3-5.7 Preparation of Labeled Nucleotides:

Nucleotides can be labeled by isotopic and non-isotopic methods.

3-5.7.1 Isotopic labeling:

Isotopes generally used for labeling nucleotides are ³²P, ³³P, ³⁵S or ³H. They can be detected directly in solution or on X-ray film using autoradiography.

Properties of radioisotopes used for labeling DNA and RNA probes:

The strength of autoradiography signal depends on intensity of radiation emitted by radioisotope and duration of exposure. ³²P emits high energy β-particles which offer high detection sensitivity. Thus, it is widely used in Southern blot hybridization, dot-blot hybridization, colony hybridization. But it is relatively unstable and when fine resolution is required to interpret results, the image is unambiguous due to its high energy β-particle emission. Due to this, ³⁵S-labeled, ³³P-labeled (moderate half-lives) and ³H-labeled nucleotides are used which emit less energetic β- radiation. They are used in DNA sequencing and in-situ hybridization. ³H requires long exposure time due to low energy β-particle emission. Due to this, ³⁵S-labeled, ³³P-labeled (moderate half-lives) and ³H-labeled nucleotides are used which emit less energetic β-radiation. They are used in DNA sequencing and in-situ hybridization. ³H requires long exposure time due to low energy β-particle emission.