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)
`
`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
`
`399
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`BD v. Enzo
`Case IPR2017-00181
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`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
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`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 con-
`centration, 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. Manufac-
`turers of hybridization equipment and reagents can often pro-
`vide strategies to optimize the performance of their products.
`
`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.
`
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`
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`

`results are unsatisfactory, a point at which it might be too late to
`determine incorporation efficiency.
`Before skipping any control steps, consider the implications.
`Minimally, measure incorporation efficiency when working with a
`new technique, a new probe, a new protocol, or a new kit. Radio-
`labeled probes need to be purified or at least Trichloroacetic acid
`(TCA) precipitated to determine labeling efficiency, as discussed
`in Chapter 7, “DNA Purification.” Determining the efficiency of
`nonradioactive labeling reactions can be more time-consuming,
`often involving dot blots and/or scanning of probe spots. Follow
`manufacturer recommendations to determine labeling efficiency
`of nonradioactive probes.
`
`Is It Necessary to Purify Every Probe?
`Unincorporated nucleotides, enzyme, crosslinking reagents,
`buffer components, and the like, may cause high backgrounds
`or interfere with downstream experiments. Hybridization experi-
`ments where the volume of the probe labeling reaction is negli-
`gible in comparison to the hybridization buffer volume do not
`always require probe cleanup. If you prefer to minimize these
`risks, purify the probe away from the reaction components.
`While there are some labeling procedures (i.e., probes gener-
`ated by random primer labeling with 32P-dCTP), where unpurified
`probe can produce little or no background (Amersham Pharma-
`cia Biotech, unpublished observations), such ideal results can’t be
`guaranteed for every probe. When background is problematic,
`researchers have the option to repurify the probe preparation.
`Admittedly, this approach wouldn’t be of much use if the experi-
`ment producing the background problem required a five day
`exposure. (Purification options are discussed in Chapter 7, “DNA
`Purification.”)
`
`HYBRIDIZATION MEMBRANES AND SUPPORTS
`What Are the Criteria for Selecting a Support for Your
`Hybridization Experiment?
`Beyond the information listed below and your personal experi-
`ence, the most reliable approach to determine if a membrane can
`be used in your application is to ask the manufacturer for appli-
`cation and or quality control data. Whether a new membrane for-
`mulation will provide you with superior results is a matter that can
`usually be decided only at the bench, and the results can vary for
`different sets of targets, probes, and detection strategies.
`
`Nucleic Acid Hybridization
`
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`

`

`Physical Strength
`Nitrocellulose remains popular for low to medium sensitivity
`(i.e., screening libraries) applications and for situations that
`require minimal handling. The greater mechanical strength of
`nylon makes it superior for situations that require repeated
`manipulation of your blot. Nylon filters may be probed 10 times
`or more (Krueger and Williams, 1995; Li, Parker, and Kowalik,
`1987). Even though nitrocellulose may be used more than once,
`brittleness, loss of noncovalently bound target during stripping,
`and decreased stability in harsh stripping solutions make nitro-
`cellulose a lesser choice for reusable blots. Glass supports and
`chips can be stripped, but stripping efficiency and aging of target
`on these supports may impair reuse of more than two to three
`cycles of stripping and reprobing. Supported nitrocellulose is stur-
`dier and easier to handle than pure nitrocellulose, but remember
`that it needs to be used in the proper orientation.
`
`Binding Capacity
`Nylon and PVDF (polyvinylidene difluoride) membranes bind
`significantly more nucleic acid than nitrocelluose; hence they can
`generate stronger signals after hybridization. Nucleic acids can be
`covalently attached to nylon but not to nitrocellulose, as discussed
`below. Positively charged nylon offers the highest binding capac-
`ities. As is the case with detection systems of greater sensitivity,
`the greater binding capacity of positively charged membranes
`could increase the risk of background signal. However, optimiza-
`tion of hybridization conditions, such as probe concentration and
`hybridization buffer composition, will usually prevent background
`problems. If such optimization steps do not prevent background,
`a switch to another membrane type, such as to a neutral nylon
`membrane, might be required. If your signal is too low, try a pos-
`itively charged nylon membrane. Positively charged nylon is often
`chosen for nonradioactive applications to ensure maximum signal
`strength. The quantity of positive charges (and potential for back-
`ground) can vary by 10-fold between manufacturers. The lower
`binding capacity of nitrocellulose decreases the likelihood of
`background problems under conditions that generate a detectable
`signal.
