4.3.1. Choice of Hybridization Membrane Immobilization and hybridization of nucleic acid was first carried out with nitrocellulose (3). However, nitrocellulose is not ideal for nucleic acid hybridization. Because the nucleic acids are attached by hydrophobic rather than covalent interactions, they are slowly leached out of the nitrocellulose matrix during hybridization and washing at high temperatures. In addition, the fragile nitrocellulose membranes cannot survive more than one or two cycles of hybridization and washing. The shortcomings of nitrocellulose have led to the development of several alternative matrices, the most versatile of which is positively charged nylon (13). Nylon membranes bind nucleic acids irreversibly and are much more durable, allowing sequential hybridizations with several different
Solutions Used in Nucleic Acid Blotting
TAE (Tris-acetate buffer)
TPE (Tris-phosphate buffer) TBE (Tris-borate buffer) MOPS buffer (3-|W-morpholino] propanesulfonic acid)
SSC (salt-sodium citrate buffer)
SSPE (salt-sodium phosphate-EDTA buffer)
DNA gel denaturation solution
Alkaline transfer buffer
Prehybridization solution (option 1)
Hybridization solution (option 1)
Prehybridization and hybridization solution (option 2) Prehybridization and hybridization solution (option 3)
DNA and RNA sample buffer
0.09 M Tris-phosphate, 0.2 M EDTA, pH 8.0 0.045 M Tris-borate, 0.1 M EDTA, pH 8.0 (5X stock) 0.1 M MOPS, pH 7.0 40 mM sodium acetate 5 mM EDTA, pH 8.0 (20X stock) 3.0 M NaCl, 0.3 M sodium citrate, pH 7.0
(20X stock) 3.6 M NaCl, 0.2 M NaH2PO4 • H2O, 20 mM EDTA, pH 7.7 0.5 M Sodium hydroxide
3 M Sodium chloride, 8 mM sodium hydroxide, pH 11.4-11.45 (50X stock)
1% Ficoll, 1% polyvinylpyrrolidine, 1% bovine serum albumin 5X SSPE, 5X Denhardt's reagent, 200 |lg/mL denatured salmon sperm DNA, 0.1% SDS, 50% formamide 5X SSPE, 2.5X Denhardt's reagent,
200 | g/mL denatured salmon sperm DNA, 0.1% SDS, 50% formamide 6X SSC, 2X Denhardt's reagent, 0.1% SDS
5X Denhardt's reagent, 0.5% SDS, 100 |g/mL denatured salmon sperm DNA
bromophenol blue, 0.25% xylene cyanol FF
Agarose gel electrophoresis; electrophoretic transfer of nucleic acids to a nylon membrane
Agarose gel electrophoresis (Southern blot) Agarose gel electrophoresis Electrophoresis of RNA through formaldehyde gels in Northern blotting
Capillary transfer of nucleic acids to nylon or nitrocellulose membrane; washing hybridized membranes Capillary transfer of nucleic acids to nylon or nitrocellulose membrane; washing hybridized membranes Denaturation of electrophoresed DNA prior to
Southern blot transfer Neutralization of DNA gels after sodium hydroxide denaturation Rapid alkaline capillary transfer of nucleic acids to nylon membranes A blocking agent added to hybridization solutions in Southern and Northern blots to reduce background Prehybridzation of Northern and Southern blots prior to hybridization with labeled probe
Hybridization of Northern and Southern blots to labeled probe
Prehybridization and hybridization of
Northern and Southern blots Useful for reducing high-background hybridization in Northern and Southern blots
Electrophoresis of RNA through formaldehyde gel probes without a loss of membrane integrity. Nylon membranes also allow highly efficient electrophoretic transfer of small amounts of nucleic acid when capillary or vacuum transfer is insufficient. The single disadvantage of nylon membranes is the propensity for higher-background hybridization, but this problem can be eliminated by increasing the amount of blocking agents during prehybridization and hybridization.
