What are DIG - labelled probes?
Answers
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.
The DIG- labelled probes is detected by an enzyme - linked immunoassay using an antibody to digoxigenin( anti - DIG ) to what alkaline phosphatase has been conjugated . A chromogenic or chemiluminogenic substrate FRIEND alkaline phosphatase can then be used to detect the DIG - labelled probes.