isolating DNA

Last reviewed 01/2018

One problem frequently encountered in genetic engineering is how best to isolate sequences of DNA from complex mixtures. There are several approaches:

• create a DNA library. This involves using a restriction endonuclease enzyme to section genomic DNA into lots of fragments which are all then introduced into vectors. The vectors are then amplified independently.

• create a complimentary DNA library. Messenger RNA from a tissue of interest is converted into cDNA by a reverse transcriptase enzyme. The cDNA is changed to DNA by a polymerase enzyme before ultimate cloning. Generally, cDNA will be different from the DNA that it originally came from as it has been been produced indirectly from mRNA lacking introns - it has been subject to post-translational modification.

• synthesise an oligonucleotide probe. This requires knowledge of the amino acid sequence of all or part of the protein product under investigation. The probe is made from base pairs that would be required as a template for the postulated protein. The radioactively labelled probe is capable of binding to its complimentary sequence amongst a library of alternatives.