Thermo Fisher Scientific UB Subject: Biotin Labeling of Oligonucleotides on a DNA/RNA Synthesizer Owner's manual

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Model: UB Subject: Biotin Labeling of Oligonucleotides on a DNA/RNA Synthesizer

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User Bulletin: ABI 392/4

Nucleic Acid Synthesizers

Subject: Biotin Labeling of

Oligonucleotides on a DNA/RNA

Synthesizer

User Bulletin - Number 70

Models 38X/39X

Biotin Labeling of Oligonucleotides

Biotin Amidite, a phosphoramidite for preparation of 5' biotin-labeled

oligonucleotides, is now available from Applied Biosystems for use on all

DNA/RNA synthesizers.

Biotin-labeled oligonucleotides are used in a diverse array of capture and

detection hybridization experiments, including PCR and sequencing. Biotin is a

small molecule with phenomenally strong and specific binding affinity for the

protein avidin. This allows for isolation of biotinylated nucleic acids from a

complex mixture. Avidin, or streptavidin, is usually immobilized on a solid matrix,

facilitating isolation and purification of the complex. The biotin/avidin complex can

be disrupted under certain nondestructive conditions, releasing the biotinylated

single- or double-stranded nucleic acids.

Chemical Structure

The chemical structure of Biotin Amidite (Figure 1) is identical to that published by

Dr. Richard T. Pon, of the University of Calgary, Canada (see the References at

the end of this User Bulletin).

Figure 1. Chemical structure of Biotin Amidite

The long linker arm between the biotin and the phosphorous atom provides for

efficient capture and binding, unperturbed hybridization and efficient

phosphoramidite coupling. Biotin Amidite contains a dimethoxytrityl (DMT) group

on the biotin ring system, which facilitates coupling yield analysis, minimizes

synthesis impurities and allows trityl-selective, post-synthesis purification by the

Oligonucleotide Purification Cartridge (OPC). Biotin Amidite labels

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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB70.html

oligonucleotides with a single biotin at the 5' end (Figure 2).

Figure 2. 5' Biotin-labeled oligonucleotide

The traditional method of biotin labeling requires the reaction of a large excess of

the biotin NHS ester with an amino-linked oligonucleotide in an off-line solution

coupling reaction. The excess biotin must be carefully removed by stringent

purification methods such as preparative HPLC or PAGE. Coupling efficiency can

be highly variable, complicating the separation of biotinylated and unlabeled

oligonucleotides. Leveraging the solid support chemistry of DNA synthesis, a DNA

synthesizer such as the Model 392 can easily wash away all excess reagent.

Installation

Biotin Amidite is available in two convenient sizes: 85 and 250 mg. The smaller

size is sufficient for producing 6-8 biotin-labeled oligonucleotides at the 0.2-µmol

scale. The larger size makes up to 18 biotin-labeled oligonucleotides at the

0.2-µmol scale, enough to satisfy the needs of large-scale users or

high-throughput laboratories.

1. For 0.2-µmol, 1-µmol and 10-µmol-scale syntheses in which a 0.1 M

solution is needed:

Dilute either 85 mg of Biotin Amidite with 1 mL of anhydrous acetonitrile, or

250 mg of Biotin Amidite with 3 mL (<90 ppm water), by the manual

method with a dry syringe. Alternatively, the 0.25-g auto-dilute procedure

can be used on Model 392 and 394 synthesizers with the 250-mg Biotin

Amidite.

For 40-nmol synthesis with Applied Biosystems polystyrene

columns:

Dilute either 85 mg of Biotin Amidite with 2 mL of anhydrous acetonitrile, or

250 mg with 6 mL. The 0.5-g auto-dilute procedure can be used on Model

392 and 394 DNA synthesizers with the 250-mg Biotin Amidite.

Biotin Amidite can be placed at any monomer position of any DNA/RNA

synthesizer. Normally, bottle positions 5-8 are used for specialty reagents.

Biotin Amidite can be placed at any monomer position of any DNA/RNA

synthesizer. Normally, bottle positions 5-8 are used for specialty reagents.

2. To conserve the reagent, create a user-defined Bottle Change Procedure

by decreasing the delivery to waste from the Biotin Amidite bottle position

to 1 second. For example, the procedure below is designed for the 392,

with Biotin Amidite at bottle position 5

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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB70.html

Step Function Num Time

1 Begin 106 0

2 Block Flush 1 5

3 18 to Waste 64 7

4 18 to 5 74 3

5 Flush to 5 10 10

6 Interrupt 104 0

7 Flush to 5 10 5

8 5 to Waste 54 1

9 18 to Waste 64 7

10 Block Flush 1 5

11 End 107 0

Remember to use the appropriate functions for steps 4, 5, 7 and 8 if you

use a base bottle other than bottle 5 for Biotin Amidite.

