PCR DIG Probe Synthesis Kit From Roche

The PCR DIG Probe Synthesis Kit is a non-radioactive system using digoxigenin (DIG), a steroid hapten, to label DNA probes for Southern hybridization. The labeled dUTP can be easily incorporated by enzymatic nucleic-acid synthesis using DNA polymerases and the polymerase chain reaction (PCR). The kit allows the amplification of minute amounts of DNA template, 10 – 100 pg of plasmid DNA or 1 – 50 ng of genomic DNA; purity of the template is not very critical. The kit is especially designed for the generation of highly sensitive hybridization probes labeled with DIG-dUTP (alkali-labile) that are suitable for the detection of low (single) copy target sequences in 10 ìg of DNA. The only prerequisites are that target sequence information is needed in order to synthesize the appropriate primers; in addition, template DNA and a thermocycler are required. Before starting PCR amplification, cycling parameters (e.g. annealing temperature, template concentration, primer sequence, and primer concentration) need to be optimized for each template and primer set in the absence of DIG-dUTP before attempting incorporation of DIG. Once the PCR conditions have been standardized, the labeling is very easy and quick. Two tubes need to be set up for the synthesis of each probe: One for labeled and another for unlabelled probe, each with the recommended concentrations of the reagents. Controls are also provided and should be used, though once the user is well-versed with the kit, the controls can be excluded. The detection sensitivity of the labeled probes can be adjusted by varying the ratio of the unlabeled nucleotides to DIG-dUTP. However, the kit contains the standard ratio of 1:3 (DIG-dUTP:dTTP), ensuring reliable detection of single-copy genes in the genomic blots following hybridization.

The main advantage of the kit is that it can be used when only a small amount of template for probe synthesis is available, or if the template DNA is not of high quality. A further advantage of this kit is that the labeling efficiency for PCR labeled probes can be determined quickly by quantitative gel electrophoresis and no special equipment or reagents are required for estimating quantity. To best estimate labeling efficiency, run a portion (5 ìl of each reaction) on an agarose mini gel and then stain the gel with ethidium bromide (EtBr).The control probe provided with the kit will have an apparent size of 500 – 550 bp. The actual size of the amplicon is 442 bp. The presence of DIG in DNA makes it run slower in the gel than unlabeled DNA. After PCR amplification, the unlabeled experimental probe should be the predicted size and labeled experimental probe should migrate slower than unlabeled probe due to the incorporation of DIG molecules. When a high ratio of DIG-dUTP:dTTP (1:3) is used, the reaction will make less labeled probe than unlabeled probe because the polymerase is slowed by the presence of the DIG hapten. I recommend gel purification of the labeled probe after PCR amplification in order to minimize background in Southern hybridization. If on the gel, the labeled PCR product intensity is high or equal to the kit control, use 1-2 ìl probe per ml hybridization buffer. If the band intensity was very faint, use up to 4 ìl probe per ml hybridization buffer. I have successfully used this kit for constructing several labeled probes of 400-500 bp and mainly for determination of GFP integration in transfected murine cell lines and murine tissue samples. I have consistently obtained good results. Though I used a higher concentration (15-30 ìg) of template DNA for Southern hybridization than mentioned in the product data sheet, it might be due to varying transfection efficiencies.

Hybridization was performed using standard procedures. Probe-target hybrids were detected with an alkaline- phosphatase-conjugated antibody either by a color reaction or by a chemiluminescence reaction. I recommend CDP-Star for chemiluminescent reactions because it saves time since light development is faster (about 10 times faster) and more intense than CSPD, thus, exposure times are significantly reduced. Multiple exposures are possible as the signal lasts for approximately 2 days. The labeled probe contains an alkali-labile DIG-11-dUTP formulation. This enables simple removal of the DIG label following chemiluminescent detection, and allows the subsequent re-hybridization of blots with multiple DIG-labeled probes. The PCR-labeled DIG probe should be stored at -15 to -25°C and is stable for at least 1 year. The probe can be used 3-4 times without any effect on the signal intensity.

Overall, this kit provides easy, simple and quick way of labeling DNA probes for Southern hybridization without involving any hazardous radioactive materials.