Chapter: Pharmaceutical Biotechnology: Fundamentals and Applications - Molecular Biotechnology

| Study Material, Lecturing Notes, Assignment, Reference, Wiki description explanation, brief detail |

DNA Hybridization

To gain insight in the DNA composition, sequencing is the final approach. There is, however, a possibility to acquire information about DNA structure by hybridization with the help of so-called DNA probes.

DNA Hybridization

 

To gain insight in the DNA composition, sequencing is the final approach. There is, however, a possibility to acquire information about DNA structure by hybridization with the help of so-called DNA probes.

In essence the probe is a specific single-stranded DNA fragment. Such a probe will form double-stranded DNA (in other words will hybridize) whenever it encounters a single-stranded complementary piece of DNA under appropriate conditions. There are many applications for DNA probes.

 

If, for example, one wishes to see which recombinant DNA molecule in an extensive DNA library harbors a gene of interest, one might use a DNA probe. DNAs from the library are converted into single-stranded DNA and then confronted with a probe that reflects a very characteristic segment of the desired gene. Hybridization will, provided that the probe has the required specificity, only occur with target DNA molecules that harbor the gene of interest.

The use of DNA hybridization probes in diag-nostic testing in humans can be illustrated by using cystic fibrosis (CF) as an example. The frequency of this heritable and deadly disease is approximately once in 2000 live births making it the most frequent genetic disorder among Caucasians. The cloning of the gene in 1989 based on its position on the human genetic map was a tour de force involving several laboratories. It enabled the molecular analysis of the genetic defect, revealing that approximately 70% of the diseased genes contain an identical mutation: a three base pair deletion in the protein coding gene resulting in the loss of a phenylalanine amino acid at position 508 of the 1480 amino acid-long protein. This mutation was named CFdel 508 and the knowledge gained was used to design oligonucleotide probes for rapid screening purposes.

These single-strand DNA probes are comple-mentary to the normal and CFdel 508 regions of the CF gene shown below. The symbols L, E, etc. represent various amino acids; F, e.g., stands for phenylalanine.

Normal:


DNA, isolated, e.g., from white blood cells of suspected carriers or amnion fluid in which embryos are suspended, is boiled to make it single stranded and then immobilized on filter paper. Next, hybridi-zation with normal and CFdel 508 specific probes clarifies whether one has the disease, in which case both the maternal and the paternal genes are affected, or whether one is a carrier of the disease in which one of the two genes is affected (heterozygosy) and the other is normal at this genomic position.

Variation on this technology allows for the automated and simultaneous screening for many genetic diseases for which the molecular lesions are known. Kits which contain all the reagents necessary for a particular test are commercially available.


Study Material, Lecturing Notes, Assignment, Reference, Wiki description explanation, brief detail


Copyright © 2018-2020 BrainKart.com; All Rights Reserved. Developed by Therithal info, Chennai.