( ...)The DIG System Nonradioactive and,,,Highly Sensitive Detection of Nucleic Acids

Roche Applied Science was one of the first companies to offer a nonradioactive technology allowing customers to move away from the use of hazardous radioactive isotopes. Even after 15 years and the increasing availability of many different competitor products and quantitative PCR-based technologies, the DIG System remains THE nonradioactive technology of choice to label and detect nucleic acids for multiple applications such as filter hybridization or in situ hybridization.

Compared to radioactive labeling and detection techniques, the DIG System has multiple advantages:

• High sensitivity (more sensitive than radioactivity)

• Short exposure times (minutes rather than hours or days)

• Safety (no contact with hazardous materials no environmental contamination)

• Probes are reusable and stable for a minimum of one year.

• Well-established protocols (many years of experience)

• Easy stripping and reprobing of Southern blots (alkali-labile DIG linker for DNA probes)

• Special protocol for easy stripping of Northern blots
  

The DIG System allows the safe and efficient labeling of DNA and RNA, or oligonucleotide probes. These probes can be used for all hybridization reactions, in particular

• Southern blotting, dot blotting,

• Northern blotting,

• arrays,

• colony hybridization,

• in situ hybridization, and

• ELISA.
  

Roche Applied Science provides a wide variety of kits and individual reagents for the labeling and detection of nucleic acids by different methods. A detailed overview can be found in the DIG Product Selection Guide or on our dedicated website at www.roche-appliedscience.com/dig/. Sideby- side comparisons of nonradioactive DIG applications versus radioactive experiments can also be found on this website.

DIG Application Hints
Some useful tips for your DIG labeling and detection experiments are outlined below.

Labeling and detection of DNA (Southern blotting or similar applications)

If only a small amount of template for probe synthesis is available, or if the template DNA is not of high quality, the PCR DIG Probe Synthesis Kit is recommended. This kit is specially designed for the generation of highly sensitive hybridization probes that are suitable for e.g., single- copy gene detection by PCR. A further advantage of this kit is that the labeling efficiency for PCR-labeled probes can be determined without a direct detection procedure (quantitative spot test). Instead, a quick estimate of labeling efficiency can be performed by quantitative gel electrophoresis.
If sufficient amounts of pure template DNA are available, the DIG High Prime DNA Labeling and Detection Starter Kits I and II, which use random priming, are recommended. These kits contain all of the reagents required for labeling, blocking of the blot membrane, hybridization, and detection. They are true all-in-one kits that contain everything you need for a successful experiment. The kits differ with respect to the detection method: the Starter Kit I contains reagents for color detection (NBT/BCIP), whereas the Starter Kit II contains reagents for chemiluminescence detection with CSPD.

Labeling and detection of RNA (Northern blotting or similar applications)

Northern blotting can be performed with either DNA or RNA probes. Usually, RNA probes are more specific and more sensitive. However, if ultimate sensitivity and specificity are not required, a DNA probe may be more convenient.

If an RNA probe is to be used, the DIG Northern Starter Kit is recommended. This kit uses linearized DNA as a template and SP6, T7, or T3 RNA polymerase for the incorporation of DIG-11-UTP into the RNA transcript. Detection is performed by a chemiluminescence reaction with CDP-Star. The DIG RNA Labeling Kit contains suitable RNA expression vectors and labeling reagents.

Quantification of a labeled probe

Since the DIG System is very sensitive, it is important to work with defined amounts of probe and template to ensure optimal results. It is also important to check the efficiency of each labeling reaction by determining the amount of DIG-labeled product via spot test. This will enable you to add the correct amount of probe to the hybridization solution (Table 1).

The consequences of using the wrong amount of probe in a hybridization are obvious. Too much probe will lead to background problems; too little probe will lead to little or no hybridization signal. Table 1 lists the suggested amount of DIG-labeled probe for common hybridizations.
The benefits of the system: DIG PCR probes may be easily estimated by loading an aliquot onto an agarose gel. RNA probes may be examined for integrity (but not quantified) by gel electrophoresis.

Hybridization conditions

Optimal hybridization conditions strongly depend on the type and GC content of the hybrids. RNARNA and RNADNA hybrids will require higher hybridization temperatures than DNADNA hybrids. In general, the relative strength of different hybrids is RNARNA hybrids > RNADNA hybrids > DNADNA hybrids. As a rule of thumb, for mammalian targets containing 40% GC, the optimal hybridization temperatures in the presence of DIG Easy Hyb or 50% formamide are:

• DNA-DNA: 3742C

• DNA-RNA: 50C

• RNA-RNA: 68C
  

We strongly recommend the use of the DIG EASY HYB buffer, which was specially developed for the DIG System and is nontoxic and guaranteed to be free of contaminants, DNase and RNase.

Amount of target nucleic acid to load on a gel

As a result of the high degree of sensitivity of the DIG System, the amount of target loaded is less than that of comparable systems (Table 2). Note that nylon membranes, positively charged, give the strongest signals and the lowest background when detecting DIG-labeled hybrids.

Detection methods

Probe-target hybrids are usually detected with an alkaline- phosphatase-conjugated antibody either by a color reaction or by a chemiluminescence reaction (Table 3). Of the many alkaline phosphatase substrates that can be used, we recommend CDP-Star or CSPD for chemiluminescence reactions and NBT/BCIP for color reactions.

Detection substrates

• CDP-Star ready to use
  

Detection of alkaline phosphatase activity with CDP-Star results in light emission that can be recorded by exposure to X-ray film or with an imaging instrument. CDPStar has the same characteristics as CSPD. However, it saves time since light development is faster (about ten times faster than CSPD) and more intense, significantly reducing exposure time. Multiple exposures are possible as the signal lasts for approximately two days. Note that the chemiluminescent substrates can only be applied on nylon membranes.

• CSPD ready to use
  

Detection of alkaline-phosphatase activity with CSPD results in light emission that can be recorded by exposure to X-ray film (e.g., Lumi-Film) or with an imaging instrument. Because of the long lasting glow, multiple exposures are easily achieved. Blots developed with CSPD or CDP-Star can be easily stripped and reprobed if nylon membranes are used.

• NBT/BCIP

The advantage of using a color substrate such as NBT/BCIP is that no X-ray film is required, and that it can be used with nitrocellulose and nylon membranes. However, if a high sensitivity is to be achieved the color reaction can take several hours. Other drawbacks are that only a single result can be obtained (no multiple exposures possible) and that reprobing of the membrane requires removal of the precipitated color with dimethylformamide.

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