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.
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;
- Selecting a hybridization assay,
- Preparation of probes,
- Labeling of probes,
- Detector system in DNA hybridization.
Materials to be required:
- Samples (DNA, RNA, Protein) to be analyzed.
- Denaturation solution,
- Neutralization solution,
- Whatman filter paper sheets,
- Nylon or nitrocellulose membrane ,
- 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.
Labelling of DNA by in vitro DNA synthesis is normally accomplished using one of the following three methods.
- PCR labeling,
- Nick translation,
- END 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.
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.
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.
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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