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Home , Electrophoresis, Gel electrophoresis

Understanding Inheritance

GEL L.

Historically electrophoresis was first (a) Conventional loaded a gel-calibration sample, a sample applied to separating essentially containing fragments of known lengths. As according to mass, but the technique was DNA fragments shown in (a), the flow of electricity through adapted to separating fragments of DNA (or the gel causes the fragments to migrate Cathode 1 gel , Anode RNA) essentially according to fragment toward the positive electrode, The shorter length. The technique works on DNA be- fragments move more easily through the cause the groups of a DNA gel and therefore travel farther. fragment are negatively charged, and there- \ fore, underthe influence of an electric field, The positions of the fragments after electro- \ Buffer solution the fragment migrates through a gel (a phoresis can be detected by soaking the gel \ Electrophoresis chamber porous, semisolid medium) in a direction in a solution of , which opposite to that of the field. Furthermore, binds strongly to DNAand emits visible light the rate at which the fragment migrates when illuminated with light. In a through the gel is approximately inversely photograph of the ultraviolet-illuminated gel, proportional to the logarithm of its length. the fragments appear as light bands, The ethidium-bromide visualization technique Gel electrophoresis of DNA is carried out makes the positions of all the fragments in with two types of electric field. Conventional the gel visible, An alternative visualization gel electrophoresis employs a field that is to separating fragments with lengths less technique detects only certain fragments temporally constant in both direction and than about a thousand base pairs and is (see ‘iHybridization Techniques”). magnitude. In contrast, pulsed-field gel elec- therefore the gel of choice for sequencing.) trophoresis employs a field that is created Conventional gel electrophoresis in an aga- The above description of gel electrophore- by pulses of current and therefore varies rose gel is illustrated in (a); details of the sis might suggest that the sample of DNA periodically from zero to some set value. technique are as follows. contains but one copy of each fragment. In More important, the direction of the electric reality the sample must contain many cop- field also varies because different pulses Agarose is dissolved in a hot buffer solution, ies of each fragment, and each band seen flow through pairs of electrodes at different and the gel solution is allowed to solidify into in the image of the length-separated frag- locations. (Note, however, that the time- a thin slab in a casting tray in which the teeth mentscontains many fragments, all of which averaged direction of the electric field is of a comb-like device are suspended. After have the same length but not necessarily along the length of the gel.) The advantage the gel has solidified, the comb is removed. the same sequence. of such a Ipulsed field is that it prevents long The “wells” formed by the teeth of the comb DNA fragments, fragments Iongerthan about are the receptacles into which the samples 50,000 base pairs, from jackknifing within of DNA are loaded. The thickness of the gel the structural framework of the gel and thus is about 5 millimeters; its length and width allowsthe long fragments tomigratethrough are much greater and vary with the purpose the gel in a length-dependent manner, just of the electrophoresis. Before being loaded as shorter fragments migrate in a constant with the DNA sample(s), the gel is im- electric field. mersed in a conducting buffer solution in an electrophoresis chamber. The gel employed is usually a solidified aqueous solution of agarose, a purified form Before a DNA sample is loaded into a well, of . 13y varying the concentration of it is mixed with a dense solution of agarose in the gel, conventional gel electro- or glycerol to prevent the DNA from escap- phoresis can be applied to samples con- ing into the buffer solution. Into one well is taining DNA fragments with average lengths between a few hundred base pairs and tens of thousands of base pairs, (Another gel used for conventional electrophoresis is , which is particularly suited

55 Understanding Inheritance

(b) Conventional Gel Electrophoresis Shown in (b) are the results of conventional (c) Pulsed-field Electrophoresis of of Fragmented Human DNA gel electrophoresis of six different samples Intact DNA Molecules of Segments of human DNA. Samples 1, 2, and 3 con- Saccharomyces cerevisiae sisted of the restriction fragments produced

-L -L by cutting the same cloned segment of . + human DNA with EcoRI alone (a 6-base cutter), with both EcoRI and I+rtdlll (an- other 6-base cutter), and with F/indlll alone, 173456 respectively, Samples4, 5, and 6consisted of the restriction fragments produced by 23.1 19.9 cutting adifferent cloned segment of human DNA again with EcoRI alone, with both 16.7 ,EcoRI and Hindlll, and with Hiridlll alone, respectively. The leftmost lane of the gel 11.8 contains fragments of the lengths indicated. lloo– Note that all the restriction fragments are

9.4 well resolved.

Shown in (c) are the results of pulsed-field 960 –

gel electrophoresis of three identical 920- samples, each containing all sixteen of the intact DNA molecules that compose the genome of the yeast Saccharomyces cerevisiae. The four longest chromosomal 800 – DNA molecules are not resolved; all four are 760 – located in the topmost band, The remaining 690 – twelve chromosomal DNA molecules, how-

ever, are well resolved. The indicated lengths 610- of the resolved DNA molecules were deter- mined from the positions, in the rightmost

lane of the gel, of the fragments in a calibra- 445 – tion sample. Even longer fragments, frag- ments with lengths up to about 5 million 360 – 2.3 base pairs, can be separated by increasing 340-

2.0 the duration of the pulses. 280- 250-

1.35

1.08

0.87

0,60

56 L(,,\