Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-47 (Electronic) 4 Nucleic Acid Hybridization Sibylle Herzer and david F Englert Planning a Hybridization Experiment The Importance of Patience 40l What Are your most essential needs? Is a More Sensitive Detection System Always Better?..401 Visualize Your Particular Hybridization Event 403 What Can You conclude from commercial Sensitivity Data? Labeling issues 403 Which Labeling Strategy ls Most Appropriate for Your situation? 403 What Criteria Could You Consider When Selecting Label? 405 Radioactive and Nonradioactive Labeling Strategies Compared What Are the Criteria for Considering direct over Indirect Nonradioactive Labeling Strategies?........... 410 What Is the st storage Stability of Labeled Probes? Should the Probe Previously Used within the Hybridization Solution of an Earlier Experiment Be Applied in a New Experiment? ...4|2 How Should a probe be denatured for reuse? Is It Essential to Determine the Incorporation Efficiency of Every Labeling Reaction? Is It Necessary to Purify Every Probe ..413
399 14 Nucleic Acid Hybridization Sibylle Herzer and David F. Englert Planning a Hybridization Experiment . . . . . . . . . . . . . . . . . . . . . 401 The Importance of Patience . . . . . . . . . . . . . . . . . . . . . . . . . . 401 What Are Your Most Essential Needs? . . . . . . . . . . . . . . . . . 401 Visualize Your Particular Hybridization Event . . . . . . . . . . . . 401 Is a More Sensitive Detection System Always Better? . . . . . 403 What Can You Conclude from Commercial Sensitivity Data? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 Labeling Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 Which Labeling Strategy Is Most Appropriate for Your Situation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 What Criteria Could You Consider When Selecting a Label? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405 Radioactive and Nonradioactive Labeling Strategies Compared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409 What Are the Criteria for Considering Direct over Indirect Nonradioactive Labeling Strategies? . . . . . . . . . . . 410 What Is the Storage Stability of Labeled Probes? . . . . . . . . . 411 Should the Probe Previously Used within the Hybridization Solution of an Earlier Experiment Be Applied in a New Experiment? . . . . . . . . . . . . . . . . . . . . . . 412 How Should a Probe Be Denatured for Reuse? . . . . . . . . . . 412 Is It Essential to Determine the Incorporation Efficiency of Every Labeling Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . 412 Is It Necessary to Purify Every Probe? . . . . . . . . . . . . . . . . . 413 Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)
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? 4l7 What Are the indicators of a functional membrane? 4l7 Can Nylon and Nitrocellulose Membranes Be Sterilized? Nucleic acid transfer 4|8 What Issues affect the transfer of nucleic acid from Agarose Gels 418 Should Membranes Be Wet or Dry Prior to Use What Can You Do to Optimize the Performance of Colony and plaque Transfers? 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 The Hybridization Reaction 424 How Do You Determine an Optimal Hybridization Te 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 opPonents 43l 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? How Do You Select the Proper Hybridization 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 440 400 Herzer and Englert
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 Detection by Storage Phosphor Imagers 44 How Do Phosphor Imagers Work? Is a Storage Phosphor Imager Appropriate for Your Research situation What Affects Quantitation? ..443 What Should You Consider When Using Screens? How Can Problems Be prevented? ....447 Troubleshooting What Can Cause the Failure of a Hybridization Experiment? h ..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 con- entration obe concentration) usually 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. Manufac- vide strategies to optimize the performance of their producilsfo. turers of hybridization equipment and reagents can often pr What Are your most essential needs? Consider your needs before you delve into the many hybridiza tion options. What criteria are most crucial for your research- speed, cost, sensitivity, reproducibility or robustness, and qualita tive 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 label ing and hybridization strategies based on your experiments Nucleic Acid Hybridization
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
