Electrophoresis Prime Principles and Techniques
Agarose, Polyacrylamide, Starch Electrophoresis of DNA and Proteins
Nov 10, 2009
From the "trial of the century" of O.J. Simpson, to nationwide epidemics, paternity suits, forensics, or basic research involving DNA or proteins, electrophoresis is a superior research tool. The principles of electrophoresis are the basis of this inquiry.
Electrophoresis Prime Principles and Techniques for Starch, Agarose, and Polyacrylamide Gels
The Greeks would call it elektron (beaming sun) phoresis (carrying) — electrophoresis is the electric field-induced movement of molecules through a gel.
When the electrophoresis gel pH is adjusted to 8.6, all proteins, or DNA molecules, placed within the wells of that gel become negatively charged. When the current is turned on, the negatively-charged molecules move towards the anode at rates dependent upon the overall charge and mass of the molecules, i.e., charge/mass ratio.
Typically, the globular proteins, when treated with sodium dodecyl sulfate (SDS) and mercaptoethanol, form uniform-shaped random coils with identical charge/mass ratios. These treated proteins then will migrate at rates inversely proportional to their molecular weights.
Either protein, or DNA molecules, can be prepared and added to reference wells (see photos 1 and 2 below). These reference wells of known marker molecules, enable scientists to calculate the actual molecular weights of all the unknown, test proteins or DNA placed in the test wells.
Electrophoresis Prime Media Gel Types — Starch, Agarose and Polyacrylamide
Three major gels are used for electrophoresis:
- Starch, a simple, modified polysaccharide – prepared as a paste from ruptured potato starch grains – is useful for proteins, especially isoenzymes.
- Agarose, another polysaccharide – derived from seaweed – is used for studies of DNA and proteins. Agarose forms clear gels, similar to those of acrylamide.
- Polyacrylamide gels, a synthetic polymer prepared by polymerizing acrylamide and bisacrylamide. Gel pore sizes and physical properties of the polymer are determined by the % gel polyacrylamide and the amount of cross-linkage. Gradient gels can be prepared with polyacrylamide, and these gels provide electrophoresis results of great definition and clarity. Denaturing, high-definition, polyacrylamide gels are used for DNA sequencing. These gels are able to resolve down to a single nucleotide, and they are useful for separating STR (Single Tandem Repeat) alleles. PAGE-SDS is also useful, as mentioned previously, for studies of proteins.
Electrophoresis Prime Principles and Techniques for DNA
- Polyacrylamide gel electrophoresis can separate and distinguish different sizes of DNA molecules. The DNA can be stained with ethidium bromide, and then detected by ultraviolet light. The bright "bands" on the gel photo (see photo 3 below) represent DNA fragments produced by treating whole DNA with a restriction endonuclease. The bands to the far left and right sides (lanes 1 and 12) are control, standardized, DNA size marker lanes. The bands in between lanes 1 and 12 represent bacterial DNA extracts treated with the same endonuclease. Careful examination of the ethidium bromide-treated gel reveals that lanes 2,3,4, 9,10, 11, all have 2 DNA bands that migrate similarly. Lane 6 contains the most unique DNA pattern, since neither of its 2 bands match any other of the test DNAs. Finally, lanes 5, 7, and 8 show another related group of DNAs that differ from the previous two DNA groups.
- For each additional nucleotide unit added to a DNA molecule, the charge is increased in proportion to the addition in mass. DNA fragments larger than about 10 bp (base pairs) possess essentially the same electrophoretic mobility. Since DNA possess a constant mass-to-charge ratio, some form matrix is needed to separate DNA fragments by molecular size. In traditional slab gel electrophoresis, a sieving matrix is obtained with polyacrylamide or agarose gels and larger DNA fragments are impeded relative to smaller DNA fragments.
- STR alleles, such as tetranucleotide and dinucleotide repeats, may be separated, resolved and typed using small (10 cm long, 1 mm thick) MetaPhor agarose gels stained by SYBR Green (White and Kusukawa in BioTechniques 1997, 22, 976-980).
The value of electrophoresis procedures including advanced 2-D electrophoresis for proteomics is well-documented, and is expected to be a valuable research tool for many years in the future.
Sources
Lodish, H. et al. 2000. Molecular Cell Biology. Fourth Ed., W. H. Freeman and Co., New York, N.Y.
Wandersee, J.H., et al., eds. 1996. Bioinstrumentation, Tools for Understanding Life. National Assoc. Biol. Teachers, Reston, Va. 329 pp
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