`
`Thickness
`Most membranes are approximately 100 to 150mm thick. Thick-
`ness influences the amount of buffer required per square
`
`414
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`

`centimeter. Thicker membranes soak up more buffer, wet more
`slowly, and dry application of thicker filters to the surface of a gel
`can be more difficult.
`
`Pore Size
`Pore size limits the size of the smallest fragment that can be
`bound and fixed onto a membrane, but bear in mind that pore size
`is an average value. In general, 0.45mm micron pore sizes can bind
`oligonucleotides down to 50 bases in length, but the more common
`working limit is 100 to 150 nucleotides or base pairs. Membranes
`comprised of 0.22mm micron pores are recommended for work
`with the smallest single- and double-stranded DNA fragments.
`Custom manufacturing of membranes with 0.1mm pore size is also
`available. Table 14.1 compares membrane characteristics.
`
`Specialized Application
`Microarrays
`Glass slides stand apart from membrane supports because glass
`allows for covalent attachment of oriented nucleic acid, is non-
`porous and offers low autofluorescence. On a nonporous support,
`buffer volumes can be kept low, which decreases cost and allows
`increased probe concentration. Unlike nylon and nitrocellulose
`membranes, background isn’t problematic under these aggressive
`hybridization conditions. Probes are labeled with different dyes
`and allow detection of multiple targets in a single hybridization
`experiment; nylon arrays are often restricted to serial or parallel
`hybridization, although examples of simultaneous detection on
`nylon membranes can be found in the literature. (Some references
`for multiple probes on nylon are Kondo et al. (1998), Holtke et al.
`(1992), and Bertucci et al. (1999).) These features make glass slides
`ideal for nonradioactive detection in micro arrays.
`
`Macroarrays
`Background problems, high buffer volumes, and hence cost,
`limit the usefulness of nonradioactive labels for macroarrays
`on nylon filters. Macroarrays employ thin charged or uncharged
`nylon membranes to reduce buffer consumption but suffer from
`low sensitivity due to the high autofluorescence of nylon. Stronger
`signals derived from enzyme-substrate driven signal amplification
`compromise resolution and quantitation. Radioactive labels such
`as 33P are preferred for macroarrays (Moichi et al., 1999; Yano
`et al., 2000; Eickhoff et al., 2000).
`
`Nucleic Acid Hybridization
`
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`
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`
`

`

`optimzed properly
`backgrounds if not
`
`Can give high
`
`detection systems
`nonradioactive
`interfere with
`membranes can
`supercharged
`systems,but some
`most nonradioactive
`usually optimal for
`
`Highest sensitivity;
`
`As for uncharged
`
`nylon
`
`signal/retention
`Poor specificity of
`
`all nylon membranes
`Lowest background of
`
`As for uncharged
`
`nylon
`
`charged nylon
`problematic with
`background is
`inappropriate and
`Where nitrocelluose
`
`often lowers signal
`many stains,staining
`
`Irreversibly binds
`
`applications
`nucleic acid
`mixed protein/
`applications,some
`some nonradioactive
`
`difficult to optimize
`that might be more
`documented method
`not so well
`
`Fairly new and hence
`
`As for unsupported
`
`nitrocellulose
`
`As for unsupported
`
`nitrocellulose
`
`applications
`sensitivity
`screens,low
`plaque lifts,library
`Radioactive colony/
`Recommended Use
`
`for RNA
`binds proteins,not
`systems because it
`not good for nonrad
`low binding capacity
`handle,flammable,
`
`More difficult to
`Limitations
`
`brittleness
`drawbacks of
`nitrocellulose
`sensitivity without
`capacity and hence
`Intermediate binding
`
`block
`background,easy to
`
