Sample tray

Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession

(anode)

Outlet Buffer

Data

Acquisition

An important difference between CE and gels is that the electric fields are on the order of 10-to-100 times stronger with CE (i.e., 300V/cm instead of 10 V/cm), which results in faster run times for CE.

Detection of the sample is performed automatically by the CE instrument through measuring the time span from sample injection to sample detection with a laser placed near the end of the capillary. Laser light is shined on to the capillary at a fixed position where a window has been burned in the coating of the capillary. DNA fragments are illuminated as they pass by this window in the capillary. As with gels, the smaller molecules will arrive at the detection point first followed by the larger molecules. Data from CE separations are plotted as a function of the relative fluorescence intensity observed from fluorescence emission of dyes passing the detector (see Chapter 13). The fluorescent emission signals from dyes attached to the DNA molecules can then be used to detect and quantify the DNA molecules passing the detector.

DNA SEPARATION MECHANISMS

Now that we have covered the two primary methods for DNA separations in use today, namely slab-gel electrophoresis and capillary electrophoresis, we will discuss briefly the theories behind DNA separations by electrophoresis.

With one phosphate group for every nucleotide unit, DNA molecules possess a constant charge-to-mass ratio. Thus, a piece of DNA that is 10 nucleotide units long will feel the same force pulling on it when an electric field is applied to it as a DNA oligomer that is 100 nucleotide units in length. In order to resolve DNA fragments that differ in size, a sieving mechanism is required. The separation of DNA is therefore accomplished with gels or polymer solutions that retard larger DNA molecules as they pass through the separation medium. The smaller molecules can slip through the gel pores faster and thus migrate ahead of longer DNA strands as electrophoresis proceeds (Figure 12.4).

Figure 12.4

Illustration of DNA separation modes in gel electrophoresis. Separation according to size occurs as DNA molecules pass through the gel, which acts as a molecular sieve (a). Ogston sieving and reptation are the two primary mechanisms used to describe the movement of DNA fragments through a gel (b).

Figure 12.4

Illustration of DNA separation modes in gel electrophoresis. Separation according to size occurs as DNA molecules pass through the gel, which acts as a molecular sieve (a). Ogston sieving and reptation are the two primary mechanisms used to describe the movement of DNA fragments through a gel (b).

In the simplest sense, a gel may be considered as a molecular sieve with 'pores' that permit the DNA molecules to pass in a size-dependent manner because larger molecules are retarded more than smaller ones. Two primary mechanisms for DNA separations through gel pores have been described: the Ogston model and reptation. These two theories are complementary as they operate in different size regimes. The Ogston model describes the behavior of DNA molecules that are smaller than the gel pores while reptation describes the movement of larger DNA molecules (Figure 12.4).

OGSTON SIEVING

The Ogston model regards the DNA molecule as a spherical particle or coil like a small tangle of thread that is tumbling through the pores formed by the gel. Molecules move through the gel in proportion to their ability to find pores that are large enough to permit their passage. Smaller molecules migrate faster because they can pass through a greater number of pores. When DNA molecules are much larger than the mesh size of the gel-sieving medium, the Ogston model predicts that the mobility (movement) of the molecules will go to zero.

REPTATION

However, gel separations have been demonstrated with DNA fragments that are much larger than the predicted pore size of the gel. The reptation model for DNA separations views the DNA molecule as moving like a snake through the gel pores. DNA molecules become elongated like a straight length of thread and enter the gel matrix end on. Separation of sample components, such as two STR alleles, occurs as the DNA winds its way through the pores of the gel matrix.

ADDITIONAL COMMENTS ON ELECTROPHETIC SEPARATIONS

Electrophoresis is a relative rather than an absolute measurement technique. The position of a DNA band on a gel has no meaning without reference to a size standard containing material with known DNA fragment sizes. Thus, samples are run on a gel side-by-side with molecular weight markers. For example, a DNA restriction digest might be used with a half-dozen or more fragments ranging in size from 100-1000 bp. A visual comparison can then be made to estimate the fragment size of the unknown sample based on which band it comes closest to since the samples were subjected to identical electrophoretic conditions. Alternatively, in multi-color fluorescent systems, an internal sizing standard labeled with a different colored dye can be run with each sample to calibrate the migration times of the DNA fragments of interest with a sample of known size (see Chapters 13 and 15).

The separation media that the DNA passes through, as well as the overall shape of the molecule and the electric field applied to the sample, influences the molecular movement (i.e., speed of separation for each component). The exact technique that one uses to separate the DNA molecules in a particular sample is dependent on the resolution required. The resolution capability of a separation system is dependent on a number of factors including the type of separation medium used and the voltage applied.

ADDITIONAL READING

Butler, J.M. (1995) Sizing and Quantitation of Polymerase Chain Reaction Products by Capillary Electrophoresis for Use in DNA Typing. PhD Dissertation, University of Virginia.

Heller, C. (ed.) (1997) Analysis of Nucleic Acids by Capillary Electrophoresis. Braunschweig: Vieweg.

Martin, R. (1996) Gel Electrophoresis: Nucleic Acids. Oxford: Bios Scientific Publishers.

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CHAPTER 13

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