Molecular biology and immunology for clinicians

Electrophoresis is a powerful tool used for the separation of proteins and nucleic acids by their size. For the latter, often very large molecules, separation techniques typically use agarose gels rather than the polyacrylamide gels used to separate proteins. Once separated and transferred to a membrane support, one can detect nucleotides by identifying unique sequences of their purine and pyrimidine bases using a complementary DNA probe specially synthesized to be specific for the nucleic acid of interest. One can electrophoretically separate and detect DNA (Southern blot analysis) or RNA (Northern blot analysis). These techniques depend on a DNA probe hybridizing with the target DNA immobilized on a membrane. Hybridization techniques are sensitive enough to detect differences (caused by either mutation or inherited differences) in the sequences of genetic material using a technique based on the digestion of nucleic acids by restriction endonucleases, which produce restriction fragment-length polymorphisms. DNA probe hybridization can be also used to detect genetic material within cells in in situ hybridization.