Electrophoresis is a technique used in genetic testing to analyze and separate proteins, DNA, and RNA. Proteins are fundamental components of all living cells and include many substances, such as enzymes, hormones, and antibodies, which are necessary for the proper functioning of an organism. Proteins are made up of amino acids that are arranged and created by the cell according to their specific DNA or RNA sequence. Deoxyribonucleic acid (DNA) is a long, double-stranded, thread-like molecule made up of large numbers of nucleotides. Nucleotides in DNA are composed of a nitrogen-containing base (base pair), a 5-carbon sugar (deoxyribose), and phosphate groups. Ribonucleic acid (RNA) is a polymeric constituent of all living cells and many viruses consisting of a long, usually single-stranded chain of nucleotides. RNA usually has a much shorter nucleoside chain than DNA.
Electrophoresis uses an electrical current to separate the molecules by electrical charge. There are two types of electrophoresis: gel and capillary. Gel electrophoresis separates the molecules by molecular weight and charge, whereas capillary only separates molecules by charge. The focus of this monograph will be primarily on gel electrophoresis, which is more widely used in research.
Gel electrophoresis uses different types of gel mediums that vary by the molecule being analyzed, as well as the desired level of resolution. Agarose and polyacrylamide gel are the most commonly used mediums for protein, DNA, and RNA separation.
Agarose is a complex sugar molecule derived from seaweed and is commonly used to analyze nucleic acids (DNA and RNA).
Polyacrylamide is a large compound that is formed from one small molecule (polymer) of the chemical acrylamide and is used in many industrial processes such as sewage and water treatment Polyacrylamide gel electrophoresis (PAGE) is frequently used to analyze proteins. Because of its capacity to separate molecules with high resolution, polyacrylamide is also used to separate nucleic acids that are small or those that differ in length by as little as one base pair.
Electrophoresis has been used in genetic screening, biochemistry, molecular biology, microbiology, and forensics. Any field of study or test that requires the analysis, separation, and quantification of proteins, RNA, and DNA may utilize electrophoresis.
General principles: The distance that the molecule will travel depends on several factors: the amount of current applied, electrical charge of the molecule, size of the pore and type of gel matrix, type of buffer solution being used, and size of the molecule. Smaller molecules move faster than larger molecules because they have less resistance when moving across the gel.
The medium that the molecules travel through is usually a gel. The gel that is used is selected based upon the size of the pores. The pore size can affect the movement of the molecules based upon their molecular weight. Changing the size of the pore allows for faster separation and better resolution.
Shape of the molecule will also affect the migration rate in a gel. Therefore, the conformations (or secondary structures) of nucleic acids and proteins are often removed before gel electrophoresis. This ensures that the fragments are separated based on size (or mass).
Proteins can also have significant secondary structure (folds and twists) that can affect migration rate. Furthermore, the amino acids have a wide range of charge. To eliminate the effects of protein charge and conformation on migration rate, proteins may be heated to undo (denature) the secondary structure. To give proteins an even charge, they may be heated in a detergent called sodium docecyl sulfate (SDS), which evens out the charge of the protein. Therefore, protein electrophoresis is frequently called SDS-polyacrylamide gel electrophoresis or SDS-PAGE.
Agarose gel electrophoresis: Agarose is a polysaccharide extracted from seaweed that is commonly used as a gel medium for DNA and RNA separation. Increasing the concentration of agarose used will give the matrix smaller pores and higher resolution of smaller DNA fragments.
Circular DNA may migrate faster than a linear fragment of the same size. Furthermore, circular DNA that is tightly wound (supercoiled) may migrate faster than relaxed or linear DNA fragments. To eliminate the effects of conformation on migration, circular DNA is usually digested (cut) with enzymes called restriction endonucleases before gel analysis.
This type of electrophoresis may be used for the analysis of polymerase chain reaction (PCR) products that have been separated for genetic fingerprinting or genetic diagnosis. PCR products are created by a technique called PCR. PCR is an enzymatic method for the repeated copying of the two strands of DNA of a particular gene sequence. It is widely used to amplify minute quantities of biologic material so as to provide adequate specimens (PCR products) for laboratory study. Agarose gel electrophoresis may also be used to analyze RNA and DNA that has been digested by restriction enzymes.
