1. Synthesis Training July, 2011
Azco Biotech, Inc.
Synthesis Training
July, 2011

2. Synthesis Training Conclusion Applications
Introduction to DNA/RNA Synthesis
Review of Synthesis Chemistry
Instrument Review
Instruments
Reagents
FAQs
Conclusion

3. Applications: Oligo Synthesis

4. Uses for Oligonucleotides Great!
Microarray
Diagnostics
DNA Forensics
Predisposition to Disease
Detection of Infectious Agents
Antisense Research
Therapeutics
Viral Infections
Immune Disorders
Cancer
Gene Therapy
Cystic Fibrosis
Diabetes
Cancer
Main Applications
Primers & Probes for PCR/RT-PCR
Synthetic Genes
- 2nd Gen Sequencing

5. Oligonucleotide Synthesis Chemistry

6. Nucleic Acid Synthesis
Developed by Marvin Carruthers, U. Colorado in 1982

7. Oligonucleotide Synthesis
Basic Synthetic Cycle Steps
(NOTE: Very little has changed since original Carruthers paper in 1982)
The Detritylation Step
The Coupling (Activation) Step
The Capping Step
The Oxidation Step
The Deprotection and Cleavage Step

8. 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.

9. 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.

10. The Coupling (Activation) Step
Water (H-O-H) Kills Coupling! B a s e D M T O 2 O O P N N C C H C H O 2 2 B a s e D M T O 2 O A C T I V A T I O N . 2 5 M E T O P B a s e N C C H C H O B a s e H O 1 2 2 O 1 O O O C P G O C P G

11. 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.

12. The Capping Step

13. 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.

14. The Oxidation Step

15. Post Synthesis

16. Deprotection and Cleavage Column 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.

17. Deprotection & Cleavage
Chip 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).
Here are the key features. Note with this system you can 1) make long oligos 2) make a lot of oligos 3) using standard phosphoramidtie chemistry. The only difference is it uses electrochemistry for deblocking the elongating oligo. It comes standard with 6 amidies and 7 liquid reagent ports, but as with most “high end” synthesizers, this can be customized to meet your needs. You can also synthesize either DNA or RNA…your choice!

18. Instruments

19. 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

20. Azco Synthesizers New Synthesizers Refurbished Synthesizers Oligo 800
OM LS2
OligoArray
Refurbished Synthesizers
ABI 391-4
ABI Expedites
BLP 96/192
Dr. Oligo 96/192
Polyplex

21. 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!

22. Oligo-800 Plumbing Diagram

23. 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

24. OligoArray In-situ Synthesizer
This is the system. It is an 8 chip system that uses syringes to push reagents across a semiconductor chip for in-situ oligo synthesis uf 8 x 12,000 or 8 x 90,000 oligos….

25. OligoArray Plumbing Diagram

26. Reagents

27. The key to quality synthesis is the reagents you select
Synthesis Reagents
Phosphoramidites
Liquid Reagents
Solid Supports and Columns or Chips
The key to quality synthesis is the reagents you select

28. DNA Phosphoramidites Standard Phosphoramidites
dA, dG, dC and T
Fast Deprotect Phosphoramidites
Acetyl - dC
dmf- dG
High Quality

29. T-CE Phosphoramidite Adds a “T” to an oligo Catalog Numbers:xx

30. dG-CE Phosphoramidite
Adds a “G” to an oligo
Catalog Numbers: xx

31. dA-CE Phosphoramidite
Adds an “A” to an oligo
Catalog Numbers: xx

32. dC-CE Phosphoramidite
Adds a “C” to
Catalog Numbers: xx

33. Acetyl-dC-CE Phosphoramidite
Fast Deprotecting “C”
Catalog Numbers: xx

34. dmf-dG-CE Phosphoramidite
Fast Deprotecting “G”
Catalog Numbers: xx

35. RNA Phosphoramidites 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!

36. Silyl RNA Amidites Popular for small RNA pH stability Ease of use
Catalog Numbers:
xx rU

37. rU-CE Phosphoramidite
Adds a “U” to an oligo
Catalog Numbers: xx

38. (Acetyl) rG-CE Phosphoramidite
Fast deprotect rG
Adds a “rG” to an oligo
Catalog Numbers: xx Ac
O-TBDMS

39. rA-CE Phosphoramidite
Adds an “rA” to an oligo
Catalog Numbers: xx
O-TBDMS

40. (Acetyl) rC-CE Phosphoramidite
Fast Deprotecting “C”
Catalog Numbers: xx
O-TBDMS

41. 2’ OMe rU-CE Phosphoramidite
RNAse Resistant
Product Numbers: xx

42. 2’ OMe (N-Ac) rG-CEPA 2’ OMe rG Fast Deprotect RNAse Resistant
Product Numbers: xx

43. 2’ OMe (N-Bz) rA-CEPA 2’ OMe rA RNAse Resistant Product Numbers:xx

44. 2’ OMe (N-Ac) rC-CE PA 2’ OMe rC Fast Deprotect RNAse Resistant
Product Numbers: xx

