Gel Electrophoresis of DNA and RNA

In Sanger sequencing four reactions are run, each designed to terminate the growing DNA chain at one of the four bases (the G reaction is shown in detail). The result is four collections of fragments whose comparative lengths indicate the positions of the four bases (the sequence) of the DNA under study. In Sanger dideoxy…

There are four chemical cleavage reactions at the core of the Maxam and Gilbert sequencing system. The figure below left shows an example from these reactions, the reaction cleaving specifically at guanine. The other three reactions cleave at G+A, C+T, or C. Guanine and cytosine, therefore, give bands in 2 lanes, adenine, and thymine in…

DNA sequences are determined by a two-step process. In the first step the sample DNA is used, either directly or as a template, to generate sets of fragments. Each set contains multiple lengths of DNA, all of which end in one (or sometimes two) of the four nucleotide bases. These fragments are generally radiolabeled to…

Molecular weight determination is the most basic use of denaturing polyacrylamide gel electrophoresis. Samples are run versus standards of known molecular weight, and a calibration curve of relative mobility (or distance migrated) versus the logarithm of the size is established. Size can be expressed as molecular weight or number of bases. It is important to…

Temperature The most critical parameter in denaturing DNA-PAGE is gel temperature. Highly concentrated urea, 6-7M, is the most commonly used denaturant, but to be fully effective, the temperature must be maintained above 40°C. Denaturing PAGE gels are generally run with a temperature in the range of 45 – 60°C, which is maintained by running the…

Sequencing gels are poured between two glass plates separated by spacers. The spacers are typically no more than 0.2mm in thickness. The extreme thinness of the gel allows air bubbles to be trapped in the gel during pouring. Such bubbles are very hard if not impossible to remove. As oxygen inhibits the polymerization process, even…

DNA samples for denaturing gel electrophoresis must be denatured prior to loading, to avoid time dependent denaturation artifacts on the gel. This is usually carried out by diluting the sample into 95% formamide and heating to 95°C (see the Dideoxy Sequencing (Taq Polymerase) Protocol for a formula for the loading buffer). Loading the proper amount…

The denaturation of DNA by urea. The electrophoretic analysis of single-stranded nucleic acids is complicated by the secondary structures assumed by these molecules. Separation on the basis of molecular weight requires the inclusion of denaturing agents which unfold the DNA or RNA strands and remove the influence of shape on their mobility. Nucleic acids form…