Basic Protocol For Hybridization

The expression hybridization refers to the (re)association of complementary nucleic acid strands (DNA, RNA, or synthetically produced primers or probes) to form double-stranded complexes. The process starts with the association of two short regions of complementary nucleic acid sequences, followed by a rapid and progressive linking of the base pairs on either end of the duplex region. The rate of hybridization and the stability of the complexes, once formed, are dependent on a number of factors. Temperature has an important role in both the rate and stability of the hybridization complex. Optimal hybridization occurs at 20-25°C and 10-15 °C below the melt temperature of DNA and RNA complexes, respectively.1-7,8-1 At suboptimal temperatures, reaction rates will be slowed and nonspecific hybridization may occur; however, if temperatures exceed the optimal range, then although stringency of the reaction may improve there may be a degradation of accurately associated dimers.

Hybridization time is also important and long incubation times may not provide the optimal results. There is a balance between achieving an optimal bind between the target and probe and the development of a nonspecific background signal or the hybridization of the probe to sequences similar to the target. Generally, incubations for less than 24 hr should not result in a high background signal, providing the probe concentration is not in excess. If the probe is in excess of the bound target, then binding to similar nontarget sequences may become problematic in the latter stages. For an overnight reaction, a usual probe concentration is 10 ng mL~1.

Mismatches between the probe and target sequence will compromise the stability of the association and this may lead to a reduction in the optimal temperature to overcome the discrepancy. Even so, a 10% mismatch is usually tolerated. However, mismatches will also have a detrimental effect on the rates of hybridization and a 10% mismatch will reduce the hybridization rate.[7]

Other factors that need to be considered include the following:

1. Salt concentrations—generally concentrations of 1.01.5 M Na+ (or equivalent, 5x SSC) are considered optimal.[9]

2. pH—reactions are usually performed at a neutral or weakly acidic pH.[4]

3. Dextran sulphate—the addition of dextran sulphate (10%) concentrates the nucleic acids by expelling them from the volume that it occupies, thus increasing the chances for the interaction between the probe and target sequence.[10]

4. Viscosity—the use of solution with a low viscosity will reduce the opportunity for the interaction between the probe and target sequence. The introduction of polyethylene glycol (PEG 8%) will increase the viscosity of less viscous solutions.[11]

5. Probe length—when probing a filter, the hybridization rate is directly proportional to the probe length when there is excess target DNA bound. Probes are usually >15 bp and <1.5 kbp[4] in length.

When performing hybridization, three steps are followed. The first stage is prehybridization, where the filter is washed with a protein, polymer, and sheared carrier DNA mix to block nonspecific binding. The next stage is hybridization and involves denaturing the probe before adding it to fresh, warm prehybridization solution, which is then applied to the filter and agitated overnight at the desired temperature. The filter is then washed for 30 min to remove any unbound or weakly bound probe using 0.2 x SSC, 0.1% SDS solution. The stringency of the wash is temperature-dependent, with washing at room temperature and close to the probe Tm considered to be moderate and high, respectively.

The most common detection methods originally utilized 32P radiolabeling of the oligonucleotide with autoradiography. The use of fluorescent dyes or lanthanide chelates, biotin-labeled nucleotide analogs, cross-linked enzymes such as horseradish peroxidase, or acridinium esters that can be detected by enhanced chemilumines-cence are just some of the currently available alterna-


Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment