Kolloquiumsunterlagen BVersion 28-8-2002

1. Background information for Southern blot analysis

Southern blotting has been one of the cornerstones of DNA analysis since its first description by E.M. Southern (1975). Southern was the first to show that immobilization of size-fractionated DNA fragments could be carried out in a reliable and efficient manner. The advent of Southern transfer and the associated hybridization techniques made it possible for the first time to obtain information about the physical organization of single and multicopy sequences in complex genomes. The term Southern blotting, now used to describe any type of DNA transfer from gel to membrane.

1.1. Critical Parameters

The amount of DNA that must be loaded depends on the relative abundance of the target sequence to which hybridization will take place. The detection limit of a radioactive probe with a specific activity of 108 to 109 dpm/µg is about 0.5 pg DNA. Thus, for human genomic DNA, 10mg – equivalent to 1.5 pg of a single-copy gene 500 bp in length – is a reasonable minimum quantity to load. Similar sensitivities can be achieved today with nonradioactive probes (see DIG system later).

Optimization of the parameters that influence Southern blotting must be carried out in conjunction with hybridization analysis, as the efficiency of transfer can be assessed only from the appearance of the x-ray film obtained after the membrane is probed. The following paragraphs describe the most important factors that should be considered.

1.2. Choice of membranes

Originally nitrocellulose membranes were used but their fragility prompted to search for alternative types of support matrix, resulting in the introduction of nylon membranes in the early 1980s. The main advantages of nylon membranes is their greater tensile strength and that DNA can be bound covalently by UV cross-linking. Nylon membranes can therefore be reprobed up to about 12 times without becoming broken or losing their bound DNA. In contrast nitrocellulose membranes are fragile (allow only 3 times reprobing) and do not bind DNA covalently: baking to immobilize the DNA leas to a relatively weak hydrophobic attachment through exclusion of water. The disadvantage of nylon membranes is the amount of background signal seen after hybridization. On the other site nylon membranes are able to bind about five times more DNA per cm2 than nitrocellulose. Nylon retains DNA fragments down to 50 nucleotides in length, but nitrocellulose is inefficient with molecules < 500 nucleotides. Hence nitrocellulose is not a good choice for restriction-digested genomic DNA or PCR fragments if the target band for hybridization probing is likely to be < 500 bp.

1.3. Transfer buffer

With nitrocellulose the transfer buffer must provide a high ionic strength to promote binding of the DNA to the membrane. Several formulations exists but 20 x SSC is recommended because it is easy to make up and can be stored for several months at room temperature. Lower SSC concentrations (e.g. 10x) should not be used with nitrocellulose as the lower ionic strength may result in loss of smaller DNA fragments during transfer. Alkaline transfer is not suitable for nitrocellulose as they do not retain DNA at pH 9.0 and fall apart after long exposure to alkali.

Nylon membranes are able to bind DNA under a variety of conditions (acid, neutral, alkaline, high or low ionic strength) but a high-salt buffer such as 20x or 10x SSC or SSPE appears to be beneficial. Positively charged nylon membranes have an additional advantage as they bind DNA covalently after transfer in an alkaline buffer (0.4 M NaOH). The only problem with alkaline blotting is that high backgrounds may result if a chemiluminescent detection system is used.

1.4. Duration of transfer

The most difficult parameter to evaluate is duration of transfer. In a capillary system, rat of transfer depends on the size of the DNA, thickness of the gel, and agarose concentration. Upward capillary transfer is slow, the architecture of the blot crushes the gel and retards diffusion of the DNA. With a high-salt buffer, it takes appr. 18 hrs to obtain acceptable transfer of a 15 kb molecule from a 5 mm thick 0.7% agarose gel; with the same gel 90% of the 1 kb molecules will be transferred in 2 hrs. This problem is partially alleviated by the depurination step, which breaks larger molecules in to fragments 1 to 2 kb in length, thereby reducing the time needed for their transfer. If the gel is thicker than 5 mm or has an agarose concentration > 1 %, it cannot be assumed that the larger fragments will have transferred to a sufficient extent after 12 hr.

