Southern blotting probe, preparation

The concentration of the sample can be estimated by examining the ethidium bromide gel or by using a spectrophotometer. The products from this particular preparation can then be used for RFLP with end labeling, for cloning, for PCR, or for making radioactive probes for Southern blots or filter lifts. The purity of the sample is essential for all applications except PCR. 

Preparation of Probe. Probes for genomic Southern blots have been made by a variety of methods and must be of high specific activity. A simple method that yields a probe with a very high specific activity utilizes the polymerase chain reaction (PCR).16 An advantage of PCR over methods based on nick translation or primer extension is that only one strand of DNA is synthesized, so that reassociation of denatured single-strand probe in solution is minimized. 

Much of the methodology for DNA preparation, and Southern blot analysis used for DNA fingerprinting, is pubhshed elsewhere (Honma et al., 1993) and the reader should refer to this for further details (particularly for steps 1-3 above) and to the procedures supplied with the Jeffreys multilocus probes). 

High specific activities of the probe can be achieved by changing to labeled nucleotide preparations of higher specific activity and reducing the labeled dNTP precursor concentration to 1-2 pM. Consequently, if the specific activity of the precursor is 1000 Ci/mmol, then 1-2 mCi should be added per ml (i.e., 25-50 pCi/25 p,l). With higher specific activities of the precursor, proportionally more radioactivity should be used to maintain the same precursor concentration (e.g., 3-6 mCi/ml if the specific activity is 3000 Ci/mmol). This can be expensive if the volume of the mixture is not scaled down, but it can be advantageous when low concentrations of probe with high specific activity are required (e.g., for certain Southern blots). The efficiency of incorporation is lower when two or more labeled nucleotides are used as precursors. 

Probes prepared with either digoxigenin- or biotin-modified nucleotides can be hybridized to Southern blots to detect target nucleic acid sequences. These methods offer an attractive alternative to radioactively tagged probes in terms of safety, cost, and efficiency. Most previous nonradioactive strategies utilized the detection of the modified base by the use of a coupled antibody- or avidin-alkaline phosphatase. The blot with the bound alkaline phosphatase was then treated with a compound, such as Nitro Blue Tetrazolium (NBT), that was converted to an insoluble, colored compound at the site of hybridization, thus facilitating visualization of the hybridized probe. 

Probing of the Northern blots and of Southern blots of restriction fragments of barley DNA was carried out using the ii cloned fragments prepared by PCR. 

Another technique is cytological H., in which radioactive RNA is hybridized with chromosomal DNA in a histological preparation. This indicates the chromosomal location of the gene(s) for the RNA in question. Conversely, cloned DNA probes can be used to show the histological distribution of mRNA corresponding to the cloned gene. Similarly, P-la-beled RNA or cDNA is used to locate complementary DNA sequences in Southern blots (see) of elec-trophoretically separated restrietion fragments. 

The main procedure of northern blotting is very similar to that of Southern blotting in that both contain steps of sample preparation, gel electrophoresis, transfer from gel to membrane, probe hybridization, and detection. Northern blotting differs from the Southern procedure in the following ways ... 

Southern s technique could not be applied to the blot transfer of RNA separated by gel electrophoresis. Alwine et al. (A2) therefore devised a procedure in which RNA bands are blot transferred from the gel onto the chemically reactive paper, where they are bound covalently. The reactive paper is prepared by diazotization of aminobenzyl oxymethyl paper, which can be prepared from Whatman paper by a series of uncomplicated reactions. Once covalently bound, the RNA is available for hybridization with radiolabelled DNA probes. Hybridizing bands are detected autoradiographically. This method is termed Northern blotting. 

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