User Bulletin
Number 86
Model 38X, 39X
Phosphalink Phosphorylation
October 1994 (updated October 2001)
SUBJECT:
Phosphalink™ Amidite for Phosphorylation of Oligonucleotides
Introduction This User Bulletin introduces Phosphalink™ (Fig. 1), a phosphorylating reagent for
efficient preparation of 5'- and/or 3'- end phosphorylated oligonucleotidess on any
Applied Biosystems DNA/RNA Synthesizer.
Figure 1.
Phosphalink
Gene construction, cloning, the oligonucleotides ligation assay (OLA), the ligation
chain reaction (LCR), and total cDNA sequencing are among the many applications of
5'-end phosphorylated oligonucleotidess. 5' Phosphorylation is also essential for the
use of λ 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
oligonucleotidess 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 oligonucleotidess.
The enzymatic methods require some post-synthesis processing and purification, and
may not be suitable for large-scale preparation of 5'-phosphorylated oligonucleotidess.
Phosphalink conducts chemical phosphorylation on any Applied Biosystems Division
nucleic acid synthesizer, with no special handling procedures required.
The 5'-phosphorylated oligonucleotides is differentiated from the non-phosphorylated
oligonucleotides by electrophoretic mobility or by HPLC retention time difference. The
difference in mobility shift or retention time between the two fragments is small for
oligonucleotidess 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 2mL 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.
Page 2 of 8
Phosphalink™ Amidite for Phosphorylation of Oligonucleotides
392/394 Bottle Change Procedure
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
Phosphalink™ Amidite for Phosphorylation of Oligonucleotides
Page 3 of 8
Synthesis 5'-Phosphorylated Oligonucleotidess
Enter the desired oligonucleotides sequence with the Phosphalink position (denoted
by the bottle position number) at the 5' end (Fig. 2).
Note 5'- and 3'-phosphorylated oligonucleotidess 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 oligonucleotidess from introduction of alkaline phosphatase.
Figure 2.
5' Phosphorylation
3'-Phosphorylated Oligonucleotidess
Enter the desired oligonucleotides 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).
Page 4 of 8
Phosphalink™ Amidite for Phosphorylation of Oligonucleotides
Figure 3.
3' Phosphorylation
Phosphalink™ Amidite for Phosphorylation of Oligonucleotides
Page 5 of 8
Consumption Phosphalink yields the following approximate number of couplings per bottle:
Synthesis Cycles
40-nmol CE
0.2-µmol CE
1-µmol CE
10−µmol CE
Couplings
12
6
4
1
Concentration
0.05 M
0.1 M
0.1 M
0.1 M
Deprotection Ammonia deprotection time for 5'- or 3'-phosphorylated oligonucleotidess prepared
with standard base-protected phosphoramidites is 4–8 h at 55 °C. For phosphorylated
oligonucleotidess made with FastPhoramidite™ reagents, the deprotection time is 1 h
at 65 °C, or 2 h at 55 °C.
Purification Phosphorylated oligonucleotidess can be purified by reverse phase HPLC,
anion-exchange HPLC or polyacrylamide gel electrophoresis. 3'-Phosphorylated
oligonucleotidess, when synthesized with trityl-on at the 5' end, can be purified by
Oligonucleotides Purification Cartridge (OPC™). 5'-Phosphorylated oligonucleotidess
cannot be purified by OPC since they do not have a trityl group.
Analysis 5'- and 3'-phosphorylated oligonucleotidess can be analyzed by reverse-phase HPLC,
anion-exchange HPLC, PAGE, and MicroGel capillary electrophoresis, using the same
conditions and methods as non-phosphorylated oligonucleotidess (Fig. 4).
Phosphorylated oligonucleotidess elute earlier than non-phosphorylated fragments by
MicroGel capillary electrophoresis and PAGE. Phosphorylated oligonucleotidess elute
slightly later than non-phosphorylated oligonucleotidess by anion exchange HPLC.
Figure 4.
Page 6 of 8
MicroGel CE of a 20-mer with the sequence: 5' phos-ACA TCT CCC CTA CCG
CTA TA-3'
Phosphalink™ Amidite for Phosphorylation of Oligonucleotides
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 oligonucleotidess with high coupling yield. Coupling can be conveniently
monitored by DMT cation assay, using either AutoAnalysis conductivity measurement
or UV absorbance.
Ordering
Information
Description
Phosphalink™
Quantity
70 mg
Phosphalink™ Amidite for Phosphorylation of Oligonucleotides
P/N
401717
Page 7 of 8
Bibliography 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 ELISA-based oligonucleotides 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., 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.
Highguchi, R. G., and Ochman, H., “Production of single-stranded DNA templates by
exonuclease digestion following the polymerase chain reaction,” Nucleic Acid Res.,
1989, 17, 5865.
Lee, L. G., Connell, C. R., and Bloch, W., “Allelic discrimination by nick-translation
PCR with flourogenic probes,” Nucleic Acid Res., 1993, 21, 3761–3766.
Andrus, A., “Oligonucleotides 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 Oligonucleotides 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.
© Copyright 2001, Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
Applied Biosystems is a registered trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
Phosphalink, FastPhoramidite, and OPC are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
Stock Number 239843-002
Information subject to change without notice.