Use this new bottle change procedure to install Biotin Amidite on base

bottle position 5, or on the appropriate bottle position if you use a different

bottle position.

Synthesis

1. To conserve the reagent, create a user-defined Begin Procedure by

decreasing the delivery to waste from the Biotin Amidite bottle position to 1

second. For example, the procedure below is for the 392 with Biotin

Amidite at bottle position 5.

Step Function Num Time

1 Begin 106 0

2 Phos Prep 101 10

3 A to Waste 50 2

4 G to Waste 51 2

5 C to Waste 52 2

6 T to Waste 53 2

7* 5 to Waste 54 1

8 Tet to Waste 58 2

9 18 to Waste 64 10

10 Block Flush 1 10

11 End 107 0

*Remember to use the appropriate function for step 7 if you do not use base bottle 5 for the

Biotin Amidite.

2. Enter the sequence of the labeled oligonucleotide, with the Biotin Amidite

position denoted by the bottle position number at the 5' end.

For example, if the desired oligonucleotide sequence is: 5' > TGT AAA

ACG ACG GCC AGT < 3', you will enter: 5' > 5TG TAA AAC GAC GGC

CAG T < 3'.

3. To create a user-defined cycle, insert an additional "wait" function step of

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120 seconds after the existing coupling wait.

Although Biotin Amidite can be used with standard cycles and coupling

times, testing shows that an additional 120-second coupling wait time

boosts coupling efficiency, especially for reagent that has been on the

synthesizer more than two days.

Below is an example of a user cycle in which Biotin Amidite is installed at

bottle position 5. Use the base specifier field to select only the base

position at which Biotin Amidite is installed.M

Step Function Num Time Base Specifier

22 B+Tet to Column 33 1.5 AGCT5

23 Push to Column 43

24 Column 2 Off 143

25 Wait 103 25.0 AGCT5

26 Wait 103 120.0 5

27 Cap Prep 102 3.0

28 18 to Waste 64 4.0

4. Select "trityl-on" for OPC purification.

Note: OPC purification is always recommended for biotin-labeled oligonucleotides.

5. Set up your instrument as follows:

For syntheses at the 40-nmol scale: Use 0.05 M phosphoramidites and

Biotin Amidite, along with the 0.2-µmol scale cycle and an additional

120-second coupling wait step.

For syntheses at the 0.2-µmol, 1-µmol and 10-µmol scales: Use 0.1 M

phosphoramidites and Biotin Amidite, along with the appropriate scale

cycle and an additional 120-second coupling wait step.

Biotin Consumption

The following are expected couplings per synthesis scale, using Biotin Amidite in

either the 85- or 250-mg bottle. The larger size reagent gives approximately three

times that of the smaller.

Size

(mg) Synthesis

Scale Approximate

Couplings

85 40 nmol 12

85 0.2 µmol 6

85 1 µmol 4

85 10 µmol 1

250 40 nmol 36

250 0.2 µmol 18

250 1 µmol 12

250 10 µmol 3

Deprotection

Biotin-labeled oligonucleotides should be deprotected in ammonia for 5-8 hours at

55 °C.

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Purification

OPC purification is conducted by the usual protocol, with the detritylation step

(including TFA) extended to 10 minutes (see either DNA Synthesis User Bulletin

59, or the OPC product tray label). The trityl-selective OPC media binds only the

oligonucleotide labeled with a Biotin that contains a DMT group. Unlabeled failure

sequences and other impurities are washed away, followed by the elution of

purified, desalted biotin-labeled oligonucleotide in 1 mL of 20% acetonitrile in

water.

Analysis

Crude or OPC-purified biotin-labeled oligonucleotide may be analyzed by the

conventional techniques of reverse-phase HPLC, PAGE or MicroGel capillary

electrophoresis (CE). Biotin increases the hydrophobicity of the oligonucleotide. A

biotin-labeled oligonucleotide will migrate approximately one base slower on

PAGE. By HPLC and MicroGel CE analysis, biotin-labeled oligonucleotides elute

later than unlabeled sequences. The sample shown in Figure 3 is the 5'

biotin-labeled M13 forward primer, analyzed by MicroGel CE after OPC

purification.

Figure 3. MicroGel CE of 5' biotin-labeled

TGT AAA ACG ACG GCC AGT 3'

Yield

For a typical synthesis of a biotin-labeled oligonucleotide at the 40-nmol scale, a

yield of 2 ODU (optical density units) of purified product (~60 µg) can be

expected.

Storage

Recommended storage conditions for Biotin Amidite powder are similar to those

for nucleoside phosphoramidites. It should be kept dry and may be stored at room

temperature. Precise shelf life is unknown. We recommend not storing the dry

reagent for more than six months.

