Detection of DNA binding ?

                   

                           Detection of DNA binding

Radioisotope: -

·       Nucleic acid probe can be labelled using radioactive isotopes.

·       Most common process but less popular today become of safety consideration as well as coast and disposal of radio waste product.

·       However, radio labelled probes are the sensitive as they provide the highest degree of resolution currently available in hybridization assay.

Non-radioactive labelled: -

·       Compared to radioactive labelled use of non-radioactive labelled have several advantages

1.    Safety.

2.    Highest stability of probe.

3.    Efficiency of labelling reaction.

4.    Detection incites.

5.    Less time taken to detect the signal.

Some example as follows Biotin- this labelled can be detect using avidin or streptavidin which have high affinity for biotin. This type of non-radioactive labelled is known as an indirect system.

Enzyme: -

Enzyme is attached to the probe and its presence is usually detected by reaction with a substrate that changes coloured used in this way the enzyme is sometimes referred to as receptor group.

For example of enzyme use include Alkenone phosphate and horse reddish peroxidase. In the presence of peroxide and per-oxidase, choloronepto chromogenic substrate for horse reddish peroxidase forms purple insoluble product.

HPR also catalyzes oxidation lumen of, chemiluminometric substrate for HPR

Chemiluminescence: -

·       Chemiluminescence chemical attach to probe and are detected by their light emission using luminometer.

Fluorescence: -

·       Fluorescence chemical attach to probe which Fluro under ultraviolet light.

Antibodies: -

·       An antibody group is coupled to a probe and presence is detected using specific antibodies.

DIG system: -

·       It is the most compulsive, convenient and effective system for labelling and detecting DNA, RNA, oligonucleotide.

DIG – Digoxigenin, like biotin can be chemically coupelled to linkers or nucleotide and DIG substituted nucleotide can be incorporated into nucleic acid probe by any of the standard enzymatic method.

For non-radioactive procedure, a deoxy or biotin molecule attach to a DNTP along is used.

Reference: -

1.   Created from PDB 1LMB

2.   ^ Created from PDB 1RVA

3.   ^ Travers, A. A. (1993). DNA-protein interactions. London: Springer. ISBN 978-0-412-25990-6.

4.   ^ Pabo CO, Sauer RT (1984). "Protein-DNA recognition". Annu. Rev. Biochem. 53 (1): 293–321. doi:10.1146/annurev.bi.53.070184.001453. PMID 6236744.

5.   ^ Dickerson R.E. (1983). "The DNA helix and how it is read". Sci Am. 249 (6): 94–111. Bibcode:1983SciAm.249f..94D. doi:10.1038/scientificamerican1283-94.

6.   ^ Zimmer C, Wähnert U (1986). "Nonintercalating DNA-binding ligands: specificity of the interaction and their use as tools in biophysical, biochemical and biological investigations of the genetic material". Prog. Biophys. Mol. Biol. 47 (1): 31–112. doi:10.1016/0079-6107(86)90005-2. PMID 2422697.

7.   ^ Dervan PB (April 1986). "Design of sequence-specific DNA-binding molecules". Science. 232 (4749): 464–71. Bibcode:1986Sci...232..464D. doi:10.1126/science.2421408. PMID 2421408.

8.   ^ Sandman K, Pereira S, Reeve J (1998). "Diversity of prokaryotic chromosomal proteins and the origin of the nucleosome". Cell Mol Life Sci. 54 (12): 1350–64. doi:10.1007/s000180050259. PMID 9893710.