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Hybridization Techniques - Hybridization is the technique in which the nucleic acid or protein immobilized on the membrane is challenged with a probe or antibody known. Hybridization is widely used to confirm the presence or absence of the DNA/RNA/protein in the unknown sample. Hybridization depends on the function of the labelled base pair between the probe and the target sequence.

After the blotting technique is completed, the membrane is placed in a solution of labelled (radioactive or non-radioactive) single stranded DNA or RNA solution. This DNA or RNA contains sequences complementary to DNA or RNA present on the membrane.

This labelled nucleic acid used to detect or locate DNA is called as probe. Conditions are chosen such that labelled DNA or RNA bind or hybridize with nucleic acid present on the membrane. If the sequence of nucleic acid in the probe is complementary to nucleotide sequence on the membrane then base pairing or hybridization will occur.

The nucleic acid present on the membrane is single-stranded and is bound on the membrane by using negative charge of the phosphate and thymine molecules. Thus when it finds a complementary strand, it forms or develop hydrogen bonds or converts into hybrid DNA (i.e., double-stranded DNA in which the two strands come from different DNA molecules).

Conditions are chosen or maintained such that there is a maximum chance of specific hybridization and minimum of non-specific hybridization. After the hybridization, the membrane is washed to remove the unbounded probes, while bound probes remain attached.

The regions of hybridization are detected by autoradiography method (if the probe is radioactively labelled) or by biotin streptavidin method (if the probe is labelled by a non-radioactive). The specificity with which a particular target sequence is detected by hybridization to a probe is called as stringency.

At high stringency, hybridization occurs when the sequence on the membrane is completely complementary to the sequence of the probe. In practice, the hybridization is carried out at low stringency level, and the membrane is washed many times such that only perfect (>80%) matches are left over.

The principle of hybridization is more or less same for all, but to differentiate and make it conceptually undoubtful, various names are given. If the probes contain or are mode of DNA, they are called as DNA probes and if they are made of RNA they are called as RNA probes.If the membrane containing immobilized DNA is challenged with DNA probe, it is called as DNA hybridization.

If the membrane containing immobilized DNA is challenged with RNA probe, then it is called as DNA: RNA hybridization. Similarly, if the membrane containing immobilized RNA is challenged with RNA probe then it is called as RNA hybridization and if challenged with DNA then it is called as RNA : DNA hybridization.

 
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