In studies of transcriptional regulation, it is often necessary to determine the structure and/or amount of a given RNA species. Several techniques have been developed making use of the fact that some nucleases will only digest single-stranded nucleic acids (ssDNA or ssRNA). Duplexes, whether DNA: DNA, RNA: RNA, or DNA: RNA, are resistant to digestion.…

The differential display is a PCR-based technique that generates a characteristic set of DNA fragments from the messenger RNA pool within a cell. The use of random hexamers (brown) in combination with oligo-dT primers (blue) allows the amplification of a population of DNA species which changes with the composition of the starting RNA pool. The…

Automated sequencing systems make use of fluorescent dye labeling, in combination with laser scanning and computerized data acquisition and processing to carry out the electrophoresis of up to 96 sequencing reactions on a single gel, and read over 1,000 bases from each reaction. A single run on an automated sequencer can thus produce as much…

Denaturing PAGE gels for DNA sequencing generally employ 6-8 M urea as their denaturant and TBE as their buffer system. They are poured as described in the section on denaturing PAGE of DNA and RNA. After a 2-2.5 hour run, a 6% polyacrylamide sequencing gel will give 200-250 bases of readable sequence starting at or…

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…