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© 1999 PE Biosystems
User Bulletin: ABI 392/4
Nucleic Acid Synthesizers
Subject: Phosphalink Amidite for
Phosphorylation of Oligonucleotides
User Bulletin - Number 86
Model 38X, 39X
Phosphalink Phosphorylation
Introduction
This User Bulletin introduces Phosphalink(Fig. 1), a phosphorylating reagent for
efficient preparation of 5'- and/or 3'- end phosphorylated oligonucleotides on any
Applied Biosystems DNA/RNA Synthesizer.
Figure 1. Phosphalink
Gene construction, cloning, the oligonucleotide ligation assay (OLA), the ligation
chain reaction (LCR), and total cDNA sequencing are among the many
applications of 5'-end phosphorylated oligonucleotides.
5' Phosphorylation is also essential for the use of lambda exonuclease to create
single-stranded sequencing templates from PCR products.
3' Phosphorylation of probes is required where DNA polymerase is present to
prevent extension of the probe when the probe anneals to its target; i.e., 3'
phosphorylation prevents a probe from serving as a primer. 3'-Phosphorylation is
also used in ligation experiments that require 3' exonuclease resistance. 5'-
and/or 3'- phosphorylated oligonucleotides can also be used for the synthesis of
stable, non-radioactive probes, to which reporter groups such as biotin,
fluorescent dyes, or spin labels are attached.
Enzymatic protocols also add phosphate groups to the 5' end of oligonucleotides.
The enzymatic methods require some post-synthesis processing and purification,
and may not be suitable for large-scale preparation of 5'-phosphorylated
oligonucleotides. Phosphalink conducts chemical phosphorylation on any Applied
Biosystems Division nucleic acid synthesizer, with no special handling procedures
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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB86.html
required.
The 5'-phosphorylated oligonucleotide is differentiated from the
non-phosphorylated oligonucleotide by electrophoretic mobility or by HPLC
retention time difference. The difference in mobility shift or retention time between
the two fragments is small for oligonucleotides longer than 10 bases.
Phosphalink bears a dimethoxytrityl (DMT) protecting group, so coupling
efficiency can be conveniently monitored using the DMT cation assay (UV
absorbance or Autoanalysis conductivity measurement). After synthesis, the
protecting group on the phosphate is deblocked rapidly in ammonia solution (1 h
at 65 °C or 2 h at 55 °C). Enzymatic digest by snake venom phosphodiesterase
and bacterial alkaline phosphatase shows no detectable base modifications.
Installation
1. Using a dry syringe, dilute Phosphalink with dry acetonitrile (>50 ppm H2O)
by the manual method.
We do not recommend autodilution for small (1-2 mL) volumes on the ABI
392/394 because a significant percentage of acetonitrile is lost during
argon bubbling.
For 0.2-µmol and 1.0-µmol scale syntheses, add 1 mL of dry
acetonitrile to prepare a 0.1 M solution of Phosphalink.
For a 40-nmol scale synthesis, add 2 mL of acetonitrile to prepare a
0.05 M solution.
2. Place the Phosphalink solution at any monomer position on your DNA/RNA
synthesizer. Typically, bottle positions 5-8 are used.
3. To conserve Phosphalink, create user-defined Bottle Change and Begin
procedures. Refer to the Functions, Cycles and Procedures section in your
User's Manual for modifying procedures.
Bottle Change Procedure
Decrease the delivery time to waste from the Phosphalink bottle position to one
second. For example, the following procedure is for the 392/394, with Phosphalink
at bottle position 5.
392/394 Bottle Change Procedure
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Step
Number Function
Number Function Name Step
Time
1 106 Begin 0
2 1 Block Flush 5
3 64 18 to Waste 7
4 74 18 to 5 3
5 10 Flush to 5 10
6 104 Interrupt 0
7 10 Flush to 5 5
8 54 5 to Waste 1
9 64 18 to Waste 7
10 1 Block Flush 5
11 107 End 0
Begin Procedure
Decrease the delivery time to waste from the Phosphalink bottle position to one
second. The procedure below, for example, is for the 392/394 with Phosphalink at
bottle position 5.
The Bottle Change and Begin procedures are modified similarly for the ABI 381A,
380B, and 391 DNA/RNA synthesizers.
392/394 Begin Procedure
Step
Number Function
Number Function Name Step
Time
1 106 Begin 0
2 101 Phos Prep 10
3 50 A to Waste 2
4 51 G to Waste 2
5 52 C to Waste 2
6 53 T to Waste 2
7 54 5 to Waste 1
8 58 Tet to Waste 2
9 64 18 to Waste 10
10 1 Block Flush 10
11 107 End 0
Synthesis
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5'-Phosphorylated Oligonucleotides
Enter the desired oligonucleotide sequence with the Phosphalink position
(denoted by the bottle position number) at the 5' end (Fig. 2).
Note: 5'- and 3'-phosphorylated oligonucleotides are
susceptible to dephosphorylation by alkaline phosphatase, a
ubiquitous environmental contaminant (from skin, bacteria)
and also a common reagent in the molecular biology
laboratory. Take extreme care to protect solutions containing
phosphorylated oligonucleotides from introduction of alkaline
phosphatase.
