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

Phvsical 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 hips can be stripped, but stripping efficiency and aging of target 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 hat 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, he 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 Thickness Most membranes are appro ness influences the amount of buffer required per square 414 Herzer and Englert

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 Herzer and Englert

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, 0.45 um 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 0.22 um micron pores are recommended for work with the smallest single- and double-stranded DNA fragments Custom manufacturing of membranes with 0. 1 um 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 macroarray 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 P are preferred for macroarrays(Moichi et al., 1999; Yano et al., 2000: Eickhoff et al., 2000) Nucleic Acid Hybridization 4l5

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 415

左 v=89=E5 §喜 2 台 三 z6M 与 E日 5= 4 16 Herzer and Engl ert

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

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 glo Membranes should be dry and uniform in appearance. They should be wrinkle- and scratch-free since mechanical damage may lead to back ground 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 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 121C 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 Nucleic Acid Hybridization 4l7

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. Nucleic Acid Hybridization 417

Treatment of membranes with 15% peroxide or 98% ethanol at oom temperature after crosslinking can also sterilize filters. Per oxide may be more harmful to nucleic acid and filter chemistry NUCLEIC ACID TRANSFER What Issues Affect the Transfer of nucleic acid from Agarose Gels? This discussion will focus on the transfer of nucleic acids from agarose gels onto a membrane via passive transfer. Details on the transfer of DNA from polyacrylamide gels are presented in Westermeyer(1997) Active or Passive Techniques Vacuum, electrophoretic, and downward gravity transfer methods are fast(less than 3 hours) and efficient (greater than 90% transfer). Transfer efficiency depends on thickness and per centage of the gel and nucleic acid concentration or size. Transfer time increases with percentage of agarose, gel thickness, and frag- ment size. Capillary blotting of RNA larger than 2.5kb takes more than 12 hours, and downward transfer only 1 to 3 hours(Ming et al., 1994; Chomczynski, 1992; Chomczynski and Mackey, 1994) Speed, low cost, no crushing of gel, and efficient alkaline transfer of RNa are the main reasons why downward transfer is gaining popularity for RNA transfer (Inglebrecht, Mandelbaum, and Mirko, 1998) Transfer Buffer Manufacturers of filter or blotting equipment provide transfer protocols that serve as a starting point for transfer buffer for mulation. If nucleic acids are of unusual size or sequence, modi fied protocols might be required. RNA, small DNA fragments (100bp), and nitrocellulose membranes usually require greater salt concentrations. Keep in mind that RNa has a very low affin ity for nitrocellulose even at high salt The effects of pH on transfer efficiency and subsequent detection of target are many and complex. Transfer buffer ph can directly affect the stabilities of the membrane and the nu cleic acid target Nitrocellulose and some nylon membranes are not stable at pH>9, and nitrocellulose will not bind DNA at pH above 9(Ausubel et al., 1993). Some nylon membranes are not stable at acidic pH(Wheeler, 2000). Transfer buffer ph 418 Herzer and Englert

Treatment of membranes with 15% peroxide or 98% ethanol at room temperature after crosslinking can also sterilize filters. Per￾oxide may be more harmful to nucleic acid and filter chemistry over time. NUCLEIC ACID TRANSFER What Issues Affect the Transfer of Nucleic Acid from Agarose Gels? This discussion will focus on the transfer of nucleic acids from agarose gels onto a membrane via passive transfer. Details on the transfer of DNA from polyacrylamide gels are presented in Westermeier (1997). Active or Passive Techniques Vacuum, electrophoretic, and downward gravity transfer methods are fast (less than 3 hours) and efficient (greater than 90% transfer). Transfer efficiency depends on thickness and per￾centage of the gel and nucleic acid concentration or size. Transfer time increases with percentage of agarose, gel thickness, and frag￾ment size. Capillary blotting of RNA larger than 2.5kb takes more than 12 hours, and downward transfer only 1 to 3 hours (Ming et al., 1994; Chomczynski, 1992; Chomczynski and Mackey, 1994). Speed, low cost, no crushing of gel, and efficient alkaline transfer of RNA are the main reasons why downward transfer is gaining popularity for RNA transfer (Inglebrecht, Mandelbaum, and Mirkov, 1998). Transfer Buffer Manufacturers of filter or blotting equipment provide transfer protocols that serve as a starting point for transfer buffer for￾mulation. If nucleic acids are of unusual size or sequence, modi- fied protocols might be required. RNA, small DNA fragments (<100bp), and nitrocellulose membranes usually require greater salt concentrations. Keep in mind that RNA has a very low affin￾ity for nitrocellulose even at high salt. The effects of pH on transfer efficiency and subsequent detection of target are many and complex. Transfer buffer pH can directly affect the stabilities of the membrane and the nu￾cleic acid target. Nitrocellulose and some nylon membranes are not stable at pH > 9, and nitrocellulose will not bind DNA at pH above 9 (Ausubel et al., 1993). Some nylon membranes are not stable at acidic pH (Wheeler, 2000). Transfer buffer pH 418 Herzer and Englert