Stringency Washes

In Situ

In Situ Hybridization

Read in this article:
Factors that Influence In Situ Hybridization
Parameters for Optimal In Situ Hybridization Conditions
Standard In Situ Hybridization Conditions
Stringency Washes

A prehybridization incubation is often necessary to prevent background staining. The prehybridization mixture contains all components of a hybridization mixture except for probe and dextran sulfate.

Various components in the hybridization solution have effects on the rate of renaturation and thermal stability of DNA hybrids free in solution. The features will be more or less identical to those of immobilized nucleic acids, such as in filter and in situ hybridizations. The largest deviation probably occurs in the kinetics.

Factors that Influence In Situ Hybridization

Hybridization depends on the ability of denatured DNA to reanneal with complementary strands in an environment just below their melting point (Tm).

The Tm is the temperature at which half the DNA is present in a single-stranded (denatured) form. The Tm value is different for genomic DNA isolated from various organisms, (e.g., for Pneumococcus DNA it is +85°C, for Serratia DNA it is +94°C). The Tm can be calculated by measuring the absorption of ultraviolet light at 260 nm. The stability of the DNA is directly dependent on the GC content. The higher the molar ratio of GC pairs in a DNA, the higher the melting point. Tm and renaturation of DNA are primarily influenced by four parameters:


The maximum rate of renaturation (hybridization) of DNA is at +25°C. However, the bell-shaped curve relating renaturation rate and temperature is broad, with a rather flat maximum from about +16 to +32°C below Tm.


From pH 5 – 9, the rate of renaturation is fairly independent of pH. Buffers containing 20 – 50 mM phosphate, pH 6.5 – 7.5 are frequently used.
Note: Higher pH can be used to produce more stringent hybridization conditions.

Concentration of monovalent cations

Monovalent cations (e.g., sodium ions) interact electrostatically with nucleic acids (mainly at the phosphate groups) so that the electrostatic repulsion between the two strands of the duplex decreases with increasing salt concentration, (i.e. higher salt concentrations increase the stability of the hybrid). Low sodium concentrations affect the Tm, as well as the renaturation rate, drastically. Sodium ion (Na+) concentrations above 0.4 M only slightly affect the rate of renaturation and the melting temperature. The following equation has been given for the dependence of Tmon the GC content and the salt concentration (for salt concentrations from 0.01 to 0.20 M):

Tm = 16.6 log M + 0.41 (GC) + 81.5:

where M is the salt concentration (molar) and GC the molar percentage of guanine plus cytosine. Above 0.4 M Na+, the following formula holds:

Tm = 81.5 + 0.41 (GC)

Free divalent cations strongly stabilize duplex DNA. Remove them from the hybridization mixture or complex them (e.g., with agents like citrate or EDTA).

Presence of organic solvents (formamide)

DNA melts (denatures) at +90 to +100°C in 0.1 – 0.2 M Na+. For in situ hybridization this implies that microscopic preparations must be hybridized at +65 to 75°C for prolonged periods. This may lead to deterioration of morphology. Fortunately, organic solvents reduce the thermal stability of double-stranded polynucleotides, so that hybridization can be performed at lower temperatures.

Formamide has for years been the organic solvent of choice. It reduces the melting temperature of DNA-DNA and DNA-RNA duplexes in a linear fashion by 0.72°C for each percent formamide. Thus, hybridization can be performed at +30 to +45°C with 50% formamide present in the hybridization mixture. The rate of renaturation decreases in the presence of formamide. The melting temperature of hybrids in the presence of formamide can be calculated according to the following equation:

For 0.01 – 0.2 M Na+:

where M is the salt concentration (molar) and GC the molar percentage of guanine plus cytosine. Above 0.4 M Na+, the following formula holds:

Tm = 81.5 + 0.41 (GC)

Tm = 16.6 log M + 0.41 (GC) + 81.5 – 0.72 (% formamide)

For Na+ concentrations above 0.4 M:

Tm = 81.5 + 0.41 (GC) – 0.72 (% formamide)

To obtain a large increase of in situ hybridization signal for rDNA, hybridize with rRNA in 80% formamide at +50 to
+55°C, instead of 70% formamide at +37°C.
Finally, it should be mentioned that during the in situ hybridization procedure, relatively large amounts of DNA can be lost (Raap et al. 1986).

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Parameters for Optimal In Situ Hybridization Conditions

Probe length

The rate of the renaturation of DNA in solution is proportional to the square root of the (single-stranded) fragment length. Consequently, maximal hybridization rates are obtained with long probes. However, short probes are required for in situ hybridization because the probe has to diffuse into the dense matrix of cells or chromosomes. The fragment length also influences thermal stability. The following formula, which relates the shortest fragment length in a duplex molecule to change in Tm, has been derived:

Change in Tm · n = 500 (n = nucleotides).

Probe concentration

The probe concentration affects the rate at which the first few base pairs are formed (nucleation reaction). The adjacent base pairs are formed afterwards, provided they are in register (zippering). The nucleation reaction is the rate limiting step in hybridization. The kinetics of hybridization is considered to be a second order reaction [r = k2 (DNA) (DNA)]. Therefore, the higher the concentration of the probe, the higher the reannealing rate.

Dextran sulfate

In aqueous solutions dextran sulfate is strongly hydrated. Thus, macromolecules have no access to the hydrating water, which causes an apparent increase in probe concentration and consequently higher hybridization rates.

