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DIG Labeling Methods

DIG DNA Labeling by PCR

PCR labeling is the preferred method for preparing DIGlabeled probes when the template is available in only limited amounts, is only partially purified, or is very short. It requires less optimization than other methods and produces a high yield of labeled probe.

In PCR labeling, a thermostable polymerase incorporates DIGdUTP as it amplifies a specific region of the template DNA. The result is a highly labeled, very specific, and very sensitive hybridization probe.

dig-labeling-methods-fig1

Reaction principle

During a standard PCR reaction Digoxigenin11dUTP is incorporated into newly synthesized DNA. The polymerase chain reaction (PCR) allows the amplification of minute amounts of DNA to levels above 1 μg. The only prerequisite is that some sequence information of the target sequence is needed in order to synthesize the appropriate primers.

The nonradioactive DIG system uses digoxigenin, a steroid hapten, to label DNA, RNA, or oligonucleotides for hybridization, and subsequent coloror luminescence detection. The digoxigenin is coupled to dUTP via an alkali-labile ester bond. The labeled dUTP can be easily incorporated by enzymatic nucleic acid synthesis using DNA polymerases. The combination of nonradioactive labeling with PCR is a powerful tool for the analysis of PCR products, and also for the preparation of labeled probes from small amounts of a respective target sequence.

Critical hints about PCR labeling

PCR conditions

  • Optimize PCR amplification parameters (cycling conditions, template concentration, primer sequence, and primer concentration) for each template and primer set in the absence of DIGdUTP before attempting incorporation of DIG.

Template

  • For best results, use cloned inserts as template. Genomic DNA can be more difficult to use.
  • Template concentration is crucial to successful production of specific probes.

Labeling

The PCR DIG Probe Synthesis Kit requires less optimisation than most labeling methods, because it contains
the Expand High Fidelity Enzyme Blend. This enzyme blend:

  • Can efficiently use GCrich regions as template
  • For most templates, requires no optimization of MgCl2 concentration; that is, most labeling reactions will work with the standard concentrations of 1.5 mM MgCl2
  • Some DNA templates (especially those with high GC content or longer templates) are not efficiently amplified in the presence of the "standard" concentration of DIGdUTP (i.e., 0.07 mM, when 5 μl PCR DIG Probe Synthesis Mix of (vial 2) is added to a 50 μl reaction). For these templates, use the nucleotide shock solution (vial 4) of the PCR DIG Probe Synthesis Kit to vary the concentration of DIGdUTP in the reaction.

DIG Random Primed DNA Labeling

Random primed labeling can label templates of almost any length.

Note: For very short sequences, use the PCR labeling method for best results.

These labeled probes are especially suitable for single copy gene detection on genomic Southern blots and in screens of recombinant libraries. Of course, they also work for dot/slot blots, and northern blots.

Since each primer has a different six-base sequence, the labeled probe product will actually be a collection of fragments of variable length. Thus, the labeled probe will appear as a smear, rather than a unique band on a gel. The size distribution of the labeled probe depends on the length of the original template.

dig-labeling-methods-fig2

Reaction principle

In random primed labeling, Klenow enzyme copies DNA template in the presence of hexameric primers and alkalilabile DIG-11-dUTP. On average, the enzyme inserts one DIG moiety in every stretch of 20-25 nucleotides. The resulting labeled product is a homogeneously labeled, sensitive hybridization probe (can detect as little as 0.10 – 0.03 pg target DNA).

Note: The spacing of the DIG molecules is very important. If DIG molecules were closer to each other, steric hindrance would prevent the large antiDIG antibody from binding to the labeled probe.

Nick Translation Labeling of dsDNA with Nick Translation Mixes for In Situ Probes

The nick translation procedure was originally described by Rigby1 and used for incorporating nucleotide analogs by Langer2. The procedure described here incorporates one modified nucleotide (DIG, biotin, fluorescein, or tetramethylrhodamine-dUTP) at approximately every 20-25th position in the newly synthesized DNA. This labeling density allows optimal enzymatic incorporation of the modified nucleotide and produces the most sensitive targets for indirect (immunological) detection. For in situ hybridization procedures, the length of the labeled fragments obtained from this procedure should be about 200-500 bases.

Note: DNA does not need to be denatured before it is labeled by nick translation.

dig-labeling-methods-fig3

Reaction principle

E. coli polymerase I and DNase I are added to a dsDNA sample to initiate a standard nick translation reaction during which digoxigenin-11-dUTP is incorporated into newly synthesized DNA. Nick translation is the method of choice for in situlabeling of nucleic acids.

References

  1. Rigby, P. W. J.; Dieckmann, M.; Rhodes, C.; Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237–241.
  2. Langer, P. R.; Waldrop, A. A.; Ward, D. C. (1981) Enzymatic synthesis of biotinlabeled polynucleotides: Novel nucleic acid affinity probes.Proc. Natl. Acad. Sci. USA 78, 6633–6637.