most appropriate probe for your research situation(Anderson 1999: Nath and Johnson, 1998; Temsamani and Agrawal, 1996: Trayhurn, 1996: Mansfield et aL., 1995; Tijssen, 2000). The questions raised in the ensuing discussions demonstrate why only the actual experiment, validated by positive and negative controls, deter mines the best choice The purpose of the following example is to discuss some of the complexities involved in selecting a labeling strategy. Suppose that you have the option of screening a target with a probe generated from the following templates: a 30 base oligo(30mer), a double stranded 800bp DNA fragment, or a double-stranded 2kb fragment 30-mer The 30-mer could be radioactively labeled at the 5 end via T4 polynucleotide kinase(PNK) or at the 3 end via Terminal deoxynucleotidyl transferase(TdT). pNK attaches a single mole cule of radioactive phosphate whereas TdT reactions are usually designed to add 10 or less nucleotides pnK does not produce the hottest probe, since only one radioactive label is attached, but the replacement of unlabeled phosphorous by P will not alter probe structure or specificity. TdT can produce a probe containing more radioactive label, but this gain in signal strength could be offset by altered specificity caused by the addition of multiple nucleotides. A 30mer containing multiple nonradioactive labels ould also be manufactured on a DNA synthesizer, but the pres ence of too many modified bases may alter the probe's hybridiza tion characteristics(Kolocheva et al., 1996) 800bp fragment The double-stranded 800 bp fragment could also be end labeled but labeling efficiency will vary depending on the presence of blunt, recessed, or overhanging termini. Since the complementary strands of the 800 bp fragment can reanneal after labeling,a reduced amount of probe might be available to bind to the target. Unlabeled template will also compete with labeled probes for target binding reducing signal output further. However, probe syn thesis from templates covalently attached to solid supports might overcome this drawback(Andreadis and Chrisey, 2000) Random hexamer- or nanomer- primed and nick translation labeling of the 800bp fragment will generate hotter probes than end labeling. However, they will be heterogeneous in size and specificity, since they originate from random location in the tem Herzer and Englert
most appropriate probe for your research situation (Anderson, 1999; Nath and Johnson, 1998; Temsamani and Agrawal, 1996; Trayhurn, 1996; Mansfield et al., 1995;Tijssen, 2000).The questions raised in the ensuing discussions demonstrate why only the actual experiment, validated by positive and negative controls, determines the best choice. The purpose of the following example is to discuss some of the complexities involved in selecting a labeling strategy. Suppose that you have the option of screening a target with a probe generated from the following templates: a 30 base oligo (30mer), a doublestranded 800bp DNA fragment, or a double-stranded 2kb fragment. 30-mer The 30-mer could be radioactively labeled at the 5¢ end via T4 polynucleotide kinase (PNK) or at the 3¢ end via Terminal deoxynucleotidyl transferase (TdT). PNK attaches a single molecule of radioactive phosphate whereas TdT reactions are usually designed to add 10 or less nucleotides. PNK does not produce the hottest probe, since only one radioactive label is attached, but the replacement of unlabeled phosphorous by 32P will not alter probe structure or specificity. TdT can produce a probe containing more radioactive label, but this gain in signal strength could be offset by altered specificity caused by the addition of multiple nucleotides. A 30mer containing multiple nonradioactive labels could also be manufactured on a DNA synthesizer, but the presence of too many modified bases may alter the probe’s hybridization characteristics (Kolocheva et al., 1996). 800bp fragment The double-stranded 800bp fragment could also be end labeled, but labeling efficiency will vary depending on the presence of blunt, recessed, or overhanging termini. Since the complementary strands of the 800bp fragment can reanneal after labeling, a reduced amount of probe might be available to bind to the target. Unlabeled template will also compete with labeled probes for target binding reducing signal output further. However, probe synthesis from templates covalently attached to solid supports might overcome this drawback (Andreadis and Chrisey, 2000). Random hexamer- or nanomer-primed and nick translation labeling of the 800bp fragment will generate hotter probes than end labeling. However, they will be heterogeneous in size and specificity, since they originate from random location in the tem- 404 Herzer and Englert
