Introduction à la biologie du développement by Thierry Darribère

By Thierry Darribère

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As a result, chain elongation is terminated whenever a ddNTP is incorporated since they have no hydroxyl groups. After incubation, the individual reactions containing one of the 4 ddNTPs are run on separate lanes of an acrylamide DNA sequencing gel and then exposed to film. The banding pattern reveals the sequence of the DNA. Figure 2 shows an example of a dideoxy sequencing reaction, with part of the corresponding sequences of two independent clones of a given stretch of DNA. Notice that one of the sequenced clones has a substitution, otherwise known as a mutation.

All other chromatographic techniques are based on retention of proteins by one or more properties. Common media for gel filtration columns include agarose-based beads, such as Sepharose and Superose, cellulose based beads, and composites (agarose and dextran = Superdex; dextran and acrylamide beads = Sephacryl). Ion exchange chromatography Ion exchange chromatography is a method used to isolate proteins based on their charge. Both anion exchange columns (positively charged columns that are used to isolate negatively charged proteins) and cation exchange columns (negatively charged columns to isolate positively charged molecules) are available.

Flow is achieved by gravity or small pumps, although specialized equipment can also be used (see HPLC and FPLC below). Material flowing through the column is collected in tubes by automated fraction collectors for further analysis. The most common forms of chromatography are summarized below. Gel filtration (or “size exclusion”) chromatography Gel filtration chromatography is based on a sieving method in which proteins are separated on the base of their size. Usually, columns are packed with a porous gel.

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