Transcriptional Labeling of RNA Probes

For some applications, DIGlabeled RNA is a more effective hybridization probe than DIG-labeled DNA. For example, DIGlabeled RNA probes can detect rare mRNAs in nanogram amounts of total RNA. These labeled RNA probes are generated by in vitro transcription from a DNA template. In the RNA transcription method, DNA is cloned into the multiple cloning site of a transcription vector between promoters for different RNA polymerases (such as T7, SP6, or T3 RNA polymerase). The template is then linearized by cleavage of the vector at a unique site (near the insert). An RNA polymerase transcribes the insert DNA into an antisense RNA copy in the presence of a mixture of ribonucleotides (including DIG-UTP). During the reaction, the DNA can be transcribed many times (up to a hundredfold) to generate a large amount of fulllength DIG-labeled RNA copies (10-20 μg RNA from 1 μg DNA in a standard reaction). DIG is incorporated into the RNA at approximately every 25-30 nucleotides.

dig-labeling-methods-fig4

Reaction principle

The DNA template to be transcribed is cloned into the polylinker site of an appropriate transcription vector, which contains promoters for SP6 and / or T3 and T7 RNA polymerases. After linearization at a suitable site, RNA is transcribed in the presence of DIG-11-UTP. Under standard conditions, approx. 10 μg of full-length DIG-labeled RNA are transcribed from 1 μg template.

Critical hints about RNA probe labeling

RNases

RNases are ubiquitous and do not require any cofactors for activity. If you want to be successful, take all possible precautions to prevent RNase contamination. For instance:

  • As far as possible, use disposable plasticware, ovenbaked glassware, or plasticware that has been decontaminated with RNase ZAP or similar reagents.
  • Prepare all solutions with water that has been treated with diethyl-pyrocarbonate (DEPC) or dimethyldicarbonate (DMDC). If possible, autoclave the solutions.
  • Wear gloves throughout the procedure. Template purity
  • Labeling efficiency depends greatly on the purity of the DNA template. Template should be highly purified.
  • The final template must be linearized, phenol/chloroform extracted and ethanol precipitated.

Template sequence

  • Some primer and/or polylinker regions in DNA templates are homologous to portions of the ribosomal 28s and 18s RNA sequences. Therefore, labeled probes may generate specific, but unwanted signals in samples that contain these prominent RNAs. To minimize this effect, remove as much of the polylinker sequences from your template as possible.
  • If you use PCR to make the DNA template, the product of the Expand High Fidelity reaction contains some fragments with a single 3´ A overhang. This overhang may produce wraparound products in the transcriptional labeling reaction.

Template length

  • Optimal template length is approximately 1 kb
  • Minimum length should be at least 200 bp

Storage of probe

  • For long term stability, RNA probes should be aliquoted and stored at -20° C or -70°C
  • DIG-labeled RNA probes are stable for at least 1 year at -20°C or -70°C in ethanol

Probe sensitivity

  • To quickly determine the sensitivity of a DIG-labeled antisense RNA probe, prepare the corresponding sense RNA (unlabeled) by in vitrotranscription. Then use the purified sense transcript at varying concentrations as target on a northern blot. From the result of the blot you can easily determine the lowest amount of target (sense transcript) that can be detected by labeled probe (antisense transcript).

DIG Oligonucleotide Labeling

For some applications, such as in situ hybridization, a DIG-labeled synthetic oligonucleotide is the best hybridization probe. In addition to in situ hybridizations, DIG-labeled oligonucleotides may be used as hybridization probes in:

  • Dot/slot blots
  • Library screening
  • Detection of repeated gene sequences on Southern blots
    Note: Oligonucleotide probes are sensitive enough to detect single copy gene sequences in complex genomes if sufficient target DNA (e.g., 10 μg human genomic DNA) is present on the blot.
  • Detection of abundant mRNAs on northern blots

Several methods are available for DIG-labeling of oligonucleotides. These are summarized below.

Labeling 5´ end with DIGNHSEster

 

dig-labeling-methods-fig5

Nucleotide needed: 100 nmol
Required time and temperature: overnight, +15 to +25°C
● Requires only a small amount of template

Labeling 3´ end with DIGddUTP

 

dig-labeling-methods-fig6

Nucleotide needed: 100 pmol
Required time and temperature: 15 minutes, +37°C
Can detect: 10 pg DNA
● Requires only a small amount of template
● Labeled probes can be used without purification
● Reaction can be scaled up indefinitely (if you increase incubation time to 1 h)

Adding a 3´ tail of DIGdUTP and dATP (approx. 40 - 50 residues)

 

dig-labeling-methods-fig7

Nucleotide needed: 100 pmol
Required time and temperature: 15 minutes, +37°C
Can detect: 1 pg DNA
● Requires only a small amount of template
● Produces more sensitive probes than end labeling
● Labeled probes can be used without purification
● Reaction can be scaled up indefinitely

Estimation of Probe Yield by the Direct Detection Procedure

To add the correct amount of probe to a hybridization, you must first determine the amount of DIG-labeled probe produced in the labeling reaction. The direct detection procedure given here compares the amount of DIG label in a series of dilutions prepared from the labeled probe with a known concentration of a DIG-labeled control nucleic acid.

Note: If you label a DNA probe by PCR, you do not need to perform a direct detection to evaluate the yield.
For PCR-labeled probes, use the gel electrophoresis evaluation method.

The direct detection involves the following steps:

  • Preparing serial dilutions of labeled probe and spotting them on a nylon membrane (time required: 15 minutes)
  • Detecting DIG in spots with chemiluminescence; time required (2-2.5 hours)

DIG is a trademark of Roche
For life science research only. Not for use in diagnostic procedures