plate Probe size can range from about 20 nucleotides to the full- length template and longer(Moran et aL., 1996; Islas, Fairley, and Morgan, 1998). However, the bulk of the probe in most random prime labeling reactions is between 200 and 500nt If the entire 800 bp fragment is complementary to the intended target, a diverse probe population may not be detrimental. If only half the template contains sequence complementary to the target then sensitivity could be reduced. Any attempt to compensate by increasing probe concentration could result in higher back grounds. However, the major concern would be for the stringency of hybridization. Different wash conditions could be required to restore the stringency obtained with a probe sequence completely complementary to the target. 2kb DNA fragment The incorporation of radioactive label into probes generated by andom-primer labeling does not vary significantly between tem plates ranging from 300bp to 2kb, although the average size of probes generated from larger templates is greater(Ambion, Inc unpublished data). Generating a probe from a larger template could be advantageous if it contains target sequence absent from a smaller template The availability of different radioactive and nonradioactive labels could further complicate the situation, but the message re mains the same Visualize the hybridization event before you go to the lab. 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. duration, and resolution of the signal. One could aloc a Lab What Criteria Could You Consider When Selecting a Label One perspective for selecting a label is to compare the strength label's effect on incorporation into the probe, and the impact of the incorporated label on the hybridization of probe to target. The quantity of label incorporated into a probe can also affect the per formance of some labels and the probe's ability to bind its target Many experienced researchers will choose at least two techniques to empirically determine the best strategy to generate a new probe (if possible) Signal Strength and Resolution Signal strength of radioactive and nonradioactive labels is iversely proportional to signal resolution. Nucleic Acid Hybridization 405
plate. Probe size can range from about 20 nucleotides to the fulllength template and longer (Moran et al., 1996; Islas, Fairley, and Morgan, 1998). However, the bulk of the probe in most random prime labeling reactions is between 200 and 500nt. If the entire 800bp fragment is complementary to the intended target, a diverse probe population may not be detrimental. If only half the template contains sequence complementary to the target, then sensitivity could be reduced. Any attempt to compensate by increasing probe concentration could result in higher backgrounds. However, the major concern would be for the stringency of hybridization. Different wash conditions could be required to restore the stringency obtained with a probe sequence completely complementary to the target. 2kb DNA Fragment The incorporation of radioactive label into probes generated by random-primer labeling does not vary significantly between templates ranging from 300bp to 2kb, although the average size of probes generated from larger templates is greater (Ambion, Inc., unpublished data). Generating a probe from a larger template could be advantageous if it contains target sequence absent from a smaller template. The availability of different radioactive and nonradioactive labels could further complicate the situation, but the message remains the same. Visualize the hybridization event before you go to the lab. 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. What Criteria Could You Consider When Selecting a Label? One perspective for selecting a label is to compare the strength, duration, and resolution of the signal. One could also consider the label’s effect on incorporation into the probe, and the impact of the incorporated label on the hybridization of probe to target.The quantity of label incorporated into a probe can also affect the performance of some labels and the probe’s ability to bind its target. Many experienced researchers will choose at least two techniques to empirically determine the best strategy to generate a new probe (if possible). Signal Strength and Resolution Signal strength of radioactive and nonradioactive labels is inversely proportional to signal resolution. Nucleic Acid Hybridization 405
