Combining Techniques to Answer Molecular Questions

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1 Combining Techniques to Answer Molecular Questions UNIT FM02 How to cite this article: Curr. Protoc. Essential Lab. Tech. 9:FM02.1-FM02.5. doi: / etfm02s9 INTRODUCTION This manual is a collection of basic techniques central to the study of nucleic acids, proteins, and whole-cell/subcellular structures. The following is an overview of how the basic techniques described in this manual can be used alone or in sequence to answer questions about the properties of proteins and nucleic acids. Flowcharts are provided to orient the novice researcher in the use of fundamental molecular techniques, and provide perspective regarding applications of the technical units in this manual. NUCLEIC ACIDS Listed below are common questions about nucleic acids and techniques used to answer them. Also refer to Figure 1. Genomic and Plasmid DNA Analyses Does a particular genomic locus or region of plasmid DNA contain a sequence of interest? Polymerase chain reaction (UNIT 10.2). What is the position of the sequence of interest relative to restriction enzyme cut sites? Restriction enzyme digestion (UNIT 10.1) Agarose gel electrophoresis (UNIT 7.2) DNA probe labeling (UNIT 8.4) Southern blot (UNITS 8.1 & 8.2). What is the sequence of a specific DNA fragment? PCR amplification, if DNA fragment is low copy (UNIT 10.2) DNA sequencing (UNIT 10.4). Gene Expression (Transcription) Analyses What is the size of a specific gene transcript? Northern blot (UNITS 8.1 & 8.2). Is a gene of interest expressed (transcribed)? Northern blot (UNITS 8.1 & 8.2) or reverse transcriptase-pcr (UNIT 10.2). Current Protocols Essential Laboratory Techniques FM02.1-FM02.5, November 2014 Published online November 2014 in Wiley Online Library (wileyonlinelibrary.com). doi: / etfm02s9 Copyright C 2014 John Wiley & Sons, Inc. FM02.1

2 Figure 1 Flowchart for answering questions related to nucleic acids. Does the transcription rate of a specific gene change (increase or decrease) with different cell treatments, mutations, or between different cell lines? Northern blot (UNITS 8.1 & 8.2) Real-time PCR (for more quantitative comparison; UNIT 10.3). What is the relative abundance of mrnas made from a specific gene compared to that from other genes? Real-time PCR (UNIT 10.3). What are characteristics (abundance and size) of rrnas or trnas? Combining Techniques to Answer Molecular Questions Northern blot (UNITS 8.1 & 8.2). FM02.2 Current Protocols Essential Laboratory Techniques

3 Figure 2 Flowchart for answering questions related to proteins. For all of the above techniques, nucleic acids (RNA or DNA, genomic or plasmid) must first be isolated and concentrated from cells (UNIT 5.2). The concentration of the nucleic acid preparation must then be determined (UNIT 2.2). The preparation can then be analyzed by gel electrophoresis (UNITS 7.1 & 7.2) with or without prior restriction enzyme digestion (UNIT 10.1), depending on the experiment. DNA preparations can also be used in other enzymatic reactions, including PCR (UNIT 10.2) to amplify specific regions for cloning, sequencing (UNIT 10.4), or labeling for various experimental applications including the generation of probes for Southern or northern blotting (UNIT 8.4). Plasmid preparations (UNIT 4.2) can be used directly to obtain sequence information. PROTEINS Listed below are common questions about proteins and techniques used to answer them. Also refer to Figure 2. In which cellular structures or organelles do specific proteins reside? Cell fractionation (UNIT 5.1) Immunoblotting of proteins from cell organelle fractions (UNITS 8.1 & 8.3) or immunofluorescence (UNIT 9.2). What is the molecular mass of a specific protein? Is it post-translationally modified? SDS-PAGE (if protein is purified) or immunoblotting (UNITS 8.1 & 8.3) if protein is in a complex mixture. How pure is a particular protein preparation? SDS-PAGE (UNIT 7.3) Staining proteins in gels (UNIT 7.4). FM02.3 Current Protocols Essential Laboratory Techniques

4 Figure 3 Techniques used to answer questions about cellular and subcellular structure. How does one isolate and analyze a particular protein? Chromatography (UNITS 6.1 & 6.2) Dialysis (UNIT 6.4) Analysis by SDS-PAGE (UNIT 7.3) Staining proteins in gels (UNIT 7.4) or immunoblotting (UNIT 8.3). For many experiments, the concentration of protein in the sample must first be quantified (UNIT 2.2). For example, this is often done prior to performing SDS-PAGE and/or an immunoblot to ensure equal loading of different protein samples for comparison. To determine the localization of specific proteins, cells can first be lysed and fractionated by centrifugation (UNIT 5.1), followed by immunoblotting of the proteins (UNIT 8.3) from fractions containing specific cell substructures. A chromatography step would further resolve proteins from the various fractions (UNITS 6.1 & 6.2). Alternatively, localization of specific proteins to distinct cellular structures can be done using the immunofluorescence technique (UNIT 9.2). WHOLE CELLS AND SUBCELLULAR STRUCTURES This manual also includes techniques for studying whole cells and their substructures. These include cell fractionation by centrifugation (UNIT 5.1), cell imaging by conventional light microscopy (UNIT 9.1), and imaging by fluorescence microscopy (UNIT 9.2). Refer to Figure 3. All of these protocols require first growing cells in culture. Protocols for culturing bacteria (UNIT 4.2), culturing mammalian cells (UNIT 4.3), and culturing yeast (UNIT 4.4) are provided. These techniques can be used to answer questions such as: Does cell morphology change under different treatment conditions? Does cell behavior change under different treatment conditions? Do genetically altered cell lines display morphological phenotypes? In which cellular substructures does a specific endogenous or altered protein reside? Combining Techniques to Answer Molecular Questions FM02.4 Conventional light microscopy can be used to image most cell organelles and structures by using the appropriate microscopy technique. Common variations and their applications are described in UNIT 9.1. Fluorescence microscopy is used to image specific organelles with fluorescent dyes, or to study the localization of specific proteins (UNIT 9.2). Current Protocols Essential Laboratory Techniques

5 GENERAL SUPPORT MATERIALS For any experiment performed, it is essential to keep thorough records in the form of a laboratory notebook. APPENDIX 2 outlines the best practices for organizing and recording experimental details to optimize their usefulness and completeness. Results from many techniques in this manual require digital imaging for documentation in a laboratory notebook and for publication. APPENDIX 3A and APPENDIX 3B present important ethical and practical considerations for capturing, manipulating, and storing digital images, as well as guidelines for preparing them for publication. Some experimental results will require statistical analyses. APPENDIX 4 provides guidelines for selecting and using appropriate statistical tests in the life sciences. FM02.5 Current Protocols Essential Laboratory Techniques