. Two formulations
TCA in dichloromethaneDCA in dichloromethane
Many switching to Tolune
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eChem Deblock Solutions
Removal of DMT group. Made prior to use, contains:
• Benzoquinone• Hydroquinone• Tetraethylammonium p-ts• Lutidine• In Methanol/Acetonitrile
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Miscellanous Reagents
2% Trifluoroacetate (17-1169-xx) 3% Trifluoroacetate (17-1172-xx) 20% Acetic Acid (17-1173-xx) 20% Acetonitrile (17-1174-xx)
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Solid Supports&
Columns
Solid Supports
PolystyreneMore non-polar, better for DNA
CPGDerivitized for almost any base or SpecialtyVarious pore sizes Uses LCAA-group to CPG
Long chain amino alkyl
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Columns
We provide various columns for DNA synthesis
Various machinesVarious scalesVarious “bases”
All filled with CPG or PS Should always consider Universal
Support
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CPG Basics
Controlled Pore Glass is essentially glass shavings that have a nucleoside based or universal group attached through linker and amine chemistry
Typically characterized by two methods• Pore Size• Nucleoside Loading
Occasionally characterized by mesh size.
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CPG Pore Size
Refers to the space between the individual shavings of the CPG particles.
Common Pore Sizes• 500 Angstroms (Standard Pore Size)• 1000 Angstroms (Long Pore Size)• 2000 Angstroms (Extra Long Pore Size)
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CPG Loading
Loading refers to the amount of nucleoside base bound to the CPG particles.• Commonly expressed as mmols/mg.• Using this unit of measure, one can
calculate the correct amount of CPG to load within a column or plate.
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CPG Loading Categories
Standard: 500Å, 25-35 mmol/mg High Load: 500Å, 45-90 mmol/mg Extra High Load: 500Å, 100-200 mmol/mg
Long : 1000Å, 25-40 mmol/mg Extra Long: 2000Å, 18-30 mmol/mg
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12K and 90K chips available
Fig. 4. 12K and 90K CustomArray microarrays after in situ synthesis and hybridization to fluorescently labeled DNA
• 12K and 94K chip formats available for making 12,000 or 94,000 oligos simultaneously
• Can use a “mask” to divide 12K chip into smaller 2K segments
• Chips are distinguished base on cleave or leave applications.
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F.A.Q.’s
Common Issues (Misconceptions)
Coupling is always regulated by reagents and water kills coupling
Average coupling efficiency is ~99%, order of efficiency T > A > C > G, so a long run of Gs will result in low quality synthesis, this dictates yield
Specialty molecules have much lower coupling efficiencies (~80 to 90%),
Purification is application dependent Synthesis is ALWAYS 3’ to 5’, can simply
purification by remembering this!
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Are the SNA reagents for use on all Oligo Synthesizers?
Yes, providing we have the follow input from the customer:• What synthesizer do you own?• What activator solutions do you use?• What packaging do you prefer?
What is the shelf life of the SNA Products?
Assuming that the products were stored properly:• For most phosphoramidites, five years dry.• For reconstituted phosphoramidites:
DNA and RNA amidites between 8-10 days. For specialty amidites 3-6 days.
• For the liquid reagents, one year.• For the solid supports, five years. It is highly unusual for us or the customer to keep
these materials for long periods of time.
What are these crystals in my dG-CE amidite?
dG amidite can crystallize under three conditions:• High water content of the ACN diluent.• High water content of the amidite itself.• A long period of reconstitution.
Can I Swap Out the Different Activator Solutions?
ETT, DCI, 5-BMT and 1-H-tet solutions generally are interchangeable.
ETT and DCI are slightly more acidic than 1-H-tet• Better for RNA synthesis.• Better for longer oligo synthesis.
My trityl monitor is showing low coupling efficiency.
The phosphoramidite lines may be clogged.• Crystals in the amidite.
Water in the diluent.Long term placement of the synthesizer.
The column is clogged.
The instrument may have issues.
The bottles you sent don’t fit my machine
Different instruments utilize bottles with different neck sizes.
This problem comes about when either the customer is unfamiliar with our catalog numbers or we don’t know what instrument they own.• Occasionally occurs with new customers.
I am having issues with base deamination in my oligo.
They oxidizer solution is too strong.• Switch to 0.02M oxidizer.
Reduce the cleavage and deprotection times.
Reduce the cleavage and deprotection temperatures.
My long oligo synthesis yielded very little material
This may actually be appropriate for the sequence.
Questions to ask:• Are you using a 1.0mmol or greater
synthesis scale?• Are you using long CPG?• Did you try to purify the material?
Azco OligoArray Synthesizer
Market and Competitors
Instruments - Market
Size $15 to $20M Annual MarketVery established and consistentNot much growthMostly a replacement market
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Instruments
Low ThroughputBioAutomation – MM4, MM12A couple in Germany
High ThroughputBiolytic – “Dr. Oligo”BioAutomation – MM96, 192, 384A Couple in Euorpe
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Instruments
Production ScaleGE Healthcare – OP10, OP100, OP400 We make custom Instruments
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Reagents - Market
Size $95 to $140M Annual Market2 companies control about 70% of this market
(Thermo Fisher and Sigma)Not much growth – same with instrumentsBig part of this market dependent on
pharmaceutical market – no successes means no growth…
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Reagents
Large Scale DNA/RNA AmiditesThermo FisherSIALChemgenesUs
Laboratory ScaleGlenn ResearchChemgenesUs
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Reagents
Specialty AmiditesGlenn ResearchChemgenesUs
Other (Dyes, Supports, etc.)Glenn ResearchUs
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NOTE
Azco is the worlds ONLY TURN-KEY SUPPLIER OF INSTRUMENTS REAGENTS, CUSTOM REAGENTS, SUPPORT, EVERYTHING NEEDED FOR DNA AND RNA SYNTHESIS!!!
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Conclusion
Conclusion
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Azco’s Core Competency
ExperienceExperience
Chemistry Chemistry
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