After it has been reconstituted in acetonitrile, Biotin Amidite should be installed on

the DNA synthesizer and used as soon as possible. Coupling efficiency will

decrease below 90% after two days. Although OPC purification will remain

effective, the yield of biotin-labeled oligonucleotides will be lessened. Regardless

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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB70.html

of care and technique, the Biotin Amidite/acetonitrile solution undergoes

significant loss of coupling efficiency when removed from the DNA synthesizer,

stored and later reinstalled.

Conclusion

Biotin Amidite makes biotin labeling of oligonucleotides more accessible, with no

complex analytical and preparative equipment required. Production of

biotin-labeled primers now rivals that of standard, unlabeled primers for ease and

simplicity.

To Order Biotin Amidite

For easy reference when ordering, most of the Applied Biosystems products

mentioned in this User Bulletin are listed below.

P/N Description Quantity

401395 Biotin Amidite 85 mg

401396 Biotin Amidite 250 mg

400771 Oligonucleotide Purification Cartridges (OPC) 10/package

400613 Triethylamine Acetate, 2.0 M 200 mL

400137 Trifluoroacetic Acid, Neat Liquid 450 mL

0652-0075 MicroGel Capillaries 3/package

References

1. Applied Biosystems DNA Sequencing User Bulletin 21, Magnetic Beads

(Dynabeads) Used as Solid Support in Purification and Cycle Sequencing

of PCR Products, April 1991.

2. Applied Biosystems DNA Synthesis User Bulletin 59, New Applications for

the Oligonucleotide Purification Cartridge, March 1991.

3. Applied Biosystems DNA Synthesis User Bulletin 61, 40-nanomole

Polystyrene: New Highly Efficient DNA Synthesis Columns, May 1991.

4. Applied Biosystems DNA Synthesis User Bulletin 67, FAM Amidite, May

1992.

5. Applied Biosystems, Evaluating and Isolating Synthetic Oligonucleotides,

1992.

6. Dubrow, R.S. "Analysis of Synthetic Oligonucleotide Purity by Capillary Gel

Electrophoresis," Amer. Laboratory, March, 1991, 64.

7. Eadie, J.S., McBride, L.J., Efcavitch, J.W., Hoff, L.B. and Cathcart, R.

"High Performance Liquid Chromatographic Analysis of

Oligodeoxyribonucleotide Base Composition," Anal. Biochem. 1987, 165,

442-447.

8. Goodchild, J. "Conjugates of Oligonucleotides and Modified

Oligonucleotides: A Review of Their Synthesis and Properties,"

Bioconjugate Chemistry, 1990, 1, 165-187.

9. Hultman, T., Stahl, S., Hornes, E. and Uhlen, M. "Direct Solid Phase

Sequencing of Genomic and Plasmid DNA Using Magnetic Beads as Solid

Support," Nucleic Acids Res. 1989, 17, 4937-4946.

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User Bulletin: ABI 392/4 Nucleic Acid Synthesizers http://www.pebio.com/ab/techsupp/doclib/nasynth/multi/ub/html/UB70.html

10. Keller, G.H. and Manak, M.M. DNA Probes; Stockton Press: Stockton,

1989.

11. McBride, L.J., McCollum, C., Davidson, S., Efcavitch, J.W., Andrus, A. and

Lombardi, S.J. "A New, Reliable Cartridge for the Rapid Purificaion of

Synthetic DNA," BioTechniques 1988, 6, 362-367.

12. Pon, Richard T. "A Long Chain Biotin Phosphoramidite Reagent for The

Automated Synthesis of 5'-Biotinylated Oligonucleotides," Tetrahedron Lett.

1991, 32(14), 1715-1718.

13. Uhlen, M. "Magnetic Separation of DNA," Nature 1989, 340(6236),

733-734.

14. Vu, H., McCollum, C., Jacobson, K., Theisen, P., Vinayak, R., Spiess, E.

and Andrus, A. "Fast Oligonucleotide Deprotection Phosphoramidite

Chemistry for DNA Synthesis," Tetrahedron Lett. 1990, 31, 7269-7272.

15. Wilchek, M., Bayer, E.A. "The Avidin-Biotin Complex in Bioanalytical

Applications," Anal. Biochem. 1988, 171, 1-32.

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/UB70.html

Thermo Fisher Scientific UB Subject: Biotin Labeling of Oligonucleotides on a DNA/RNA Synthesizer Owner's manual

Brand: Thermo Fisher Scientific

Model: UB Subject: Biotin Labeling of Oligonucleotides on a DNA/RNA Synthesizer

Type: Owner's manual

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Thermo Fisher Scientific UB Subject: Biotin Labeling of Oligonucleotides on a DNA/RNA Synthesizer Owner's manual

Thermo Fisher Scientific UB Subject: Biotin Labeling of Oligonucleotides on a DNA/RNA Synthesizer Owner's manual

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