Alkaline blots are more rapid, with most of the DNA being transferred during the first 2 hrs. More rapid transfer can be achieved with the downward capillary blot procedure

1.5. Transfer method

Capillary transfer is still the most popular method of Southern blotting. Its advantages are simplicity, reliability and the lack of special equipment requirements. Alternative transfer methods include electroblotting, originally developed for protein transfer. This method does not work with high-salt buffers and so is less suitable for nitrocellulose.

The second alternative to capillary transfer is vacuum or positive pressure blotting. The vacuum/pressure must be controlled carefully to avoid compressing the gel and retarding transfer.

1.6. Immobilization techniques

The aim of immobilization is to attach as much of the transferred DNA as possible to the membrane as tightly as possible. The standard methods for nitrocellulose is baking in a vacuum, for nylon membranes – UV irradiation and for positively charged nylon membranes, immobilization during alkaline transfer or UV irradiation.

Baking and alkaline transfer are straightforward procedures that cannot be improved on if carried out according to the instructions. UV cross-linking is more variable. Calibration of the UV source is essential since to long UV treatment can result in a loss of DNA.

2. Background for Hybridization Analysis

The principle of hybridization analysis is that a single-stranded DNA or RNA molecule of defined sequence (the "probe") can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target"). The stability of the hybrid depends on the extent of base pairing that occurs. The analysis is usually carried out with a probe that has been labeled and target DNA that has been immobilized on a membrane support. The two critical parameters are sensitivity (sufficient probe must anneal to the target to produce a detectable signal after exposure) and specificity (the probe must be attached only to the desired target sequence).

2.1. Factors influencing sensitivity

The sensitivity of hybridization analysis is determined by how many labeled probe molecules attach to the target DNA. The greater the number of labeled probe molecules that anneal, the greater is the intensity of the hybridization signal. The specific activity is a measure of the incorporation of labeled nucleotides/probe. Modern labeling procedures as nick translation, random priming, PCR or in vitro RNA synthesis routinely provide probes with high specific activity.

The amount of target DNA is also essential and was already mentioned under chapter 1.1.

The kind of labeling is another factor influencing sensitivity. Traditionally 32P or 35S labeled probes were used. Today nonradioactive probes are becoming increasingly popular as they have many advantages (see chapter 3).

2.2. Factors influencing specificity

The hybridization incubation is carried out in a high-salt solution that promotes base-pairing between probe and target sequences. Hybridization is normally carried out below the Tm for the probe/target and the specificity of the experiment is the function of post-hybridization washes. The critical parameters are the ionic strength of the final wash solution and the temperature at which this wash is done.

The highly stringent wash conditions should destabilize all mismatched heteroduplexes, so that hybridization signals are obtained only from sequences that are perfectly homologous to the probe.

2.3. Factors influencing hybrid stability and hybridization rate

A.)Hybrid stability

Ionic strengthTm increases 16.6 °C for each 10-fold increase in monovalent cations between 0.01 and 0.4 M NaCl.

base compositionAT are less stable than GC base pairs in aqueous solutions containing NaCl

destabilizing agentsEach 1% formamide reduces the Tm by about 0.6 °C for a DNA-DNA hybrid. 6 M urea reduces the Tm by about 30 °C.

mismatched base pairsTm (melting temperature) is reduced by 1°C for each 1% of mismatching

B.)Hybridization rate

temperature Maximum rate occurs at 20-25°C below the Tm for DNA-DNA hybrids, 10-15°C below Tm for DNA-RNA hybrids

Ionic strengthOptimal hybridization rate at 1.5 M Na+

destabilizing agents50% formamide has not effect, but higher or lower concentrations reduce the hybridization rate

mismatched base pairseach 10% of mismatching reduces the hybridization rate by a factor of two

duplex lengthhybridization rate is directly proportional to duplex length

viscosityincreased viscosity increases the rate of membrane hybridization, 10% dextran sulfate increases rate by factor ten.

probe complexityrepetitive sequences increase the hybridization rate

base compositionlittle effect

pHlittle effect between pH 5.0 and pH 9.0.

3. General Consideration using nonradioactive nucleic acid labeling and detection systems

Nonisotopically labeled probes have become increasingly popular in recent years and replace radioactive probes in techniques such as in situ hybridization, Southern, Northern and Western blot analysis. The advantages of these techniques are:

  • the technologies are safe
  • no hazardous radioactive waste accumulations
  • Probes can be stored for at least a year.
  • Hybridization solutions can be reused several times.
  • shorter exposure times

Types of nonradioactive labeling and detection systems used include the biotin-streptavidin and the digoxigenin –antidigoxigenin system.

