Non-Radio Isotopic Procedure of Hybridization

Introduction

Radio isotopic hybridization techniques have been used for many years for the detection of DNA and RNA fragments from a mixture of molecules. But radio isotopic hybridization techniques are more hazardous as they cause harm to the person in the vicinity by activating cancerous cells and causing harm to the DNA.

Therefore, to avoid this problem safer alternatives have been searched and researchers have come up with a comparatively new technique in which enzyme-labeled probes react with the florescent substrate to colored products.

Non-Radioactive Hybridization

The non-radio isotopic hybridization method helps in the detection of the DNA fragment of interest by using an enzyme that acts on the chemiluminescent or chromogenic substrate. This substrate gets converted into a product that emits light.

This method is comparatively safe as no radioactive materials are used so no harmful radiation will be produced.

Types of Non-Radio Isotopic Hybridization

There are two types of no-radio isotopic hybridization methods −

• Direct non-radioactive method.

• Indirect non-radioactive method.

Direct Non-Radio Isotopic Method

In this method, the reaction mixture is incorporated with a nucleotide which is attached to a labeled group. The group which is labeled is called the fluorophore. A fluorophore is a group that fluoresces or emits light when exposed to light of a particular wavelength.

Some of the common examples of fluorophores are biotin which emits red color, fluorescein emits green color. Rhodamine emits red color, etc.

Indirect Non-Radio Isotopic Method

• In this method a reporter is chemically bound to a nucleotide. The reported labeled nucleotide attaches itself to the DNA. The marker molecule is attached to the affinity molecule which has a very high specificity for the reporter. The conjugated marker molecule can be detected by assaying and then added to the reaction mixture.

• As the affinity molecule can easily detect the reporter molecule it will get bound to it. The unbound molecules can be removed by washing. The affinity molecules which are generally used are antibodies or a specific ligand.

• The assays used for the detection of the marker molecule depend on the group attached to it. For example, if a specific fluorescent dye is used it can be detected by using fluorometry and if an enzyme is attached to the marker molecule, then colorimetry is used to identify the colored product formed in the end.

Types of Indirect Non-Radio Isotopic Method

There are two types of indirect systems based on the ligands attached or conjugated −

• Biotin streptavidin system

• Digoxigenin system

Biotin Streptavidin System

• In this system two ligands are conjugated to each other; one is biotin which is a commonly found vitamin and second is the streptavidin or avidin. Streptavidin is a bacterial protein and avidin is a protein found in the egg white.

• Streptavidin acts an affinity molecule which is highly specific for the biotin which acts as a reporter molecule.

• Firstly, nucleotides are conjugated to the biotin molecules which act as a probe. This probe is then hybridized to the DNA fragment of interest.

• Then streptavidin is added to this mixture followed by the addition of the biotin-labeled enzyme which is an alkaline phosphatase enzyme conjugated to the biotin.

• Then the substrate for the labeled enzyme is added and the product formed by the enzymatic reaction is assayed depending on the substrate used.

• Streptavidin has a very peculiar feature of four biotin-binding sites. So a single streptavidin molecule can bind to a biotin-labeled probe and biotin-labeled enzyme simultaneously. The activity of the enzyme is not interrupted by the biotin labeling or streptavidin binding.

Digoxigenin System

• Digoxigenin is obtained from the plant Digitalis and is a steroid produced by the flowers and leaves of this plant.

• Digoxigenin is bound to a specific antibody which is then used for the detection of the nucleotide fragment of interest.

• Here digoxigenin bound antibody act as affinity molecules and digoxigenin acts as a reporter molecule.

• Firstly, the nucleotide is conjugated with digoxigenin, this conjugate is then hybridized with the DNA fragment.

• To this the affinity molecule that is the digoxigenin bound to the antibody is added. Now digoxigenin-labeled by the enzyme is formed by conjugating alkaline phosphatase to digoxigenin.

• Substrate is added to it the product formed is then detected using colorimetry or other techniques depending on the substrate used.

Depending on the substrate used for amplification of the signal non-radio isotopic method is of two types:

• Chemiluminescence method: In this, a chemiluminescent substrate is used which emits light after conversion into a specific product which can be measured using a luminometer.

• Chromogenic method: In this method a chromogenic substrate is used which changes color when a suitable enzyme acts on it. This change in color can be measured using a colorimeter.

Advantages of Non-Radio Isotopic Hybridization

• They are more sensitive as compared to the radio isotopic method. For example, the chemiluminescence method is highly sensitive and produces accurate results.

• They pose less impact on nature because it does not use biohazardous radioactive material. They can be disposed of easily and do not require special methods.

• It is a very fast method as the results can be obtained within a few hours of the procedure.

• Probes are more stable. For example, the biotin-labeled probe remains stable at room temperature for more than one year.

Conclusion

Although most of the research laboratories use radio isotopic hybridization techniques which involve the binding of DNA of interest to a solid support and the use of radioactive material as probes. But, because of their hazardous nature and the need for sophisticated disposal equipment, they have been replaced by non-radioactive techniques which use eco-friendly substrates and probes for detection.