`Low cost,low
`
`block
`background,easy to
`
`Low cost,low
`
`Benefits
`
`hydrolyze the target
`conditions that don’t
`Up to 10 times if under
`
`water,formamide
`Yes,strip with SDS,
`
`Possibly 2–6 times via
`
`gentle conditions
`
`Strip or Reprobe
`
`and physical strength
`solvent resistance
`membrane of good
`hygroscopic
`
`Hydrophilic,
`
`and physical strength
`solvent resistance
`membrane of good
`hygroscopic
`
`Hydrophilic,
`
`charged
`positively
`
`Nylon
`
`charged
`negatively
`
`Nylon
`
`and physical strength
`solvent resistance
`membrane of good
`hygroscopic
`
`Nylon neutralHydrophilic,
`
`stability <135C
`with a thermal
`physical strength
`solvent resistance/
`membrane of fair
`
`Hydrophobic
`
`PVDF
`
`Nitrocellulose
`
`supported
`
`NitrocelluloseHydrophilic membrane Not recommended
`Membrane
`
`physical strength
`resistance and
`of low solvent
`
`Characteristics
`
`Physical
`
`Table 14.1Characteristics of Membranes Used in Nucleic Acid Blotting Applications
`
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`

`Which Membrane Is Most Appropriate for
`Quantitative Experiments?
`The size of the nucleic acid being transferred, the physical
`characteristics of the membrane, and the composition of transfer
`buffer affect the transfer efficiency. There is no magic formula
`guaranteeing linear transfer of all nucleic acids at all times.
`Linearity of transfer needs to be tested empirically with dilution
`series of nucleic acid molecular weight markers.
`
`What Are the Indicators of a Functional Membrane?
`Membranes will record every fingerprint, drop of powder,
`knick, and crease. Always handle membranes with plastic forceps
`and powder-free gloves.
`Membranes should be dry and uniform in appearance. They
`should be wrinkle- and scratch-free since mechanical damage
`may lead to background problems in these affected areas. Mem-
`branes should wet evenly and quickly. If membranes do appear
`blotchy or spotty, or seem to have different colors, it is best not to
`use them. Membranes are hygroscopic, light sensitive, and easily
`damaged, but as long as membranes are properly stored, may
`remain functional for years. Please note that manufacturers only
`guarantee potency for shorter time periods, usually six to twelve
`months. If the vitality of the membrane is in doubt, a quick dot
`blot or test of the binding capacity may help. Manufacturers can
`provide guidelines for assessing binding capacity. Including an
`untreated, target-free piece of membrane to evaluate background
`in a given hybridization buffer or wash system can help to
`troubleshoot background problems.
`
`Can Nylon and Nitrocellulose Membranes Be Sterilized?
`Researchers performing colony hyrbidizations often ask about
`membrane sterilization. While membranes might not be supplied
`guaranteed to be sterile, they are typically produced and packaged
`with extreme care, minimizing the likelihood of contamination.
`Theoretically it is possible to autoclave membranes, but cycles
`should be very short (two minutes at 121°C in liquid cycle). Note
`that such short durations cannot guarantee sterility. Membranes
`should be removed as soon as the autoclave comes down to a safe
`temperature, and dried at room temperature. Multiple membranes
`should be separated by single sheets of Whatman paper. Note that
`filters may turn brown, become brittle, may shrink and warp and
`become difficult to align with plates, but this does not interfere
`with probe hybridization.
`
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`

`One reference cited decreased shelf lives for storage at room
`temperature (Giusti and Budowle, 1992). Blots maintained
`dry (desiccant for long-term storage), dark, and protected from
`mechanical damage may be stored safely for 6 to 12 months.