Agarose gels are prepared by heating agarose in a buffer solution. Molten agarose is poured into a gel mold and a comb is inserted to form sample wells along the top of the gel.
The gels are submerged into buffer solutions, which contain salts that allow an electric current to pass through the solution. Commonly used buffers include tris-acetate-EDTA (TAE) or tris-borate-EDTA (TBE). The properties of the buffer allow for different voltages to be used, time to run the procedure, and better resolution depending on the size of the molecules being analyzed.
DNA (or RNA) samples are mixed with loading buffer, which consists of a dense material (glycerol) that allows the sample to settle into the sample well. Loading buffers also contain dyes that move at specific rates. These dyes are used to track the migration of the DNA in the gel. Xylene cyanol is a color marker that moves at about the same rate as a 4,000 base pair DNA fragment. Bromophenol blue (about 500 base pairs), Cresol red (125 base pairs), and Orange G (50 base pairs) are also used. The rate of movement is an approximation and may vary depending on the dye used, the buffer concentration, and the concentration of agarose gel.
In order to visualize the sample, a fluorescent dye such as ethidium bromide is used to stain nucleic acid. The ethidium bromide forms a bond with the DNA or RNA and moves with the molecules through the gel matrix. Ethidium bromide can be seen under UV light.
Agarose gel electrophoresis has a large range of separation but a low resolving power, making it more useful in separating DNA molecules with large variations in size. Typically, agarose gel electrophoresis only separates nucleic acids up to 50,000 base pairs in length. However, by using a pulsing electric current, DNA that is up to 3,000 kilobase pairs long can be isolated. This technique, called pulse-field gel electrophoresis (PFGE), uses an alternating electrical current that changes strength and location. This alternation of current causes a snaking effect (reptation). As the process runs longer, the gel becomes more liquefied and the larger molecules move more easily. This method has been used to separate out whole genes for analysis.
Very small nucleic acids (less than approximately 500 base pairs) are separated with better resolution using polyacrylamide gel electrophoresis (PAGE). These gels can also separate nucleic acids that differ in length by as little as one base pair. Therefore, PAGE is often used in DNA sequencing, which analyzes DNA one base pair at a time.
Polyacrylamide gel electrophoresis (PAGE): Polyacrylamide is a polymer of acrylamide used to form a gel matrix in electrophoresis. The length of the polymer chain or amount of crosslinking is determined by the concentration of acrylamide used. Using a higher concentration of acrylamide results in more crosslinking and subsequently smaller pores in the gel. PAGE is typically used for the separation of proteins because of its small range of separation. However, PAGE does have a high resolving power and can be used to separate DNA fragments that vary in length by only one base pair.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): This form of PAGE is commonly used in genetics, biochemistry, and molecular biology to analyze proteins. Sodium dodecyl sulfate is a negatively charged compound that is added to a protein solution to give the proteins a negative charge. This negative charge allows the proteins to move through the gel matrix according to their molecular weight. Without the negative charge, some proteins with the same molecular weight might move differently due to other properties that proteins possess.
Two-dimensional polyacrylamide gel electrophoresis (2D PAGE): This technique uses two dimensions to separate proteins. The first dimension uses isoelectric focusing (IEF), a process that separates proteins by their charge (isoelectric point). The second dimension uses SDS-PAGE to separate the proteins by molecular weight. This process is particularly useful for the identification of a large amount of proteins that are then further analyzed by mass spectroscopy.
Two-dimensional agarose gel electrophoresis: This method is similar to 2D PAGE except that the molecule being analyzed is a DNA fragment and the gel medium is agarose. The first dimension is run at a low voltage in a low agarose concentration gel that allows for the separation of DNA according to its mass. The second dimension is run at a higher voltage and uses a higher agarose concentration gel that separates the DNA by shape.
