武汉大学生命科学学院：《分子生物学》（英文版）Nucleic Acid Hybridization.pdf

Hybridization Membranes and Supports . . . . . . . . . . . . . . . . . . 413 What Are the Criteria for Selecting a Support for Your Hybridization Experiment? . . . . . . . . . . . . . . . . . . . . . . . . . . 413 Which Membrane Is Most Appropriate for Quantitative Experiments? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 What Are the Indicators of a Functional Membrane? . . . . . 417 Can Nylon and Nitrocellulose Membranes Be Sterilized? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 Nucleic Acid Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418 What Issues Affect the Transfer of Nucleic Acid from Agarose Gels? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418 Should Membranes Be Wet or Dry Prior to Use? . . . . . . . . 420 What Can You Do to Optimize the Performance of Colony and Plaque Transfers? . . . . . . . . . . . . . . . . . . . . . . . 421 Crosslinking Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422 What Are the Strengths and Limitations of Common Crosslinking Strategies? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422 What Are the Main Problems of Crosslinking? . . . . . . . . . . . 423 What’s the Shelf Life of a Membrane Whose Target DNA Has Been Crosslinked? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 The Hybridization Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 How Do You Determine an Optimal Hybridization Temperature? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424 What Range of Probe Concentration Is Acceptable? . . . . . . 425 What Are Appropriate Pre-hybridization Times? . . . . . . . . . 426 How Do You Determine Suitable Hybridization Times? . . . 426 What Are the Functions of the Components of a Typical Hybridization Buffer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 What to Do before You Develop a New Hybridization Buffer Formulation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430 What Is the Shelf Life of Hybridization Buffers and Components? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431 What Is the Best Strategy for Hybridization of Multiple Membranes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 Is Stripping Always Required Prior to Reprobing? . . . . . . . . 432 What Are the Main Points to Consider When Reprobing Blots? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433 How Do You Optimize Wash Steps? . . . . . . . . . . . . . . . . . . . 434 How Do You Select the Proper Hybridization Equipment? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435 Detection by Autoradiography Film . . . . . . . . . . . . . . . . . . . . . . 436 How Does an Autoradiography Film Function? . . . . . . . . . . . 436 What Are the Criteria for Selecting Autoradiogaphy Film? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438 Why Expose Film to a Blot at -70°C? . . . . . . . . . . . . . . . . . 440 400 Herzer and Englert

Helpful Hints When Working With Autoradiography Film . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 Detection by Storage Phosphor Imagers . . . . . . . . . . . . . . . . . . 441 How Do Phosphor Imagers Work? . . . . . . . . . . . . . . . . . . . . . 441 Is a Storage Phosphor Imager Appropriate for Your Research Situation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 What Affects Quantitation? . . . . . . . . . . . . . . . . . . . . . . . . . . . 443 What Should You Consider When Using Screens? . . . . . . . . 445 How Can Problems Be Prevented? . . . . . . . . . . . . . . . . . . . . . 447 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 What Can Cause the Failure of a Hybridization Experiment? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453 PLANNING A HYBRIDIZATION EXPERIMENT Hybridization experiments usually require a considerable investment in time and labor, with several days passing before you obtain results. An analysis of your needs and an appreciation for the nuances of your hybridization event will help you select the most efficient strategies and appropriate controls. The Importance of Patience Hybridization data are the culmination of many events, each with several effectors. Modification of any one effector (salt concentration, temperature, probe concentration) usually impacts several others. Because of this complex interplay of cause and effect, consider an approach where every step in a hybridization procedure is an experiment in need of optimization. Manufacturers of hybridization equipment and reagents can often provide strategies to optimize the performance of their products. What Are Your Most Essential Needs? Consider your needs before you delve into the many hybridization options. What criteria are most crucial for your research— speed, cost, sensitivity, reproducibility or robustness, and qualitative or quantitative data? Visualize Your Particular Hybridization Event Consider the possible structures of your labeled probes and compare them to your target(s). Be prepared to change your labeling and hybridization strategies based on your experiments. Nucleic Acid Hybridization 401