`
`THE HYBRIDIZATION REACTION
`The hybridization step is central to any nucleic acid detection
`technique. Choices of buffer, temperature, and time are never
`trivial because these effectors in combination with membrane,
`probe, label, and target form a complex network of cause and
`effect. Determining the best conditions for your experiment will
`always require a series of optimization experiments; there is no
`magic formula. The role of the effectors of hybridization, recom-
`mended starting levels, and strategies to optimize them will be the
`focus of this section. Readers interested in greater detail on the
`intricacies and interplay of events within hybridization reactions
`are directed to Anderson (1999), Gilmartin (1996), Thomou and
`Katsanos (1976), Ivanov et al. (1978), and Pearson, Davidson, and
`Britten (1977).
`
`How Do You Determine an Optimal Hybridization
`Temperature?
`Hybridization temperature depends on melting temper-
`ature (Tm) of the probe, buffer composition, and the nature of the
`target:hybrid complex. Formulas to calculate the Tm of oligos,
`RNA, DNA, RNA-DNA, and PNA-DNA hybrids have been de-
`scribed (Breslauer et al., 1986; Schwarz, Robinson, and Butler,
`1999; Marathias et al., 2000). Software that calculates Tm is
`described by Dieffenbach and Dveksler (1995).
`The effects of labels on melting temperatures should be taken
`into consideration. While some claim little effect of tags as large
`as horseradish peroxidase on hybrid stability/Tm (Pollard-Knight
`et al., 1990a), others observed Tm changes with smaller base mod-
`ifications (Pearlman and Kollman, 1990). It will have to suffice that
`nonradioactive tags may alter the hybridization characteristics of
`probes and that empiric determination of Tm may be quicker than
`developing a formula to accurately predict hybridization behav-
`ior of tagged probes. Hybridization temperatures should also take
`into account the impact of hybridization temperature on label sta-
`bility. Alkaline phosphatase is more stable at elevated tempera-
`tures than horseradish peroxidase. Thermostable versions of
`enzymes or addition of thermal stabilizer such as trehalose
`
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`

`(Carninci et al., 1998) may provide alternatives to hybridization
`at low temperatures.
`When switching from a DNA to an RNA probe, hybridization
`temperatures can be increased due to the increased Tm of RNA-
`DNA heteroduplexes. Because of concerns about instability of
`RNA at elevated temperatures, an alternative approach with
`RNA probes is the use of a denaturing formamide or urea buffer
`that allows hybridization at lower temperature.
`A good starting point for inorganic (nondenaturing) buffers are
`hybridization temperatures of 50 to 65°C for DNA applications
`and 55 to 70°C for RNA applications. Formamide buffers offer
`hybridization at temperatures as low as 30°C, but temperatures
`between 37 and 45°C are more common. Enzyme-linked probes
`should be used at the lowest possible temperature to guarantee
`enzyme stability.
`After hybridization and detection has been performed at the
`initially selected hybridization temperature, adjustments may be
`required to improve upon the results. A hybridization tempera-
`ture that is too low will manifest itself as a high nonspecific back-
`ground. The degree by which the temperature of subsequent
`hybridizations should be adjusted will depend on other criteria
`discussed throughout this chapter (GC content of the probe and
`template, RNA vs. DNA probe, etc.), and thus hybridization tem-
`perature can’t be exactly predicted. Most hybridization protocols
`employ temperatures of 37°C, 42°C, 50°C, 55°C, 60°C, 65°C, and
`68°C.
`Note that sometimes a clean, strong, specific signal that is totally
`free of nonspecific background cannot be obtained. Background
`reduction, especially through the use of increased hybridization
`temperatures, will result in the decrease of specific hybridization
`signal as well. There is often a trade-off between specific signal
`strength and background levels. You may need to define in each
`experiment what amount of background is acceptable to obtain
`the necessary level of specific hybridization signal. If the results
`are not acceptable, the experiment might have to be redesigned.
`
`What Range of Probe Concentration Is Acceptable?
`Probe concentration is application dependent. It will vary with
`buffer composition, anticipated amount of target, probe length
`and sequence, and the labeling technique used.
`Background and signal correlate directly to probe concentra-
`tion. If less probe than target is present, then the accuracy of band
`quantities is questionable.