4.3.2. Methods of DNA Transfer Single-stranded (denatured) DNA can be transferred to a solid support, such as nitrocellulose of nylon membrane. If nitrocellulose membranes are used, the recommended method of transfer is capillary transfer. With the more versatile nylon membrane, several methods of transfer are available, including capillary transfer, electrotrans-fer, and vacuum transfer. Although nitrocellulose can be used for vacuum transfer or electrotransfer, these methods are optimal with nylon membranes. Regardless of the method of transfer, it is recommended that the gel and the membrane be equilibrated in transfer buffer prior to transfer. The composition of buffer is dependent on the method of transfer employed (see Table 2).
In capillary transfer (1), nucleic acid fragments are eluted from the gel and deposited onto the membrane by transfer buffer that is drawn through the gel by capillary action. Rate of transfer is dependent on the size of the fragments, with larger fragments transferring less efficiently. The disadvantage of traditional capillary transfer is the length of time required for efficient transfer of large nucleic acid fragments which transfer less efficiently (usually overnight). When large (>5 kb) fragments of DNA are to be analyzed, many protocols recommend depurinating the electrophoresed DNA prior to denaturation by soaking the gel in 0.2 M hydrochloric acid for 5-15 min (14). Depurination, along with denaturation, leads to the breakdown of long DNA fragments into shorter pieces, which transfer more efficiently. However, this nicking of the DNA has been reported to reduce the final hybridization signal significantly and decrease the clarity of bands on the autoradiograph (5). Downward alkaline capillary transfer, which can be completed
in 1-3 h, offers a fast and efficient alternative to traditional capillary transfer (15,16) and does not require special equipment (see Fig. 3).
Electrophoretic transfer (17) was developed as a faster alternative to capillary transfer. Size-separated nucleic acid fragments are transferred onto a membrane (preferably nylon) by placing the gel between porous pads that are inserted between parallel electrodes in a large buffer tank. These apparatuses are available from several manufacturers (BioRad [Hercules, CA], Schleicher & Schuell [Keene, NH]). Electroblotting can be quite efficient, with complete transfer of high-molecular-weight nucleic acids in 2-3 h. However, the electrophoresis apparatus must be equipped with a cooling mechanism for maintaining an acceptable buffer temperature, and large buffer volumes are required. The need for special equipment has limited the practical application of the elec-troblot to situations in which capillary transfer or vacuum transfer is not sufficient.
Vacuum blotting was introduced as another alternative to capillary transfer (18,19), and involves the application of negative pressure to the gel. Nucleic acids are eluted by buffer that is drawn through the gel by application of negative pressure (a vacuum). Transfer of nucleic acids by vacuum blotting is more efficient than capillary transfer and has been reported to result in a twofold to threefold increase in final hybridization signal obtained (20). Vacuum blotting devices are commercially available (Bio-Rad) and work well when the vacuum is applied evenly over the gel surface. However, efficiency of transfer could be reduced if the vacuum exceeds 60 cm of water (6), because of compression of the gel. When carried out properly, the vacuum blot provides a means for fast (approx 4 h), efficient, highly reproducible transfer of nucleic acids.
4.3.3. Fixing DNA onto Nitrocellulose or Nylon Membranes After transfer, the DNA must be adhered stably to the membrane to ensure that it remains in place during hybridization and washing. If a nitrocellulose membrane was used, the DNA can be affixed by baking the damp membrane at 80°C for 2 h in a vacuum oven. Alternatively, nylon membranes can be exposed (DNA side up) to low-level ultraviolet
(UV) irradiation at 254 nm. Irradiation of the membrane results in crosslinks between the nucleic acid residues and positively charged amine groups on the membrane surface (21). Overirradiation of the membrane could cause covalent attachment of a high percentage of the nucleic acid residues, resulting in a decreased hybridization signal. Special ovens for the irradiation of membranes are commercially available, and most manufacturers recommend 1.5 J/cm2 for damp membranes and 0.15 J/cm2 for dry membranes. Optimally, the ideal amount of irradiation should be determined empirically. Baked or irradiated Southern blots can be stored at room temperature until they are ready to be hybridized to a labeled nucleic acid probe.
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