Radioactive Isotope signal strength diminishes in the order: P>>P>S> 3H. When sensitivity is the primary concern, as when searching for a low-copy gene, P is the preferred isotope Tritium is too weak for most blotting applications, but a nucleic acid probe labeled with multiple tritiated nucleotides can produce a useful, highly resolved signal without fear of radiolytic degradation of the probe H and"S are used for applications such as in situ hybridiza tion(ish) where resolution is more essential than sensitivity. The resolution of P is similar to 3S, but Ausubel et al. ( 1993)cites an improved signal-to-noise ratio when"P is applied in ISH Signal strengths of nonradioactive labels are difficult to compare. It is more practical to assess sensitivity instead of signal strength. The resolution of nonradioactive signals is also more omplicated to quantify because resolution is a function of signal strength at the time of detection and most nonradioactive signals weaken significantly over time. Therefore the length of exposure to film must be considered within any resolution discussion Background fluorescence or luminescence from the hybridi zation membrane has to be considered as well. Near-infrared dyes are superior due to low natural near-infrared occurrence (Middendorf, 1992). Some dyes emit in the far red 2700nm (Cy7, Alexa Fluor 549, allophycocyanin) Older nonradioactive, colorimetric labeling methods suffered from resolution problems because the label diffused within the membrane. Newer substrates, especially some of the precipitating chemifluorescent substrates, alleviate this problem. Viscous components such as glycerol are often added to substrates to limit diffusion effects. Colorimetric substrates and some chemilumi nescent substrates will impair resolution if the reaction proceeds beyond the recommended time or when the signal is too strong Hence background can increase dramatically due to substrate diffusion Mohandas ghandi said that there is more to life than increas- its speed(John-Roger and McWilliams, 1994), and the sam olds true for detection systems. Most nonradioactive systems deliver a signal within minutes or hours, but this speed is useless if the system cant detect a low-copy target Searching for a single copy gene with a P labeled probe might require an exposure of several weeks, but at least the target is ultimately identified 406 Herzer and Englert
Radioactive Isotope signal strength diminishes in the order: 32P > 33P > 35S > 3 H. When sensitivity is the primary concern, as when searching for a low-copy gene, 32P is the preferred isotope. Tritium is too weak for most blotting applications, but a nucleic acid probe labeled with multiple tritiated nucleotides can produce a useful, highly resolved signal without fear of radiolytic degradation of the probe. 3 H and 35S are used for applications such as in situ hybridization (ISH) where resolution is more essential than sensitivity. The resolution of 33P is similar to 35S, but Ausubel et al. (1993) cites an improved signal-to-noise ratio when 33P is applied in ISH. Nonradioactive Signal strengths of nonradioactive labels are difficult to compare. It is more practical to assess sensitivity instead of signal strength. The resolution of nonradioactive signals is also more complicated to quantify because resolution is a function of signal strength at the time of detection, and most nonradioactive signals weaken significantly over time. Therefore the length of exposure to film must be considered within any resolution discussion. Background fluorescence or luminescence from the hybridization membrane has to be considered as well. Near-infrared dyes are superior due to low natural near-infrared occurrence (Middendorf, 1992). Some dyes emit in the far red ≥700nm (Cy7, Alexa Fluor 549, allophycocyanin). Older nonradioactive, colorimetric labeling methods suffered from resolution problems because the label diffused within the membrane. Newer substrates, especially some of the precipitating chemifluorescent substrates, alleviate this problem. Viscous components such as glycerol are often added to substrates to limit diffusion effects. Colorimetric substrates and some chemiluminescent substrates will impair resolution if the reaction proceeds beyond the recommended time or when the signal is too strong. Hence background can increase dramatically due to substrate diffusion. Detection Speed Mohandas Ghandi said that there is more to life than increasing its speed (John-Roger and McWilliams, 1994), and the same holds true for detection systems. Most nonradioactive systems deliver a signal within minutes or hours, but this speed is useless if the system can’t detect a low-copy target. Searching for a singlecopy gene with a 32P labeled probe might require an exposure of several weeks, but at least the target is ultimately identified. 406 Herzer and Englert