`
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`
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`In the absence of rate-accelerating “fast” hybridization buffers,
`probe concentration is typically 5 to 10ng/ml of buffer. Another
`convention is to apply 2 to 5 million counts/ml of hybridization
`buffer, which may add up to more than 10ng/ml if the probe
`was end-labeled, as compared to a random primer-generated
`probe. The use of rate accelerators or “fast” hybridization buffers
`requires a reduction in probe concentration to levels of 0.1 to
`5ng/ml of hybridization buffer.
`Another approach to select probe concentration is based on the
`amount of target. A greater than 20¥ excess of probe over target
`is required in filter hybridization (Anderson, 1999). Solution
`hybridization may not require excess amounts for qualitative
`experiments. To determine if probe is actually present in excess
`over target, perform replicate dot or slot blots containing a dilu-
`tion series of immobilized target and varying amounts of input
`probe (Anderson, 1999). If probe is present in excess, the signal
`should reflect the relative ratios of the different concentrations of
`target. If you do not observe a proportional relationship between
`target concentration and specific hybridization signal at any of
`the probe concentrations used, you may need to increase your
`probe concentration even higher. Probe concentration cannot be
`increased indefinitely; a high background signal will eventually
`appear.
`
`What Are Appropriate Pre-hybridization Times?
`Prehybridization time is also affected by the variables of
`hybridization time. For buffers without rate accelerators, prehy-
`bridization times of at least 1 to 4 hours are a good starting point.
`Some applications may afford to skip prehybridization altogether
`(Budowle and Baechtel, 1990). Buffers containing rate accelera-
`tors or volume excluders usually do not benefit from prehy-
`bridization times greater than 30 minutes.
`
`How Do You Determine Suitable Hybridization Times?
`Hybridization time depends on the kinetics of two reactions or
`events: a slow nucleation process and a fast “zippering” up. Nucle-
`ation is rate-limiting and requires proper temperature settings
`(Anderson, 1999). Once a duplex has formed (after “zippering”),
`it is very stable at temperatures below melting, given that the
`duplex is longer >50bp. Hybridizing overnight works well for a
`wide range of target or probe scenarios. If this generates a dissat-
`isfactory signal, consider the following.
`There are several variables that affect hybridization time.
`Double-stranded probes (i.e., an end-labeled 300bp fragment)
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`require longer hybridization times than single-stranded probes
`(end-labeled oligonucleotide), because reassociation of double-
`stranded probes in solution competes with annealing events of
`probes to target. At 50% to 75% reassociation, free probe con-
`centration has dwindled to amounts that make further incubation
`futile. Hybridization time for a double-stranded probe can there-
`fore be deduced from its reassociation rate (Anderson, 1999).
`Glimartin (1996) discusses methods to predict hybridization times
`for single-stranded probes, as does Anderson (1999). Other vari-
`ables of hybridization time include probe length and complexity,
`probe concentration, reaction volume, and buffer concentration.
`Buffer
`formulations
`containing higher
`concentrations
`(≥10ng/ml) of probe and/or rate accelerators or blots with high
`target concentrations may require as little as 1 hour for hybridiza-
`tion. Prolonged hybridization in systems of increased hybridiza-
`tion rate will lead to background problems. The shortest possible
`hybridization time can be tested for by dot blot analysis. Standard
`buffers usually require hybridization times between 6 and 24
`hours. Plateauing of signal sets the upper limit for hybridization
`time. Again, optimization of hybridization time by a series of dot
`blot experiments, removed and washed at different times, is rec-
`ommended. Plaque or colony lifts may benefit from extended
`hybridization times if large numbers of filters are simultaneously
`hybridized.
`
`What Are the Functions of the Components of a Typical
`Hybridization Buffer?
`Hybridization buffers could be classified as one of two types:
`denaturing buffers, which lower the melting temperatures (and
`thus hybridization
`temperatures) of nucleic acid hybrids
`(i.e., formamide buffers), and salt/detergent based buffers, which
`require higher hybridization temperatures, such as sodium phos-
`phate buffer (as per Church and Gilbert, 1984).