In the biotin system biotin is incorporated in the probe by using biotinylated dNTPs during probe synthesis. The incorporated biotin is detected directly by avidin or streptavidin or an anti-biotin antibody conjugated to a fluorochrome or an enzyme such as alkaline phosphatase or horseradish peroxidase. Avidin is a 68 kD glycoprotein derived from egg white and streptavidin is a 60 kD protein from Streptomyces avidinii.


The digoxigenin-anti-digoxigenin system uses digoxigenin (DIG), a cardenolide steroid isolated from Digitalis plants.

In both systems the probe is detected witheither chromogenic (colorimetric) substrates, fluorescence or chemiluminescence (see later).

4. The Digoxigenin System


The DIG System is an effective system for the labeling and detection of DNA, RNA, and oligonucleotides. The protocols for labeling with digoxigenin (Figure 1) and subsequent detection are based on well-established, widely used methods. DNA, RNA, and oligonucleotide probes are labeled according to the methods (usually enzymatic) used for preparing radioactive probes. Hybridization of digoxigenin-labeled probes (e.g., to target DNA or RNA on a Southern or Northern blot) is also carried out according to standard protocols, except that a special blocking reagent is used to eliminate background. The signal on the nucleic acid blot is detected according to the methods developed for western blots. The incorporation and spacing of digoxigenin in DNA, RNA, and oligonucleotides can be varied by using different labeling protocols.

4.1. DIG Labeling protocols

  • PCR Labeling: DIG probe synthesis incorporates Digoxigenin-11-dUTP by the polymerase chain reaction. (1 every 10–20 nucleotids)
  • Random-primed labeling method: Digoxigenin-11-dUTP is incorporated (1 every 20-25 nucleotides) by using the Klenow Polymerase and hexaoligonucleotides as primers.
  • T7/SP6-mediated transcription for the synthesis of strand-specific RNA probes. Using the RNA polymerases T7 or SP6 a DIG labeled RNA probe can be synthesis during transcription by incorporating Digoxigenin-11-UTP
  • 3'-End Labeling: Terminal transferase adds a single Digoxigenin-11-ddUTP to the 3'-end of the oligonucleotide.
  • DIG Oligonucleotide Tailing: Terminal transferase adds a string of Digoxigenin-11-dUTP interspersed with unlabeled dATP to the 3'-end of oligonucleotides.
  • DIG Oligonucleotide 5'-End Labeling: DIG-NHS ester labels the 5' end.
  • DIG labeling by nick translation: Uses DNA Polymerase I to create singel-stranded nicks in double stranded DNA. The 5'-3' exonuclease activity of E.coli Polymerase I enters the nicks and removes stretches of single-stranded DNA; the degraded DNA is than replaced by the 5'-3' polymerase activity.
  • cDNA synthesis: Using AMV (Avian Myelobastrosis Virus) reverse transcriptase the cDNA is labeled by incorporation of Digoxigenin-11-dUTP during cDNA synthesis.

The yield of the labeling reaction should be estimated to ensure the success of the reaction and to approximate the amount of probe to be used in the hybridization experiment. A simple dot blot method is used to estimate probe yield;

4.2. DIG Detection

Several alternatives are available for the detection of digoxigenin (or biotin)-labeled probes.



  • luminescent detection uses the chemiluminescent alkaline phosphatase substrate CSPD® to produce a light signal, which is detected by exposing the membrane to an X-ray film.

Figure 3:Schematic of the CSPD reaction: Enzymatic dephosphorilation of the dioxetane CSPD by alkaline phosphatase leads to the metastable phenolate anion, which decomposes and emits light at 477 nm.


  • colorimetric detection uses the substrates NBT and BCIP to generate purple/brown precipitate directly on the membrane.

Figure 4: Schematic of the NBT/BCIP reaction: when alkaline phosphatase removes the phosphate group of BCIP (5-bromo-4-chloro-3-indolyl-phosphate) the resulting molecules dimerizes under oxidating conditions to give the blue precipitate (5, 5'-dibromo-4,4'-dichloro-indigo). During the reaction with BCIP, NBT (nitroblue tetrazolium) is reduced to its colored form to give an enhanced color reaction.