Will you need to detect a signal from your blot several times over a period of hours or days? Are you pursuing a low-copy target that requires an exposure time of days or weeks? Would ou prefer a short-lived signal t id stripping a blot pri re-probing? Some nonradioactive detection systems allow for the quick inactivation of the enzyme that generates the signal, eliminating the stripping step prior to re-probing(Peterhaensel, Obermaier, and Rueger, 1998). The effects and implications of stripping are discussed in greater detail later in the chapter The practical lifetime of common radiolabels is several days to weeks, and is dependent on the label, the ligand, and its environ ment, as discussed in Chapter 6, Working Safely with Radioactive Materials. Some nonradioactive systems based on alkaline phos phatase can generate signals lasting 10 days without marked reduction(personal observation). Some chemifluorescent systems generate a fluorescent precipitate capable of producing a cumu lative signal, much like isotopes. The functional lifetime of fuorescent labels will vith the chemical nature of the fluorescent tag and the methodology of the application. For example, signal duration of a fluorescent tag could be defined by the number of times the chromophore can be excited to produce a fluorescent emission. Some tags can only be excited/scanned once or a few times, while others are much more stable. Consult the manufacturer of the labeled product for this type of stability information. In systems where an enyzme cat alyzes the production of a reagent required for fluorescence, the enzyme's half-life and sufficient presence of fresh substrate can limit the duration of the sign Will the Label Be efficiently Incorporated into the Probe? The effects of label size, location, and linkage method on the corporation of nucleotides into DNA or RNA are enzyme dependent and can be difficult to predict. Small side chains can inhibit nucleic acid synthesis(Racine, Zhu, and Mamet-Bratley, 993), while larger groups such as biotin might have little or no effect(Duplaa et al., 1993; Richard et al., 1994).In general, nucleotides labeled with isotopes of atoms normally present in nucleotides(P, P,H, C)will be incorporated by DNA and RNa polymerases more efficiently than nucleotides labeled with isotopes of nonnative atoms. Commercial polyermases are frequently engineered to overcome such incorporation bias Nucleic Acid Hybridization 407
Signal Duration Will you need to detect a signal from your blot several times over a period of hours or days? Are you pursuing a low-copy target that requires an exposure time of days or weeks? Would you prefer a short-lived signal to avoid stripping a blot prior to re-probing? Some nonradioactive detection systems allow for the quick inactivation of the enzyme that generates the signal, eliminating the stripping step prior to re-probing (Peterhaensel, Obermaier, and Rueger, 1998). The effects and implications of stripping are discussed in greater detail later in the chapter. The practical lifetime of common radiolabels is several days to weeks, and is dependent on the label, the ligand, and its environment, as discussed in Chapter 6,“Working Safely with Radioactive Materials.” Some nonradioactive systems based on alkaline phosphatase can generate signals lasting 10 days without marked reduction (personal observation). Some chemifluorescent systems generate a fluorescent precipitate capable of producing a cumulative signal, much like isotopes. The functional lifetime of fluorescent labels will vary with the chemical nature of the fluorescent tag and the methodology of the application. For example, signal duration of a fluorescent tag could be defined by the number of times the chromophore can be excited to produce a fluorescent emission. Some tags can only be excited/scanned once or a few times, while others are much more stable. Consult the manufacturer of the labeled product for this type of stability information. In systems where an enyzme catalyzes the production of a reagent required for fluorescence, the enzyme’s half-life and sufficient presence of fresh substrate can limit the duration of the signal. Will the Label Be Efficiently Incorporated into the Probe? The effects of label size, location, and linkage method on the incorporation of nucleotides into DNA or RNA are enzymedependent and can be difficult to predict. Small side chains can inhibit nucleic acid synthesis (Racine, Zhu, and Mamet-Bratley, 1993), while larger groups such as biotin might have little or no effect (Duplaa et al., 1993; Richard et al., 1994). In general, nucleotides labeled with isotopes of atoms normally present in nucleotides (32P, 33P, 3 H, 14C) will be incorporated by DNA and RNA polymerases more efficiently than nucleotides labeled with isotopes of nonnative atoms. Commercial polyermases are frequently engineered to overcome such incorporation bias. Nucleic Acid Hybridization 407
