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General Procedures
Chapter 5
General Procedures Weimin Sun, Ph.D., ABMG, CGMB, CLSP(MB) Quest Diagnostics Nichols Institute, Molecular Genetics Department, San Juan Capistrano, CA 92675
I. Introduction Some of the classical molecular biology methods are also used routinely in clinical molecular diagnostic laboratories, either as part of patient testing procedures or as quality control and/or quality assurance measures. These procedures may also serve as building blocks of more sophisticated molecular diagnostic procedures. Although important molecular biology methods such as plasmid construct preparation, bacterial transformation, or transfection of mammalian cells are rarely if ever used in a molecular diagnostic laboratory, others such as gel electrophoresis, polymerase chain reaction (PCR, which is described in more detail in Chapter 4, “Nucleic Acid Extraction and Amplification”), and ultraviolet (UV) spectrometry are commonly used.
II. Gel electrophoresis Charged biomolecules such as proteins, peptides, and nucleic acids can migrate through a gel matrix under the influence of electric fields. Hence, using appropriate buffers and gel compositions, biomolecules can be separated according to their charges, sizes, shapes, and other physical properties. Nucleic acid molecules are negatively charged. Singlestranded RNA, double-stranded DNA, or small oligonucleotide molecules can all be analyzed by gel electrophoresis. As an analytical tool, gel electrophoresis can be used to separate a complex group of nucleic acids or to identify the presence of specific nucleic acid molecules by size or mobility characteristics. In addition, it can also be used as a preparative method to purify and concentrate a specific nucleic acid molecule. As the most commonly used analytical procedure in a molecular pathology laboratory, gel electrophoresis allows us to visualize patient nucleic acid specimens or their assay products directly. Qualitative as well as quantitative information can be obtained from this procedure. Oftentimes, it is part of an assay procedure either as a quality control Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory 1 ed. Copyright © 2010 by Academic Press. Inc. All rights of reproduction in any form reserved.
(QC) measure or as a detection method (Godde et al., 2006). Dependent on application needs, different gels or gel formats, such as agarose versus polyacrylamide, or slab gel versus capillary gel, can be used.
A. Agarose Gel Electrophoresis Agarose gel electrophoresis used to be the essential gel electrophoresis method for any molecular diagnostic laboratory due to its simple procedure, wide applicability, and inexpensive apparatus. A standard agarose gel apparatus is also referred to as a submarine apparatus, where a tray containing solidified agarose gel is submerged in buffer solution during electrophoresis. A gel is prepared by dissolving a set amount of agarose powder in gel buffer (see Table 5.1) by heating the mixture in a microwave oven. The agarose gel solution is cooled to 60°C or so and then poured into a sample preparation tray with sample comb(s) inserted (Fig. 5.1). Particular care needs to be taken to pierce the bubbles in the gel molten with clean pipet tips or they will cause distorted nucleic acid migration during electrophoresis (There are more bubbles in higher percentage gels, such as 3%). After a gel is fully solidified, sample comb(s) can then be carefully removed to leave well-formed sample wells. Nucleic acid specimens need to be mixed with a loading buffer that contains glycerol (Ficoll or sucrose can also be used to increase sample density) and tracking dye(s) such as bromophenol blue or xylene cyanol FF before loading. In addition to serving as an indicator that a nucleic acid sample has been properly loaded into a sample well, the dyes are also crude tracking devices that monitor the progress of electrophoresis. To allow nucleic acid specimens to be visualized by UV transillumination, ethidium bromide (EtBr) is added either to the agarose gel solution right before pouring or to the tank buffer, or both. Gloves need to worn at all times when handling gels or buffers containing EtBr as it is a potent DNA intercalator and a known mutagen. 49
General Procedures Weimin Sun, Ph.D., ABMG, CGMB, CLSP(MB) Quest Diagnostics Nichols Institute, Molecular Genetics Department, San Juan Capistrano, CA 92675
I. Introduction Some of the classical molecular biology methods are also used routinely in clinical molecular diagnostic laboratories, either as part of patient testing procedures or as quality control and/or quality assurance measures. These procedures may also serve as building blocks of more sophisticated molecular diagnostic procedures. Although important molecular biology methods such as plasmid construct preparation, bacterial transformation, or transfection of mammalian cells are rarely if ever used in a molecular diagnostic laboratory, others such as gel electrophoresis, polymerase chain reaction (PCR, which is described in more detail in Chapter 4, “Nucleic Acid Extraction and Amplification”), and ultraviolet (UV) spectrometry are commonly used.
II. Gel electrophoresis Charged biomolecules such as proteins, peptides, and nucleic acids can migrate through a gel matrix under the influence of electric fields. Hence, using appropriate buffers and gel compositions, biomolecules can be separated according to their charges, sizes, shapes, and other physical properties. Nucleic acid molecules are negatively charged. Singlestranded RNA, double-stranded DNA, or small oligonucleotide molecules can all be analyzed by gel electrophoresis. As an analytical tool, gel electrophoresis can be used to separate a complex group of nucleic acids or to identify the presence of specific nucleic acid molecules by size or mobility characteristics. In addition, it can also be used as a preparative method to purify and concentrate a specific nucleic acid molecule. As the most commonly used analytical procedure in a molecular pathology laboratory, gel electrophoresis allows us to visualize patient nucleic acid specimens or their assay products directly. Qualitative as well as quantitative information can be obtained from this procedure. Oftentimes, it is part of an assay procedure either as a quality control Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory 1 ed. Copyright © 2010 by Academic Press. Inc. All rights of reproduction in any form reserved.
(QC) measure or as a detection method (Godde et al., 2006). Dependent on application needs, different gels or gel formats, such as agarose versus polyacrylamide, or slab gel versus capillary gel, can be used.
A. Agarose Gel Electrophoresis Agarose gel electrophoresis used to be the essential gel electrophoresis method for any molecular diagnostic laboratory due to its simple procedure, wide applicability, and inexpensive apparatus. A standard agarose gel apparatus is also referred to as a submarine apparatus, where a tray containing solidified agarose gel is submerged in buffer solution during electrophoresis. A gel is prepared by dissolving a set amount of agarose powder in gel buffer (see Table 5.1) by heating the mixture in a microwave oven. The agarose gel solution is cooled to 60°C or so and then poured into a sample preparation tray with sample comb(s) inserted (Fig. 5.1). Particular care needs to be taken to pierce the bubbles in the gel molten with clean pipet tips or they will cause distorted nucleic acid migration during electrophoresis (There are more bubbles in higher percentage gels, such as 3%). After a gel is fully solidified, sample comb(s) can then be carefully removed to leave well-formed sample wells. Nucleic acid specimens need to be mixed with a loading buffer that contains glycerol (Ficoll or sucrose can also be used to increase sample density) and tracking dye(s) such as bromophenol blue or xylene cyanol FF before loading. In addition to serving as an indicator that a nucleic acid sample has been properly loaded into a sample well, the dyes are also crude tracking devices that monitor the progress of electrophoresis. To allow nucleic acid specimens to be visualized by UV transillumination, ethidium bromide (EtBr) is added either to the agarose gel solution right before pouring or to the tank buffer, or both. Gloves need to worn at all times when handling gels or buffers containing EtBr as it is a potent DNA intercalator and a known mutagen. 49