De-oxyribonucleic acid (DNA) hybridization

De-oxyribonucleic acid (DNA) hybridization, What is hybridization? Hybridization is the process of establishing and non-covalent, sequence-specific interaction between two or more complementary strains of nucleic acid into a single hybrid. Under normal conditions when oligonucleotide DNA or RNA will bind to their complement nucleotide sequence, they will readily bind to each other.

DNA hybridization:

The principle of DNA hybridization is that a single stranded DNA molecule recognises and specifically bind to a complementary DNA strand in a mixture of other DNA strands.

Steps in hybridization assay:

Following are the steps involved in hybridization assay;

  1. Selecting a hybridization assay,
  2. Preparation of probes,
  3. Labeling of probes,
  4. Detector system in DNA hybridization.

Materials to be required:

  1. Samples (DNA, RNA, Protein) to be analyzed.
  2. Denaturation solution,
  3. Neutralization solution,
  4. Whatman filter paper sheets,
  5. Nylon or nitrocellulose membrane ,
  6. Probe,
  7. Probe detector.


Probe is a synthetic single stranded DNA molecule that can recognise and specifically bind to a target DNA by complementary base pairing in a mixture of biomolecule.

Preparation of nucleic acid probes:

DNA is prepared from cell based cloning or by PCR. Probe is double stranded. Then it is labelled by DNA polymerase-based DNA synthesis.

It is prepared by transcription from DNA cloned in an expression vector. Probe is single stranded and is labelled by “run-off” transcription

It is prepared by chemical synthesis. Probe is single stranded and is labelled by end labelling.

Labeling the probe:

The probe must be labelled so that it can be detected after hybridization has occurred under test conditions.

Labeling DNA:

Labelling of DNA by in vitro DNA synthesis is normally accomplished using one of the following three methods.

  1. PCR labeling,
  2. Nick translation,
  3. END labeling.

PCR labeling:

It is a straightforward and now very popular method of incorporating labeled nucleotides in to a probe. Design primers that will amplify a sequence you wish to use as a probe and simply include a lebeled dNTP during PCR.

Nick translation:

Action of 2 enzymes on dsDNA you wish to label: DNasel and DNA polymerase 1.

DNasel will randomly introduce a few nicks (that is; single strand breaks) in the DNA backbone. These nicks are repaired by DNA polymerase 1. DNA polymerase 1 synthesize DNA in 5′-3′ direction. It also has exonuclease activity. These two activities act together to remove and then replace a few nucleotides down from each nick. The newly formed sequence bits are now labelled.

End labeling:

Single stranded oligonucleotide are usually end-labelled using polynucleotides kinase. Label is provided in the form of a ³²P at the Y-phosphate position of ATP. The polynucleotide kinase catalyses an exchange reaction with the 5′-terminal phosphates. The same procedure can also be used for labeling double stranded DNA.

Labeling of nucleic acids:

The nucleic acids can be labelled by; isotropic and non-isotropic methods. The non-isotropic method is done either by; direct non-isotopic labelling or by indirect non-isotopic labelling.

Isotopic labeling:

The nucleotides containing radioisotopes are normally labelled by isotopic labeling. Often l³²P, ³³P, ³⁵S or ³H are used. These can be detected specifically in solution or, but more commonly, within a solid specimen.

Direct non-isotopic labeling:

It is incorporated with modified nucleotides containing a fluorophore- a chemical group which can fluoresce when exposed to light of a certain wavelength.

Indirect non-isotopic labeling:

It is carried out by binding of affinity group to the reporter. This is then detected by detecting the marker fluorometric assay or enzymatic assay.

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