Figure 2. 5' Phosphorylation
3'-Phosphorylated Oligonucleotides
Enter the desired oligonucleotide sequence with an extra base at the 3' end after
the Phosphalink position (denoted by the bottle position number). For example, to
synthesize 5'- TAT CA phos-3', enter the sequence as 5'-TAT CA5 T-3', where 5
is the Phosphalink bottle position. The extra T at the 3' end will be removed during
deprotection (Fig. 3).
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Figure 3. 3' Phosphorylation
Consumption
Phosphalink yields the following approximate number of couplings per bottle:
Synthesis Cycles Couplings Concentration
40-nmol CE 12 0.05 M
0.2-µmol CE 6 0.1 M
1-µmol CE 4 0.1 M
10-µmol CE 1 0.1M
Deprotection
Ammonia deprotection time for 5'- or 3'-phosphorylated oligonucleotides prepared
with standard base- protected phosphoramidites is 4 - 8 h at 55 °C. For
phosphorylated oligonucleotides made with FastPhoramidite reagents, the
deprotection time is 1 h at 65 °C, or 2 h at 55 °C.
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Purification
Phosphorylated oligonucleotides can be purified by reverse phase HPLC,
anion-exchange HPLC or polyacrylamide gel electrophoresis. 3'-Phosphorylated
oligonucleotides, when synthesized with trityl-on at the 5' end, can be purified by
Oligonucleotide Purification Cartridge (OPC). 5'-Phosphorylated oligonucleotides
cannot be purified by OPC since they do not have a trityl group.
Analysis
5'- or 3'-phosphorylated oligonucleotides can be analyzed by reverse-phase
HPLC, anion-exchange HPLC, PAGE, and MicroGel capillary electrophoresis,
using the same conditions and methods as non-phosphorylated oligonucleotides
(Fig. 4). Phosphorylated oligonucleotides elute earlier than non-phosphorylated
fragments by MicroGel capillary electrophoresis and PAGE. Phosphorylated
oligonucleotides elute slightly later than non-phosphorylated oligonucleotides by
anion exchange HPLC.
Figure 4. MicroGel CE of a 20-mer with the sequence: 5'
phos-ACA TCT CCC CTA CCG CTA TA-3'
Storage
Store bottled Phosphalink desiccated at -20 °C. Use Phosphalink as soon as
possible after dissolution in dry acetonitrile and installation on the DNA
synthesizer. Coupling efficiency may decrease after approximately nine days.
Conclusion
Phosphalink allows the convenient addition of a phosphate group at the 5' and/or
3' ends of oligonucleotides with high coupling yield. Coupling can be conveniently
monitored by DMT cation assay, using either AutoAnalysis conductivity
measurement or UV absorbance.
Ordering Information
Description Quantity P/N
Phosphalink 70 mg 401717
Bibliography
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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB86.html
Horn, T., and Urdea, M. S., "A Chemical 5'-phosphorylation of
oligonucleotides that can be monitored by trityl cation release,"
Tetrahedron Lett., 1986, 27, 4705-4708.
Modrich, P. and Lehman, I. R., "Deoxyribonucleic acid ligase: A steady
kinetics analysis of the enzyme from Escherichia coli," J. Biol. Chem.,
1973, 248, 7502-7511.
Landegren, U., Kaiser, R., Sanders, J. and Hood, L., "A ligase-mediated
gene detection technique," Science, 1988, 241, 1077-1080.
Nickerson, D. A., Kaiser, R., Lappin, S., Stewart, J., Hood, L. and
Landegren, U., "Automated DNA diagnostics using an ELISA-based
oligonucleotide ligation assay," Proc. Natl. Acad. Sci., 1990, 87,
8923-8927.
Barany, F., "Genetic disease detection and DNA amplification using cloned
thermostable ligase," Proc. Natl. Acad. Sci., 1991, 88, 189-193.
Barany, F., "The ligase chain reaction in a PCR world," PCR Methods and
Applications, 1991, 1, 5-16.
Winn-Deen, E. S. and Iovannisci, D. M., "Sensitive fluorescence method
for detecting DNA ligation amplification products," Clinical Chemistry, 1991,
37, 1522-1523.
Winn-Deen, E. S. and Iovannisci, D. M., Brinson, E. C. and Eggerding, F.
A., "Application of DNA probe ligation detection to genetic disease
analysis," Clinical Chemistry, 1993, 39, 727-728.
Iovannisci, D. M. and Winn-Deen, E. S., "Ligation amplification and
fluorescence detection of Mycobacterium tuberculosis DNA," Molecular
and Cellular Probes, 1993, 7, 35-43.
Highuchi, R. G. and Ochman, H., "Production of single-stranded DNA
templetes by exonuclease digestion following the polymerase chain
reaction," Nucleic Acids Res., 1989, 17, 5865.
Lee, L. G., Connell, C. R., and Bloch, W., "Allelic discrimination by
nick-translation PCR with fluorogenic probes," Nucleic Acids Res., 1993,
21, 3761-3766.
Andrus, A., "Oligonucleotide analysis by gel capillary electrophoresis." In
Methods: A Companion to Methods in Enzymology, Ed., Wiktorowicz, J.,
1992, 4, 213-226.
Andrus, A., "Gel capillary electrophoresis analysis of oligonucleotides," In
Protocols for Oligonucleotide Conjugates. In Methods in Molecular Biology,
Ed., Agrawal, S., 1994, 26, 277-300.
Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A
Laboratory Manual, 1989, Cold Spring Harbor Press, Cold Spring Harbor,
NY.
Information subject to change without notice.
Last updated May 1996
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