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DNA gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via polymerase chain reaction (PCR), but may be used as a preparative technique for other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or southern blotting for further characterization.
Electrophoresis techniques used in the assessment of DNA damage include alkaline gel electrophoresis and pulsed field gel electrophoresis. For short DNA segments such as 20 to 60 bp double stranded DNA, running them in polyacrylamide gel (PAGE) will give better resolution (native condition). [ 1 ]
A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix.
Kenneth and Noreen Murray introduced this technique as Southern. The second innovation is the gel electrophoresis that is based on separation of mixtures of DNA, RNA, or proteins according to molecular size, which was also developed at Johns Hopkins University, by Daniel Nathans and Kathleen Danna in 1971.
The DNA bands may then be visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. Part of a radioactively labelled sequencing gel. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths.
Pulsed-field gel electrophoresis (PFGE) is a technique used for the separation of large DNA molecules by applying an electric field that periodically changes direction to a gel matrix. [ 1 ] [ 2 ] Unlike standard agarose gel electrophoresis , which can separate DNA fragments of up to 50 kb, PFGE resolves fragments up to 10 Mb. [ 1 ]
Agarose gel has large pore size and good gel strength, making it suitable as an anticonvection medium for the electrophoresis of DNA and large protein molecules. The pore size of a 1% gel has been estimated from 100 nm to 200–500 nm, [ 4 ] [ 5 ] and its gel strength allows gels as dilute as 0.15% to form a slab for gel electrophoresis. [ 6 ]
Following the restriction reaction, the mixture of fragments is separated using either capillary or polyacrylamide electrophoresis in a DNA sequencer and the sizes of the different terminal fragments are determined by the fluorescence detector. Because the excised mixture of amplicons is analyzed in a sequencer, only the terminal fragments (i.e ...