Recombinant DNA Technology Problem Set

Problem 3: Southern Technique

Tutorial to help answer the question

The "Southern" technique involves:
the detection of DNA fragments on membranes by a radioactive DNA probe.


Separating DNA by gel electrophoresis

DNA is taken from individuals A and B.

It is digested with restriction endonuclease.

Fragments are applied onto an agarose gel for electrophoresis.

DNA has a negative charge, and in an electric field migrates towards a positive electrode.

The rate of migration through a gel is proportional to the size of the fragment.


Transferring DNA fragments to nitrocellulose sheets

DNA fragments are transferred to nitrocellulose sheets where they bind.

DNA fragments are denatured and separated by gel electrophoresis.

Fragments are blotted onto a sheet of nitrocellulose and fixed by heating.

Blot is reacted with a radioactive probe of RNA or DNA which binds to complementary DNA.

Autoradiography is used to detect radioactive fragments.

Fixing the fragments of DNA

The denatures fragments of DNA are fixed by baking.

A radioactive probe is added. It can hybridize with a gene sequence in the DNA.

The sheet is rinsed and placed next to X-ray film for autoradiography.

The Biology Project
University of Arizona
Updated: July 15, 1999
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