Question

A chromatographic column in which oligo-dT is linked to an inert substance is useful in separating eukaryotic mRNA from other RNA molecules. On what principle does this column operate?

Answer 1

oligo-dT is single stranded Deoxythymindine (dT) sequence. Oligo-dT is used during column chromatography to isolate or extract mRNA from a sample. mRNA has poly-A tail at it's 3' end so this poly-A tail is hybridized with oligo-dT which is attached to the column.

Other materials present in the sample like proteins, DNA, dsRNA, rRNA or any other moiety lacking poly-A tail are not attached to the column and only RNA having poly-A tail are attached to the column.

So, Oligo dT is a chromatography column with covalently bounded oligo-dT ligands on its surface. In a high salt concentration buffer a sample containing mRNA which are polyadenylated is loaded onto the column. The electrostatic repulsion between the negatively charged backbones are screened by salt ions and allow interaction between the poly-adenylated tail of mRNA and oligo-dT. All other contaminants which do not contain poly-A tail are flow through the column.

After that before elution of the product, a wash step is done at reduced salt concentration to remove unspecifically bound contaminants. Then under mild conditions in low conductivity buffer at neutral pH elution of mRNA occurs. When the salt is absent, electrostatic repulsion between the negatively charged backbones of Oligo dT and poly-adenine destabilises the T–A pairs and releases mRNA from the column. By this means oligo-dT column are used for extraction of mRNA from a sample.

The oligo-dT column thus operates on the principle of hybridization or nucleotide base pairing i,e A-T base pairing.