The technique provides labeling of DNA by radioactive or non-radioactive labeling molecules. The reaction involves two enzyme action where, one of it produces nick in single/double strand and another enzyme extend the DNA strand from free 3’ -OH group of nick end and it extend and replace the existing nucleotide from 5’ side of the nicked
DNA fragment. The pancreatic DNase I makes a nick and Escherichia coli DNA polymerase I (Klenow fragment) to extend and labeled nucleotide into DNA strand. The technique produces the dsDNA probe. The reaction prolong for 1-2hrs depending up on type of enzymes and their efficiency. The reaction mixture consist of 1U of DNase I, 5-15U of DNA polymerase I, 1x concentration compatible buffer, nuclease free water, 0.25 – 1 µg of template and dNTP mixture. For radioactive labeling the three dNTPs
(dATP, dTTP and dGTP) are mixed in equal concentration and *α -32P] dCTP (3000 Ci/mmol, 10 µCi/µL) are mixed and for non-radioactive labeling the *α -32P] dCTP is substituted with fluorescent molecule labeled nucleotide such as biotin-11-dUTP, fluorescein-12-dUTP, DIG-dUTP or aminoallyl-dUTP molecule is used. During the reaction along with non-labeled nucleotides (1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP) mixed with 0.35 mM DIG-11-dUTP (molar ratio of 1:3-1:5). Incubate the reaction mixture at 15oC for 2 hrs and later it is stopped by addition of 1μL of 0.5 M EDTA, pH 8.0. These labeled molecules are extracted from reaction mix by DNA precipitation method.
The reaction is a normal PCR method, where it includes the one of the nucleotide is labeled and polymerase enzyme is Klenow fragment. The reaction involves the denaturation of double strand DNA into single strand DNA and the random primer (hexamer) of 6-10 base long inallowed for annealing. The one of the labeled dNTP such as biotin labeled dCTP or DIG labeled dUTP incorporated in to synthesizing DNA strand. The incorporation take place by complementary base pairing method hence, the incorporation of labeled molecule take place all along the DNA strand.
The nucleic acid’s either 5’ or 3’ ends of the strands are labeled. The 3’ end labeling involves the tailing reaction by terminal transferase, which is template independent DNA polymerase. The normal PCR runs with unlabeled nucleotide along with labeled fluorescent labeled biotin/ DIG-11-dCTP molecule. The terminal transferase incorporates this labeled molecule at the end of the 3’ end of the oligonucleotide. Similarly the 5’ end can be labeled with radioactive/ non-radioactive molecules. The 5' end labeling in a two-step synthesis with first an amino linker residue on the 5' end of the oligonucleotide, and then after purification, a digoxigenin-N-hydroxy-succinimide ester is covalently linked to the free 5'-amino residue. Apart from this, it can also be labeled with radioisotopes by transferring the γ-32P from *γ-32P] ATP to the 5' end using the enzyme bacteriophage T4 polynucleotide kinase. Before addition the template strand is dephosphorylated by calf intestinal alkaline phosphatase and a labeled phosphate molecule incorporated at this site by polynucleotide kinase.
This method includes labeling of RNA molecule by either radioactive or non-radioactive molecules. The target DNA (cDNA) fragment need to be labeled is cloned in to a bacterial vector system and expressed under the viral promoters like T7 or SP6 promoters. This molecule is expressed under in vitro condition by use of viral RNApolymerase specific to target promoter. During transcription process the labeled molecules incorporated in to RNA molecule. For SP6 and T7 promoters their respective RNA Polymerases are incubated with 40mM Tris-HCl (pH 7.9), 10mM NaCl, 6mM MgCl2, 10mM DTT, 2mM spermidine, 0.05% Tween®-20, 0.5mM each of dATP, dGTP, dCTP and dUTP, 0.5µCi [3H] dCTP and 1-2 µg of the linearized template DNA/ vector molecule. The reaction is allowed to take place at 37oC for 1hr.
Copyright © 2018-2020 BrainKart.com; All Rights Reserved. Developed by Therithal info, Chennai.