Thermo Fisher Scientific Subject: Phosphalink Amidite Owner's manual

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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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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB86.html

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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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB86.html

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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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB86.html

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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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB86.html

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

6 of 7 11/23/99 8:09 AM

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,"

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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

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8923-8927.

Barany, F., "Genetic disease detection and DNA amplification using cloned

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Barany, F., "The ligase chain reaction in a PCR world," PCR Methods and

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Winn-Deen, E. S. and Iovannisci, D. M., "Sensitive fluorescence method

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Winn-Deen, E. S. and Iovannisci, D. M., Brinson, E. C. and Eggerding, F.

A., "Application of DNA probe ligation detection to genetic disease

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Iovannisci, D. M. and Winn-Deen, E. S., "Ligation amplification and

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Highuchi, R. G. and Ochman, H., "Production of single-stranded DNA

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Lee, L. G., Connell, C. R., and Bloch, W., "Allelic discrimination by

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Andrus, A., "Oligonucleotide analysis by gel capillary electrophoresis." In

Methods: A Companion to Methods in Enzymology, Ed., Wiktorowicz, J.,

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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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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB86.html

Thermo Fisher Scientific Subject: Phosphalink Amidite Owner's manual

Brand: Thermo Fisher Scientific

Model: Subject: Phosphalink Amidite

Type: Owner's manual

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Thermo Fisher Scientific Subject: Phosphalink Amidite Owner's manual

Thermo Fisher Scientific Subject: Phosphalink Amidite Owner's manual

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