`
`Denaturants
`Denaturing buffers are preferred if membrane, probe, or label
`are known to be less stable at elevated temperatures. Examples
`are the use of formamide with RNA probes and nitrocellulose
`filters, and urea buffers for use with HRP-linked nucleic acid
`probes. Imperfectly matched target:probe hybrids are hybridized
`in formamide buffers as well.
`For denaturing, 30% to 80% formamide, 3 to 6M urea, ethyl-
`ene glycol, 2 to 4M sodium perchlorate, and tertiary alkylamine
`
`Nucleic Acid Hybridization
`
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`

`chloride salts have been used. High-quality reagents, such as
`deionized formamide, sequencing grade or higher urea, and
`reagents that are DNAse- and/or RNAse-free are critical.
`Formamide concentration can be used to manipulate stringency,
`but needs to be >20%. Hybrid formation is impaired at 20% for-
`mamide but not at 30 or 50% (Howley et al., 1979). 50% to 80%
`formamide may be added to hybridization buffers. 50% is rou-
`tinely used for filter hybridization. 80% formamide formulations
`are mostly used for in situ hybridization (ISH) where temperature
`has the greatest influence on overall stability of the fixed tissue
`and probe, and in experiments where RNA:DNA hybrid forma-
`tion is desired rather than DNA:DNA hybridization. In 80% for-
`mamide, the rate of DNA:DNA hybridization is much lower than
`RNA:DNA hybrid formation (Casey and Johnson 1977). Phos-
`phate buffers are preferred over citrate buffers in formamide
`buffers because of superior buffering strength at physiological pH.
`In short oligos 3M tetramethylammonium chloride (TMAC)
`will alter their Tm by making it solely dependent on oligonu-
`cleotide length and independent of GC content (Bains, 1994;
`Honore, Madsen, and Leffers, 1993). This property has been
`exploited to normalize sequence effects of highly degenerate
`oligos, as are used in library screens. Note that some specificity
`may be lost.
`
`Salts
`Binding Effects
`Hybrid formation must overcome electrostatic repulsion forces
`between the negatively charged phosphate backbones of the
`probe and target. Salt cations, typically sodium or potassium, will
`counteract these repulsion effects. The appropriate salt concen-
`tration is an absolute requirement for nucleic acid hybrid
`formation.
`Hybrid stability and sodium chloride concentration correlate in
`a linear relation in a range of up to 1.2M. Stability may be
`increased by adding salt up to a final concentration of 1.2M, or
`decreased by lowering the amount of sodium chloride. It is the
`actual concentration of free cations, or sodium, that influences sta-
`bility (Nakano et al., 1999; Spink and Chaires, 1999). Final con-
`centrations of 5 to 6¥ SSC or 5 to 6¥ SSPE (Sambrook, Fritsch,
`and Maniatis, 1989), equivalent to approximately 0.8 to 0.9M
`sodium chloride and 80 to 90mM citrate buffer or 50mM sodium
`phosphate buffer, are common starting points for hybridization
`buffers. At 0.4 to 1.0M sodium chloride, the hybridization rate of
`
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`DNA:DNA hybrids is increased twofold. Below 0.4M sodium
`chloride, hybridization rate drops dramatically (Wood et al., 1985).
`RNA:DNA and RNA:RNA hybrids require slightly lower salt
`concentrations of 0.18 to 1.0M to increase hybridization by
`twofold.
`
`pH Effects
`Incorrect pH may impair hybrid formation because the charge
`of the nucleic acid phosphate backbone is pH dependent. The pH
`is typically adjusted to 7.0 or from 7.2 to 7.4 for hybridization
`experiments. Increasing concentrations of buffer substances may
`also affect stringency. EDTA is sometimes added to 1 to 2mM to
`protect against nuclease degradation.
`
`Detergent
`Detergents prevent nonspecific binding caused by ionic or
`hydrophobic interaction with hydrophobic sites on the membrane
`and promote even wetting of membranes. 1% to 7% SDS, 0.05%
`to 0.1% Tween-20, 0.1% N-lauroylsarcosine, or Nonidet P-40 have
`been used in hybridization buffers. Higher concentrations of SDS
`(7%) seem to reduce background problems by acting