  • multicolordetection comprises three naphthol-AS-phosphate/diazonium salt combinations for the visualization of a green, red, or blue hybridization signal.

Figure 5: Principle of the multicolor detection system: detection of the different labels is performed by binding the respective antibody- or streptavidine-alkaline phosphatase conjugate. The reactions are performed consecutively with heat-inactivationof the alkaline phosphatase between the detections. Hybrids must be stabilized by UV-crosslinking if mulitcolor detection is used.

  • There is also a wide range of alternative anti-digoxigenin conjugates available, such as anti-digoxigenin-peroxidase, anti-digoxigenin-gold (for electron microscopic studies), anti-digoxigenin-fluorescein and anti-digoxigein-rhodamine (for fluorescence detection in the microscope or with a fluorimeter).

4.3. General Considerations for Hybridization with DIG-probes

Please review this section of general hybridization considerations before proceeding with the DIG-system. Several points are critical for successful use of the DIG-system, especially when performing chemiluminescent detection.

4.3.1. Membrane Selection

For best results, use positively charged Nylon membranes for the transfer. Uncharged membranes can also be used with the DIG-system. Their binding capacity is lower and therefore a lower maximum sensitivity can be achieved.

Nitrocellulose membranes cannot be recommended in combination with the DIG-System. They can only be used when colorimetric detection will be performed and no stripping and reprobing is planned.

4.3.2. Probe Concentration

In the following chapters we give recommendations for probe concentrations in the different applications. These recommendations refer to newly synthesized, DIG-labeled probe. The labeling efficiency has always to be confirmed by estimating the yield of a labeling reaction. The recommended probe concentration must be regarded as a starting point for your hybridization.


Note: If chemiluminescent detection is performed, a too high probe concentration will often lead to background. Therefore, the probe concentration should not be increased above the recommended concentrations.

Figure 7: Mock hybridization and effect probe concentration. Naked pieces of membrane were incubated with the indicated amounts of DIG-labeled DNA probe and detected with chemilumininescence.

4.3.3. Optimization of the probe concentration – the “mock” hybridization

To prevent background problems as a result of a too high probe concentration, we recommend optimizing the probe concentration in a mock hybridization before the actual hybridization is performed.

The mock hybridization is carried out by incubating small membrane pieces (without DNA transferred to it) with different probe concentrations in the hybridization solution and subsequent detection with the procedure of choice.

For example, the highest probe concentration that gives an acceptable background should be used for the hybridization experiment (see figure 7, 25 ng/ml).
4.3.4. Hybridization and Washing Conditions

We have found that DIG-labeled probes demonstrate the same hybridization kinetics as radiolabeled probes. Hybridization and washing conditions for DIG-labeled probes do not differ substantially from those of radiolabeled probes.

The optimal hybridization and wash conditionsfor each probe must be determined experimentally. In this User’s Guide, we provide recommendations for hybridization and washing conditions. Use the conditions given as a starting point. It may then be necessary to optimize conditions to obtain maximum sensitivity with your probe.

Labeled probes can hybridize non-specifically to sequences that bear homology but are not entirely homologous to the probe sequence. Such hybrids are less stable than perfectly matched hybrids. They can be dissociated byperforming washes of various stringencies. Thestringency of washes can be manipulated byvarying the salt concentration and temperature.For some applications, the stringency of the washes should be higher. However, we recommend that you hybridize stringently rather than wash stringently.

4.3.5. Prehybridization/Hybridization solutions

Several hybridization buffers can be used with the DIG-System. The main difference with hybridization buffers described elsewhere, is the presence of Blocking Reagent. The protein in Blocking Reagent reduces the non-specific binding of probe to the membrane filter.

DIG Easy Hyb* Standard buffer Standard buffer High SDS buffer

+ 50% formamide (Church buffer)

ready-to-use 5 x SSC, 0.1% (w/v) 50% formamide, deionized, 7% SDS, 50% formamide,

solution, non- N-lauroylsarcosine, 5 x SSC, 0.1% (w/v) N-lauroyl- deionized, 5 x SSC, 2%

toxic used like 0.02% (w/v) SDS, sarcosine, 0.02 (w/v) SDS, Blocking Reagent, 50 mM