What Do You know About Your Target? The sensitivity needs of your system are primarily determined by the abundance of your target, which can be approximated according to its origin. Plasmids, cosmids, phagemids as colony lifts or dot blots, and PCr products are usually intermediate to high-abundance targets. Genomic DNa is considered an intermediate to low-abundance target. Most prokaryotic genes are present as single copies, while genes from higher eukaryotes can be highly repetitive, of intermediate abundance, or single copy(Anderson, 1999). However, sensitivity requirements for single-copy genes should be considered sample dependent be cause some genes thought to be single copy can be for und as multiples. Lewin(1993) provides an example of recently poly ploid plants whose genomes are completely repetitive. The RNA situation is more straightforward; 80% of RNa transcripts are present at low abundance, raising the sensitivity requirements for most Northerns or nuclease protection assays(Anderson 1999) If you're uncertain about target abundance, test a series of different target concentrations(van Gijlswijk, Raap, and Tanke 1992; De Luca et aL., 1995). Manufacturers of detection systems often present performance data in the form of target dilution series. Known amounts of target are hybridized with a probe to show the lowest detection limit of a kit or a methe limic this experimental approach to determine your sensitivity requirements and the usefulness of a system. This strategy requires knowing the exact amount of target spotted onto the What Do You Know about Your Probe or Probe template? The more sequence and structural information you know about your probe and target, the more likely your hybridization will deliver the desired result(Bloom et aL., 1993). For example, the size and composition of the material from which you will gener ate your probe affects your choice of labeling strategy and hybridization conditions, as discussed in the question, Which Labeling Strategy Is Most Appropriate for Your Situation? GC content, secondary structure, and degree of homology to the target should be taken into account, but the details are beyond the scope of this chapter. (See Anderson, 1999; Shabarova 1994; Darby, 1999; Niemeyer, Ceyhan, and Blohm, 1999; and http://www2.cbm.uam.es/ilcastrillo/lab/protocols/hybridn.htmfor 402 Herzer and Englert

What Do You Know About Your Target? The sensitivity needs of your system are primarily determined by the abundance of your target, which can be approximated according to its origin. Plasmids, cosmids, phagemids as colony lifts or dot blots, and PCR products are usually intermediate to high-abundance targets. Genomic DNA is considered an intermediate to low-abundance target. Most prokaryotic genes are present as single copies, while genes from higher eukaryotes can be highly repetitive, of intermediate abundance, or single copy (Anderson, 1999). However, sensitivity requirements for single-copy genes should be considered sample dependent because some genes thought to be single copy can be found as multiples. Lewin (1993) provides an example of recently polyploid plants whose genomes are completely repetitive. The RNA situation is more straightforward; 80% of RNA transcripts are present at low abundance, raising the sensitivity requirements for most Northerns or nuclease protection assays (Anderson, 1999). If you’re uncertain about target abundance, test a series of different target concentrations (van Gijlswijk, Raap, and Tanke, 1992; De Luca et al., 1995). Manufacturers of detection systems often present performance data in the form of target dilution series. Known amounts of target are hybridized with a probe to show the lowest detection limit of a kit or a method. Mimic this experimental approach to determine your sensitivity requirements and the usefulness of a system. This strategy requires knowing the exact amount of target spotted onto the membrane. What Do You Know about Your Probe or Probe Template? The more sequence and structural information you know about your probe and target, the more likely your hybridization will deliver the desired result (Bloom et al., 1993). For example, the size and composition of the material from which you will generate your probe affects your choice of labeling strategy and hybridization conditions, as discussed in the question, Which Labeling Strategy Is Most Appropriate for Your Situation? GC content, secondary structure, and degree of homology to the target should be taken into account, but the details are beyond the scope of this chapter. (See Anderson, 1999; Shabarova, 1994; Darby, 1999; Niemeyer, Ceyhan, and Blohm, 1999; and http://www2.cbm.uam.es/jlcastrillo/lab/protocols/hybridn.htm for in-depth discussions.) 402 Herzer and Englert

