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Chapter 3:
Manipulating Nucleic Acids and Proteins
Abstract
Since nucleic acids have an inherent negative charge, an electric field can be used to apply a force to the molecule and move it through a medium like gel. By selecting a propagation medium that provides appropriate "€œfriction,"€ the nucleic acids can be sorted by size (length). Depending on the application, different gels, electric fields, and instruments are needed. DNA sequencing usually requires single-base resolution for DNA fragments with 20 to 1,000 bases. The details of this will be discussed later. DNA "€œsizing" may be done in simple gel boxes with large electrodes (see Fig. 3.1). The samples are introduced into the gel at one end near the cathode and propagate toward the anode at the other end of the gel. At a fixed time, DNA molecules of similar size will collocate in a band in the gel. Shorter DNA fragments move more quickly through the gel. These bands can be visualized with dyes, optical labels, or radioactive labels. An example is shown in Fig. 3.2 for a gel using myotonic dystrophy studies. Proteins are more complex, but are approached in a similar manner. An entire gel can be imaged at one point in time to produce a two-dimensional map of the gel with DNA bands. It is also possible to monitor the gel with a detector at a fixed distance from the cathode or loading well. The bands of DNA move past the detector and produce a one-dimensional time plot. The time series approach is particularly useful for molecules separated by a gel in glass capillaries. Samples are introduced at one end of the capillary and the molecules are detected after being driven down the capillary by an electric field. A synthetic set of data for capillary collection of the gel in Fig. 3.2 is shown in Fig. 3.3.
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CHAPTER 3
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