45. 2’ F rU-CE Phosphoramidite
RNAse Resistant
Product Numbers: xx

46. 2’ F (N-Ac) rC-CE PA 2’ F (N-Ac) rC RNAse Resistant Product Numbers:xx

47. Specialty Phosphoramidites
Fluorescent Phosphoramidites
TF and TQ dyes and quenchers
Biotin
Linkers and Spacers
Modified and non-natural Bases

48. Specialty Phosphoramidites
Phosphorylation Reagent
Amino Modifiers
Thiolation Reagent
Cholesterol
Custom Phosphoramidites

49. 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

50. Deprotection Depends on Amidite
Amidtite
Deprotect Soln
Time
DNA All Standard
Ammonium Hydroxide
10 hours at 60C
14 hours at 50 C
DNA All Fast
AMA (1:1 MeNH4/NH4OH)
15 minutes at 55C
DNA w/ dmf-dG
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
7 hours at 65C
14 hours at 55C
Fast for Oligo Pool
Same as above…

51. Liquid Reagents

52. 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.

53. 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: xx xx

54. Other Activators DCI, 5-BMT
Slight acidic conditions excellent for DNA and RNA synthesis.

55. Cap A Solution
One of Two reagents used to stop the elongation of failure sequences.
Components ( xx):
Tetrahydrofuran
Acetic Anhydride
Lutidine
Other formulations: ( xx)

56. Cap B Solution The second solution used to form the chemical cap.
Components ( xx):
Tetrahydrofuran
N-Methylimidazole
Pyridine
Other Formulations: ( xx)

57. 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

58. Deblock Solutions Removal of DMT group. Two formulations
TCA in dichloromethane
DCA in dichloromethane
Many switching to Tolune

59. eChem Deblock Solutions
Removal of DMT group.
Made prior to use, contains:
Benzoquinone
Hydroquinone
Tetraethylammonium p-ts
Lutidine
In Methanol/Acetonitrile

60. Miscellanous Reagents
2% Trifluoroacetate ( xx)
3% Trifluoroacetate ( xx)
20% Acetic Acid ( xx)
20% Acetonitrile ( xx)

61. Solid Supports & Columns

62. Solid Supports Polystyrene CPG More non-polar, better for DNA
Derivitized for almost any base or Specialty
Various pore sizes
Uses LCAA-group to CPG
Long chain amino alkyl

63. Columns We provide various columns for DNA synthesis
Various machines
Various scales
Various “bases”
All filled with CPG or PS
Should always consider Universal Support

64. 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.

65. 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)

66. 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.

67. CPG Loading Categories
Standard: 500Å, mmol/mg
High Load: 500Å, mmol/mg
Extra High Load: 500Å, mmol/mg
Long : 1000Å, mmol/mg
Extra Long: 2000Å, mmol/mg

68. 12K and 90K chips available
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.
Fig K and 90K CustomArray microarrays after in situ synthesis and hybridization to fluorescently labeled DNA
Chips are available in 12K and 90K formats. You can use a “mask” to further subdivide the chip into 1/4ths so 3K for the 12K chip and 23K for the 90K chip. The chips are single use, but with the mask can be reused until all spots have had an oligo made on them. The list price for these chips is $300 for the 12K chip and $550 for the 90K chip. With the 12K chip, the total cost of synthesizing 12,000 oligos is ~$360 for 100mers, or 0.03 cents per coupling!!!

69. F.A.Q.’s
The other new product from Azco…the OligoArray synthesizer.

70. 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!

71. 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?

72. 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.

73. 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.

74. 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.

75. 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.

76. 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.

77. 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.

78. 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?

79. Azco OligoArray Synthesizer
The other new product from Azco…the OligoArray synthesizer.

80. Market and Competitors

81. Instruments - Market Size $15 to $20M Annual Market
Very established and consistent
Not much growth
Mostly a replacement market

82. Instruments Low Throughput High Throughput BioAutomation – MM4, MM12
A couple in Germany
High Throughput
Biolytic – “Dr. Oligo”
BioAutomation – MM96, 192, 384
A Couple in Euorpe

83. Instruments Production Scale GE Healthcare – OP10, OP100, OP400
We make custom Instruments

84. Reagents - Market Size $95 to $140M Annual Market
2 companies control about 70% of this market (Thermo Fisher and Sigma)
Not much growth – same with instruments
Big part of this market dependent on pharmaceutical market – no successes means no growth…

85. Reagents Large Scale DNA/RNA Amidites Laboratory Scale Thermo Fisher
SIAL
Chemgenes Us
Laboratory Scale
Glenn Research

86. Reagents Specialty Amidites Other (Dyes, Supports, etc.)
Glenn Research
Chemgenes Us
Other (Dyes, Supports, etc.)

87. NOTE
Azco is the worlds ONLY TURN-KEY SUPPLIER OF INSTRUMENTS REAGENTS, CUSTOM REAGENTS, SUPPORT, EVERYTHING NEEDED FOR DNA AND RNA SYNTHESIS!!!

88. Conclusion

89. Conclusion

90. Azco’s Core Competency
Experience
Support
Chemistry