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Features

Non-radioactive labeling of DNA.
Efficient priming of labeling reactions with random decamers.
High yields with Klenow Fragment, exo-: no degradation of a labeled probe during reaction.

Applications

Generation of biotin-labeled DNA probes for a variety of non-radioactive hybridization experiments, including Southern and Northern blots, colony/plaque hybridizations, dot/slot blots and in situ hybridizations.

Description

The Biotin DecaLabel™ DNA Labeling Kit is an advanced system for the efficient synthesis of biotin-labeled DNA probes, based on an improved random-primed labeling method originally developed by Feinberg and Vogelstein (1, 2). The primary improvement over traditional random-primed method involves the use of random decamers instead of hexamers to ensure more efficient annealing with DNA at 37°C. Klenow Fragment, exo- is also included in the kit; this genetically engineered enzyme has no detectable exonuclease activity. Therefore, the enzyme does not degrade the labeled probe during reaction, which results in a high labeling yield even with low amounts of template. As a result, DNA fragments of any length can be uniformly labeled. Biotin-labeled DNA is detected with the Biotin Chromogenic Detection Kit or with conventional biotin-avidin or biotin-streptavidin detection systems.

Principle

Random decamers are annealed to a denatured template DNA molecule and new strands are synthesized by the Klenow Fragment, exo- in the presence of biotin-dUTP. During this reaction, the biotinylated nucleotides are incorporated into the newly synthesized complementary DNA strand (see Fig.1).

Quality Control

The kit is tested in a control labeling reaction. The biotin-labeled DNA probe is used in dot-blot. Low amounts of homologous DNA (0.1-0.03 pg) are detected after 16 hours of color development with Biotin Chromogenic Detection Kit.

Components

Klenow Fragment, exo-
Decanucleotides in Reaction Buffer
Biotin Labeling Mix
Control Template
Biotin-labeled DNA
Water, nuclease-free
Detailed Protocol

Figure 1. DNA labeling by the random primed method.
* [α-³²P]-dNTP, [α-³³P]-dNTP, biotin-dUTP, fluorescein-, aminoallyl- or DIG-dUTP can be used.

Figure 2. Dot-blot hybridization with a biotin-labeled probe.
Lambda DNA/HindIII was biotin-labeled with the Biotin DecaLabel™ DNA Labeling Kit and used as a hybridization probe in a dot-blot of the homologous DNA on SensiBlot™ Plus Nylon Membrane. The blot was developed with the Biotin Chromogenic Detection Kit.

Patents, Licenses, Trademarks

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Southern Blotting. Dot Blotting

Required solutions

Denaturation solution: 1.5 M NaCl, 0.5 M NaOH.
Neutralization solution: 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2), 1 mM EDTA.
20X SSC, pH 7.0 (blotting buffer): 3 M NaCl, 0.3 M sodium citrate.
100X Denhardt\'s solution: 2% (w/v) BSA, 2% (w/v) Ficoll™, 2% (w/v) PVP (polyvinylpyrrolidone).
Pre-hybridization solution: 6X SSC, 5X Denhardt's solution, 50% formamide, 0.5% SDS.

Electrophoresis
Load genomic DNA probes along with the marker (e.g. DNA Markers for Genomic DNA analysis) on 0.7% agarose gel (20 cm length). Run for 18 hours at 3 V/cm in 1X TAE buffer.
Southern Blotting

Rinse the gel in deionized water, add Denaturation solution and shake for 30 min at room temperature. Rinse the gel in deionized water and add Neutralization solution. Shake for 15 min at room temperature. Repeat neutralization procedure.
Fill the glass dish with 20X SSC blotting buffer. Make a platform and cover it with a sheet of Whatman™ 3 MM filter paper, saturated with the blotting buffer (see picture below).
Place the gel upside down on the filter and avoid trapping air bubbles beneath it.
Cut a sheet of SensiBlot™ Plus Nylon Membrane to match the size of the gel and place it on the top of the gel. Avoid trapping air bubbles beneath the membrane.
Cut 2-3 sheets of Whatman™ 3 MM filter paper to the size, wet with blotting buffer and place on the top of the membrane.
Place a stack of absorbent paper towels on top of the 3 MM paper, place a glass plate on the top of the paper towels and put a 0.5 kg weight on the top.
Allow upward capillary transfer of DNA at room temperature for 18 hours.
Wash the membrane in 2X SSC buffer to remove any residual agarose, dry at room temperature and fix for 2 min under UV-light.

Dot Blotting

Prepare several dilutions of the labeled probe (from 1 ng/µl to 10 fg/µl) and spot 1 µl of each dilution onto a nylon membrane strip.
Air-dry the spotted membrane at room temperature for 30-45 minutes or at 80°C for 10 minutes.
Place the membrane on a UV trans-illuminator (spotted side down) and cross link the probe to the membrane for 1-5 minutes.

Note

The spotted membrane can be stored at 4°C or at room temperature in a plastic bag until needed.
Generation of Labeled Probes
Two labeled probes are prepared using Biotin DecaLabel™ DNA Labeling Kit, DecaLabel™ DNA Labeling Kit or using protocol for random-primed labeling.

Hybridization probe for the genomic DNA (test probe).
Hybridization probe for visualization of DNA Marker (e.g. DNA Markers for Genomic DNA analysis). 50 ng of marker is sufficient for generation of radioactively labeled probe for 3-5 hybridization reactions.

Hybridization

Prepare 30 ml of the pre-hybridization solution.
Denature sonicated salmon sperm DNA solution (10 mg/ml) by heating at 100°C for 5 min. Chill on ice and add to the pre-hybridization solution to a final concentration of 50-100 µg/ml.
Place the membrane into the hybridization container, add pre-hybridization solution with the denatured salmon sperm DNA (0.2 ml/cm² of membrane) and pre-hybridize for 2 hours at 42°C with shaking.
Prepare the hybridization solution:
- mix the two prepared probes: labeled probe for the DNA marker and probe for genomic DNA. Denature by heating at 100°C for 5 min and chill immediately on ice.
Add the following amounts of the probe mixture to the pre-hybridization solution:
- to 10 ng/ml (1/5 of probe mix) if specific activity is 10⁸ dpm/µg,
- to 2 ng/ml (1/25 of probe mix) if specific activity is 10⁹ dpm/µg,
- to 25-100 ng/ml if non-radioactively labeled probes.
Discard the pre-hybridization solution (from step 3) and add the prepared hybridization solution to the hybridization bag (60 µl/cm²). Incubate for at least 12 hours at 42°C.
Carry out the following washes of the membrane:
- twice in 2X SSC + 0.1% SDS for 10 min at room temperature,
- twice in 0.1X SSC + 0.1% SDS for 10 min at 65°C (for high stringency).
Dry the membrane using sheets of Whatman™ 3 MM paper.

Autoradiography
Wrap the dried membrane with Saran Wrap™ and expose to a phosphoimager or a film with an intensifying screen.

Figure. Upward capillary transfer of DNA from agarose gels.

Feinberg, A.P., Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Biochem., 132, 6-13, 1983.
Feinberg, A.P., Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Addendum, Biochem., 137, 266-267, 1984.