Base mismatch

Mismatching of base pairs results in reduction of both hybridization rates and thermal stability of the resulting duplexes. To discriminate maximally between closely related DNA sequences, hybridize under fairly stringent conditions (e.g., at Tm -15°C). On the average, the Tm decreases about 1°C per % (base mismatch) for large probes. Mismatching in oligonucleotides greatly influences hybrid stability; this forms the basis of point mutation detection.

Use of single-stranded versus double-stranded probes

A number of competing reactions occur during in situ hybridization with double-stranded probes. These include:

  • Probe renaturation in solution
  • In situ hybridization
  • In situ renaturation (possibly, for ds targets)

Consequently, the use of single-stranded probes has advantages for in situ hybridization. Such probes can be made by using the single-stranded M13 (or similar bacteriophage cloning vectors) as template, or by using transcription vectors which permit the production of large amounts of single-stranded RNA.

Competition in situ hybridization

Recombinant DNA isolated from eukaryotic DNA often contains genomic repetitive sequences (e.g., the Alu sequence in humans). In situ hybridization to chromosomes with a probe which contains repetitive DNA usually results in uniform staining. However, unlabeled competitor DNA (usually total genomic DNA) prevents the repetitive probe sequences from annealing to the target and leads to stronger in situ hybridization signals from the unique sequences in the probe. (This approach was first described for in situ hybridization by Landegent et al., 1987; Lichter et al., 1988a; and Pinkel et al., 1988). Obviously, the greater the complexity of probe (plasmids < phages < cosmids < yeast artificial chromosomes < chromosome libraries), the greater the need for competition in situ hybridization. This approach has proven particularly useful for in situ hybridization with DNA isolated from chromosome-specific libraries (CISS-hybridization); a specific chromosome can be fluorescently labeled over its full length (Lichter et al., 1988a,b; Cremer et al., 1988; Pinkel et al., 1988).

Oligonucleotide hybridization

The rules given for hybrid stability and kinetics of hybridization can probably not be extrapolated to hybridization with oligodeoxynucleotides. For in situ hybridization, the advantages of oligonucleotides include their small size (good penetration properties) and their single-strandedness (to prevent probe reannealing). The small size, however, is also a disadvantage because it covers less target. The nonradioactive label should be positioned at the 3´ or the 5´ end; internal labeling affects the Tm too much. In an experiment with 20-mers of 40 – 60% GC content, start with the hybridization conditions described below. Depending on the results obtained, you may decide to use other stringency conditions.

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Standard In Situ Hybridization Conditions

(Department of Cytochemistry and Cytometry, University of Leiden, Netherlands)

For “large” DNA probes (≥ 100 bp)

  • 50% deionized formamide
  • 2x SSC1
  • 50 mM NaH2PO4 / NaH2PO4 buffer; pH 7.0
  • 1 mM EDTA
  • carrier DNA/RNA (1 mg/ml each)
  • probe (approx. 20 – 200 ng/ml)

Optional components:

  • 1x Denhardt’s2
  • dextran sulfate, 5 – 10%

Temperature: +37 to +42°C
Hybridization time: 5 minutes – 16 hours

For synthetic oligonucleotides

  • 25% formamide
  • 4x SSC)1
  • 50 mM NaH2PO4 / NaH2PO4 buffer; pH 7.0
  • 1 mM EDTA
  • carrier DNA/RNA (1 mg/ml each)
  • probe (approx. 20 – 200 ng/ml)
  • 5x Denhardt’s2

Temperature: +15 to +25°C
Hybridization time: 2 -16 hours

1SSC : 1x SSC = 150 mM NaCl, 15 mM sodium citrate; pH 7.0: Make a 20x stock solution (3 M NaCl, 0.3 M sodium citrate)
250x Denhardt’s solution = 1% Ficoll (type 400), 1% polyvinylpyrrolidone, and 1% bovine serum albumin

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Stringency Washes

Labeled probe can hybridize non-specifically to sequences which are partially but not entirely homologous to the probe sequence. The extent to which the latter occurs can be manipulated to some extent by varying the stringency of the hybridization reaction. Such hybrids are less stable than perfectly matched hybrids. They can be dissociated by performing washes of various stringencies.

The stringency of the washes can be manipulated by varying

  • formamide concentration
  • salt concentration
  • temperature

Often a wash in 2x SSC containing 50% formamide suffices. For some applications the stringency of the washes should be higher.

To remove the background associated with nonspecific hybridization, wash the sample with a dilute solution of salt. The lower the salt concentration and the higher the wash temperature, the more stringent the wash. In general, greater specificity is obtained when hybridization is performed at a high stringency and washing at similar or lower stringency, rather than hybridizing at low-stringency and washing at high stringency.

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  • Casey, J.; Davidson, N. (1976) Rates of formation and thermal stabilities of RNA-DNA and DNA-DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 4, 1539–1552.
  • Cox, K. H.; DeLeon, D. V.; Angerer, L. M.; Angerer, R. C. (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Develop. Biol. 101, 485–503.
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  • Lichter, P.; Cremer, T.; Borden, J.; Manuelidis, L.; Ward, D. C. (1988a) Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80, 224–234.
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One response to “Stringency Washes

  1. In general, greater specificity is obtained when hybridization is performed at a high stringency and washing at similar or lower stringency, rather than hybridizing at low-stringency and washing at high stringency.


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