Is a More Sensitive Detection System Always Better? Greater sensitivity can solve a problem or create one. The more sensitive the system, the less forgiving it is in terms of background a probe that generates an extremely strong signal may require an extremely short exposure time on film, making it difficult to capture signal at all or in a controlled fashion. Femtogram sensitivity is required to detect a single-copy gene and represents the lower detection limit for the most sensitive probes. Methods at or below femtogram sensitivity can detect 1 to 5 molecules, but this increases the difficulty in discerning true pos itive signals when screening low-copy targets(Klann et al., 1993 Rihn et aL, 1995). Single-molecule detection is better left to tech niques such as nuclear magnetic resonance or mass spectrometry The pursuit of hotter probes for greater sensitivity can be an unnecessary expense. Up to 56% of all available sites in a 486 nucleotide (nt) transcript could be labeled with biotinylated dUTP, but 8% was sufficient to achieve similar binding levels of Streptavidin than higher-density labeled probes(Fenn and Herman, 1990). Altering one or more steps of the hybridization process might correct some the above-mentioned problems. The key is to evaluate the true need, the benefits and the costs of increased sensitivity What Can You Conclude from Commercial Sensitivity Data? Manufacturers can accurately describe the relative sensitivities of their individual labeling systems. Comparisons between label- ing systems from different manufacturers are less reliable becaus each manufacturer utilizes optimal conditions for their system. Should you expect to reproduce commercial sensitivity claims? Relatively speaking, the answer is yes, provided that you optimize your strategy. However, with so many steps to a hybridization ex periment (electrophoresis, blotting, labeling, and detection) quantitative comparisons between two different systems are imperfect Side-by-side testing of different detection systems uti lizing the respective positive controls or a simple probe/target system of defined quantities (e. g, a dilution series of a house- keeping gene)is a good approach to evaluation. LABELING ISSUES Which Labeling Strategy Is Most Appropriate for Your situation? Each labeling strategy provides features, benefits, and limita- tions, and numerous criteria could be considered for selecting the Nucleic Acid Hybridization 403

Is a More Sensitive Detection System Always Better? Greater sensitivity can solve a problem or create one. The more sensitive the system, the less forgiving it is in terms of background. A probe that generates an extremely strong signal may require an extremely short exposure time on film, making it difficult to capture signal at all or in a controlled fashion. Femtogram sensitivity is required to detect a single-copy gene and represents the lower detection limit for the most sensitive probes. Methods at or below femtogram sensitivity can detect 1 to 5 molecules, but this increases the difficulty in discerning true positive signals when screening low-copy targets (Klann et al., 1993; Rihn et al., 1995). Single-molecule detection is better left to techniques such as nuclear magnetic resonance or mass spectrometry. The pursuit of hotter probes for greater sensitivity can be an unnecessary expense. Up to 56% of all available sites in a 486 nucleotide (nt) transcript could be labeled with biotinylated dUTP, but 8% was sufficient to achieve similar binding levels of Streptavidin than higher-density labeled probes (Fenn and Herman, 1990). Altering one or more steps of the hybridization process might correct some the above-mentioned problems. The key is to evaluate the true need, the benefits and the costs of increased sensitivity. What Can You Conclude from Commercial Sensitivity Data? Manufacturers can accurately describe the relative sensitivities of their individual labeling systems. Comparisons between labeling systems from different manufacturers are less reliable because each manufacturer utilizes optimal conditions for their system. Should you expect to reproduce commercial sensitivity claims? Relatively speaking, the answer is yes, provided that you optimize your strategy. However, with so many steps to a hybridization experiment (electrophoresis, blotting, labeling, and detection), quantitative comparisons between two different systems are imperfect. Side-by-side testing of different detection systems utilizing the respective positive controls or a simple probe/target system of defined quantities (e.g., a dilution series of a housekeeping gene) is a good approach to evaluation. LABELING ISSUES Which Labeling Strategy Is Most Appropriate for Your Situation? Each labeling strategy provides features, benefits, and limitations, and numerous criteria could be considered for selecting the Nucleic Acid Hybridization 403