Agarose Gel Electrophoresis for DNA Analysis

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During Lecture 9 and 10 we have studied basics of protein electrophoresis. Recalling our discussion during lecture, protein needs to be boiled with SDS to give uniform negative charge. This enables protein to move toward positive electrode when electric field is applied. Polyacrylamide was used as solid support for separation. We also discussed why a solid support is needed for electrophoratic separation (please refer our earlier lecture to refresh the concepts).

Let us see how DNA electrophoresis differs from protein electrophoresis.

•  Basic theory of the electrophoresis is valid in case of DNA electrophoresis as well.

•  Generally agarose is used for DNA separation. As size of the DNA is lot bigger than proteins, a stable solid support with bigger pore size is required. Size of polyacrylamide pores is smaller for this purpose. Note: Agarose gel can also be used for separation of very large protein/protein complex. Agarose gel electrophoresis is most suitable for separation of DNAs/RNAs in the range of 100bp to about 15kb. Polyacrylamide gels matrix may be used for separation of small DNAs or RNAs.

•  DNA already has uniform negative charge. If you recall DNA structure from our last class, two nucleotides are connected together by a phosophodiester bond which gives one negative charges.

Here, we are ignoring one negative charge due to phosphate at 5' end as this is very small compare to net charge provided by phosophodiester bonds in a polynucleotide.