Radiolabeling Technique

Under The Supervision of Prof. S. M. Prasad

Presented By Arun Singh Yadav

Radiolabeling TechniqueRadiolabeling (or isotopic labelling)

is a technique used to track the passage of an isotope (an atom with a detectable variation) through a reaction, metabolic pathway, or cell, called radiolabeling technique.

Atoms that have same atomic number, but have different masses are known as isotopes. Some isotope of an atom have unstable nuclei which after nuclear reaction emit characteristic radiation.

These isotopes are called Radioisotopes, or more commonly radionuclides.

Labeling of nucleic acid-Nucleic acid may be modified with

tags that enable detection or purification.

Resulting nucleic acid can be used to identify or recover other interesting molecules.

Nucleic acid can be labeled by isotopic and non-isotopic labeling methods.

Isotopic Labeling-Isotopic labeling of nucleic acids has

been conducted by incorporating nucleotides containing radioisotopes.

Such radiolabeled proofs contain nucleotides with a radioisotop (often 32P, 33P, 35S or 3H) , Which can be detected specifically in solutions or much more commonly within a solid specimen.

In molecular biology, two or specially important- The radioactive isotop of Phosphorus, 32P, and Sulfur, 35S. Since sulfur is not a normal component of DNA or RNA, we use Phosphorothioate derivatives.

A normal phosphate group has four oxygen atoms around the central phosphorus.

In phosphorothioate, one of these is replaced by sulfur. To introduce 35S into DNA or RNA, phosphorothioate groups containing radioactive sulfur atoms are used to link together the nucleotides.

Radioisotopes which are commonly used in biological research ISOTOPE HALF-LIFE32P 14 days131I 8.1 days35S 87 days14C 5570 years

45Ca 164 days3H 12.3 years

The intensity of signal produced by radioisotopes is dependent on the intensity of the radiations emitted by isotopes , and the time of exposure , which may often be long ( one or more days, are even weeks in some applications) .

32p has been used widely in southern blot hybridization , dot blot hybridization, colony and plaque hybridizations, because it emits high energy beta particles which provide a high degree of sensitivity of detection . The incorporation of radioisotopes in a sample can be detected by two common methods.

Autoradiography- Autoradiography is a technique which is

used for detecting radioisotopes present in a solid sample on gels or membranes. It involves the production of an image in a photographic emulsion.

Such emulsion consist of silver halide crystals in gelatin base. When a beta-particles or gamma ray from a radionuclide passes through the emulsion, the silver ions are converted to silver atoms.

The resulting latent image can then be converted to a visible image once the image is developed, an amplification process in which entire silver halide crystal are reduce to give metallic silver.

Scintillation Counting- Relies on a special chemicals called scintillants.

The scintillants molecules absorb the beta particles emitted by the radioisotopes in the samples, and in turn emit a flash of light.

The light pulses from the scintillants are detected by photocell.

To use the scintillation counter (machine that detects and counts pulses of light), radioactive samples to be measured are added to a viable containing scintillant fluid and located in to counter the counter prints out the number of light flashes it detects within a designated time.

End-Labeling of DNAEnd labeling can be performed at the 3’ or

5’-end. 3’-End labeling-Template- dependent polymerization of [α-32P]

NTP to the 3’ terminus of DNA is catalise by calf thymus terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase [TdT] is a template independent polymerase that incorporates dNTPs to the 3’ hydroxyl end of the single or double stranded DNA and RNA in an irreversible manner.

This enzyme is used for the formation of homo or hetero polymeric heteropolymeric tail at the 3’-end and also for incorporating a single nucleiotide analog such as [α- 32p ] cordycepin- 5’ triphosphate.

Terminal dideoxynucleotidyl transferase labels 3’ protruding ends more efficiently than blunt end.

5’- Labeling- 5’-Labeling is performed by enzymatic method (T4 polynucleotide kinase ), by chemical modification sensitized oligonucleotides with phosphoromidite, or by combined methods. 5’-end labeling is usually performed using polynucleotide kinase ( kinase end-labeling).

The polynucleotide kinase utilize two types of reactions.

Forward reaction-A hydroxyl group is first created by removing the unlabeled phosphate residue from the 5’-end of the DNA with an alkaline phosphates.

T4 polynucleotide kinase is then used to transfer the labeled gamma phosphate from ATP to the 5’-end of DNA.

Exchange reaction-Polynucleotide kinase first transfer the phosphate from the 5’-end of DNA to ADP, forming ATP and leaving a dephosphorylated target.

Then enzyme performs a forward reaction and transfers a labeled gamma phosphate from ATP onto the target DNA.

Hershey-Chase Experiment 1952