Some applications will exploit impaired incorporation of labeled nucleotides(Alexandrova et aL., 1991). Fluorescein attached to position 5 of cytosine in dCTP inhibits terminal transferase and causes addition of only one to two labeled dNTPs at the 3 end of DNA. Fluorescein-or biotin-riboUTP have been similarly applied Igloi and Schiefermayr, 1993) If no specific data exist regarding incorporation efficiency of your labeled nucleotide-labeling enzyme combination, contact the manufacturer of both products. They will likely be able to provide you with a starting point from which you can optimize your labeling reactions. Will the Label Interfere with the Probe's ability to bind to the Target? Hybridization efficiency can be altered by a labels chemical structure,its location within the probe, the linker that connects he label to the ligand, and the quantity of label within the probe. Isotopes of elements present in nucleic acids in vivo might not directly alter the probe's structure, but as described below(and Chapter 6), a label's radioactive emissions can fragment a probe The importance of label location is demonstrated by comparing the hybridization efficiencies of probes labeled with Cy5Morigi nating at either C5 or the primary amine attached to C4 of dCTP. Probes labeled throughout their sequence with the C5-linked label hybridize efficiently, and are commonly applied in micro- array applications(Lee et aL., 2000). Probes containing the label attached to the amine group at C4 do not hybridize efficiently to their targets. The purpose of the C4 amine-label is the addition of a single molecule of Cys dctP to the 3 end of a sequencing primer(Ansorge et al., 1992). A molecular model to accurately predict the effects of labels on analog conformation, hydrogen bonding, stacking interactions, and hybrid helical geometry has been proposed(Yuriev, Scott, and Hanna, 1999) The C5 position is also preferred for dUTP(Petrie, Oshevskil, Kumarev, and grachev, 1989). C5 is such an attractive labeling site because it does not contribute to base-pairing by hydrogen bonding, and at least some linkers seem to allow posi tioning of the label attached in position 5 so that helix formation is not impaired. But bulky tags linked to pyrimidines in the C5 position still interfere to some degree with hybridization because of steric hindrance. Other sites on purines and pyrimidines have been used as tag or label attachment points. However, they have only been shown to work as primers, not for internal labeling strategies(Srivastava, Raza, and Misra, 1994) Herzer and Englert
Some applications will exploit impaired incorporation of labeled nucleotides (Alexandrova et al., 1991). Fluorescein attached to position 5 of cytosine in dCTP inhibits terminal transferase and causes addition of only one to two labeled dNTPs at the 3¢ end of DNA. Fluorescein- or biotin-riboUTP have been similarly applied (Igloi and Schiefermayr, 1993). If no specific data exist regarding incorporation efficiency of your labeled nucleotide–labeling enzyme combination, contact the manufacturer of both products. They will likely be able to provide you with a starting point from which you can optimize your labeling reactions. Will the Label Interfere with the Probe’s Ability to Bind to the Target? Hybridization efficiency can be altered by a label’s chemical structure, its location within the probe, the linker that connects the label to the ligand, and the quantity of label within the probe. Isotopes of elements present in nucleic acids in vivo might not directly alter the probe’s structure, but as described below (and in Chapter 6), a label’s radioactive emissions can fragment a probe. The importance of label location is demonstrated by comparing the hybridization efficiencies of probes labeled with Cy5TM originating at either C5 or the primary amine attached to C4 of dCTP. Probes labeled throughout their sequence with the C5-linked label hybridize efficiently, and are commonly applied in microarray applications (Lee et al., 2000). Probes containing the label attached to the amine group at C4 do not hybridize efficiently to their targets. The purpose of the C4 amine-label is the addition of a single molecule of Cy5 dCTP to the 3¢ end of a sequencing primer (Ansorge et al., 1992). A molecular model to accurately predict the effects of labels on analog conformation, hydrogen bonding, stacking interactions, and hybrid helical geometry has been proposed (Yuriev, Scott, and Hanna, 1999). The C5 position is also preferred for dUTP (Petrie, 1991; Oshevskii, Kumarev, and Grachev, 1989). C5 is such an attractive labeling site because it does not contribute to base-pairing by hydrogen bonding, and at least some linkers seem to allow positioning of the label attached in position 5 so that helix formation is not impaired. But bulky tags linked to pyrimidines in the C5 position still interfere to some degree with hybridization because of steric hindrance. Other sites on purines and pyrimidines have been used as tag or label attachment points. However, they have only been shown to work as primers, not for internal labeling strategies (Srivastava, Raza, and Misra, 1994). 408 Herzer and Englert