Electrophoretic mobility shift assay (EMSA): This test is used to identify the interaction of proteins with DNA. A radiolabeled DNA fragment with a specific DNA site is isolated and combined with proteins and then analyzed using gel electrophoresis that does not break down (denature) the protein. Protein-DNA bound complexes move slower than unbound DNA through the gel that can then be further analyzed. EMSA is useful for studying a protein's DNA binding ability and the strength of that binding as well as comparing several proteins' affinities for the same DNA binding site.
Blotting techniques: Blotting is the transfer of separated material from the gel matrix to a solid membrane like nitrocellulose paper or nylon. This is performed after electrophoresis to help visualize and quantify the results of the separation of molecules.
Southern blot: This technique is used for the detection of specific DNA fragments. The DNA fragments of interest are separated by gel electrophoresis and blotted (transferred) on to a plastic sheet (typically nitrocellulose or nylon). The fragments are then detected by a technique called hybridization that uses a radioactive probe with a matching DNA sequence. Hybridization is the joining of the probe with the fragment, which allows the target molecule to be analyzed. The radioactive DNA is then visualized by autoradiography, which develops film exposed to radiation (similar to an X-ray).
Northern blot: Northern blot uses the same technique as the southern blot, but instead of DNA, it detects RNA. Once the sample of RNA goes through electrophoresis, it is labeled with a radioactive probe containing a DNA fragment with a matching sequence.
Western blot: Also known as immunoblotting, western blot is similar to southern and northern blotting except it is used to identify specific proteins. The probe consists of a specific antibody to the protein of interest. The antibody may be labeled with a molecule that can expose film, which is then developed (similar to a southern or northern blot).
General: Researchers often have a large sample with multiple proteins, DNA, RNA, or any combination of these products. These samples can be simplified through separation using electrophoresis. The separated product can then be analyzed using other techniques (mass spec, PCR).
Known mutations or defects in protein, DNA, and RNA may be screened for using gel electrophoresis techniques.
The combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-page) and western blot has been used to analyze specific protein to protein interactions and protein activity. There are specific antibodies for identifying phosphorylated proteins. Phosphorylated proteins are a marker for protein activity.
Conformation-sensitive gel electrophoresis (CSGE): Researchers have used CSGE to screen for RET proto-oncogene germline mutations (RET). RET gene mutations are inherited and have been associated with a rare disease called multiple endocrine neoplasia type 2 (MEN2). CSGE was shown to be a fast, cost-effective, simple, and sensitive technique to identify RET gene mutations. Research in cancer has used CSGE to identify mutations in BRCA1/2 genes that are associated with an increased risk of breast and ovarian cancer.
Proteomics: Proteomics is the study of the creation and action of proteins in the body and cells. In proteomics two-dimensional electrophoresis has been widely used to simplify samples in order to make protein analysis easier. This technique has limited reproducibility and sensitivity. Researchers have used difference gel electrophoresis to give better reproducibility and sensitivity. Difference gel electrophoresis uses multiple fluorescent dyes that are attached to specific proteins of interest.
Southern blot: Southern blot hybridization has been used in combination with polymerase chain reaction (PCR) to detect human papillomavirus (HPV). Some forms of HPV have been associated with the development of cervical cancer. PCR may be more sensitive at detecting certain types of HPV but less specific. Southern blot requires a larger DNA sample and is a harder test to perform. PCR is often the method of choice and has become more readily available than southern blot.
Electrophoresis is one of many steps in isolating the molecules of interest in genetics (proteins, DNA, and RNA). The technique remains a well-established method of effectively simplifying study samples by a specific protein or DNA sequence.
Gel electrophoresis is limited to the resolution power of the gel being used. Agarose gels can be used to detect DNA with much longer base pairs than with acrylamide gels. Polyacrylamide gels are more useful in the analysis of proteins.
Electrophoresis is often used with other tests, and its utility is restricted by the limitations of the other tests.
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The information in this monograph is intended for informational purposes only, and is meant to help users better understand health concerns. Information is based on review of scientific research data, historical practice patterns, and clinical experience. This information should not be interpreted as specific medical advice. Users should consult with a qualified healthcare provider for specific questions regarding therapies, diagnosis and/or health conditions, prior to making therapeutic decisions.