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Southern Blots


"Southern blots" is a short-hand term for a rather lengthy procedure that determines specific sequences in DNA. In the FISH section, you learned that small, fluorescent-tagged stretches or "oligomers" of DNA can detect specific sequences in chromosomes; Southern blots use similar oligomers to detect specific sequences on naked DNA.

In preparation for a Southern blot procedure, DNA is extracted from cells and purified. This DNA is then cleaved with restriction enzymes. A restriction enzyme is an enzyme that sits down on a DNA strand only when it recognizes a specific sequence of nucleotides. Once it sits down on the DNA, it cleaves it.

You can think of restriction enzymes as proteins that only recognize certain arrangements of letters or "words" on the DNA. One might only recognize the word "black", one only the word "elephant". If "black" and "elephant" are randomly placed on a very, very long sentence ("DNA"), the black restriction enzyme would chop the sentence into a different number and size of pieces than would the elephant restriction enzyme. Discovered in the 1970s, restriction enzymes are a routine tool in the molecular biological laboratory.

When a person's germline sample of DNA is treated or "digested" with a particular restriction enzyme, it results in a reproducible set of DNA fragments.

Using the analogy above, every DNA sentence in a person's germline DNA would have the word black in exactly the same position and exactly the same number of times; therefore cleavage with black restriction enzyme would always produce the same number and size of smaller pieces.

The next step requires these DNA fragments to be separated according to size. First, each different restriction enzyme digest of cut DNA pieces is loaded onto a separate "lane" on a thin slab of agarose gel. Then an electric current is placed across the gel, causing the DNA fragments to move down the gel in their lane - this is called "electrophoresis". The rate with at which each fragment moves depends on its size. When the electrophoresis process is complete, the result is a pattern of bands of larger-to-shorter DNA down the lanes of the gel.

The bands of DNA in the gel are then transferred from the gel to a thin membrane. The particulars of this transfer vary in the choice of nylon-like membrane material, the method of DNA denaturation, and the method of fixing the DNA to the membrane. (1)

The final step is to incubate the sized-DNA pieces on the membrane with single-stranded DNA oligomers, or probes. These probes are labeled, usually with a radioisotope like 32P. The sequence of the DNA probes depends on the specific sequence of interest. After the probes hybridize to the bands of DNA, the gel is developed by placing a photographic film on it. Distinct bands show up wherever the radioactive probe has annealed to a band of DNA.

Southerns in leukemia

Southern blots are used to detect clonal leukemia populations. First, they isolate DNA from both germline and suspected leukemia populations. They then separate each type (germline and suspected leukemic) into three tubes, and add the restriction enzyme Eco RI to one tube, HindIII to another, and Bam HI to the third. All six tubes are incubated to give the restriction enzymes time to do their work. Then they apply them to an agarose gel, electrophorese, and transfer to a membrane which is treated with labeled, single-stranded DNA probes.

You learned in the clonality section that the regions coding for Ig and TCR are of interest to leukemia researchers because in normal B-cells, the DNA of each and every B-cell in these regions is unique to that cell. This means that if this region is treated with "black" restriction enzyme, a random population will give a large spread of different sized pieces, since the word "black" would sometimes be close together, and sometimes far apart. Sometimes the word "black" is even eliminated from the DNA in this region.

On the other hand, if a B-cell population is clonal, the DNA in this region will be the same in every DNA molecule in this clone. Therefore, treatment with "black" restriction enzyme would give many fragments that are exactly the same size.

Thus, digestion with restriction enzymes should show a new, novel, intense band if the population is clonal; digestion of a normal population will give many bands that are of too low an intensity to be detected.

The visualization of the restriction enzyme digests is facilitated by labeled, single stranded DNA probes used in the last step of the Southern procedure. For the detection of clonal B-cells, they choose oligomers which will stick to specific segments in the Ig and/or TCR regions. Even though much of the DNA in the super gene varies in all cells, adjacent to the variable DNA regions is a "consensus" region. For instance, almost all B-cells have the same short sequences in the J (joining) region, designated "JH" and "Jkappa". These two sequences, among others, are used as probes to search for leukemia clones.

A hypothetical Southern of 3 restriction enzyme digests - Bam HI, EcoRI, and HindIII - of both germline and a population with expected leukemic cell DNA probed with oligomers representing consensus regions is shown below. Normal, germline cells show a characteristic pattern for each enzyme digest in lanes 1, 3, 5. DNA from the suspected leukemia cell containing population is in lanes 2, 4, and 6. Lanes 2 and 4 show the germline band plus a new, smaller band. Lane 6 shows no novel bands.

The interpretation of these results would be that the population being tested is a clonal leukemic population, because the BamHI and EcoRI digests show a novel band corresponding to a large population with a same-size V-D-J region, as would happen if this region were rearranged with deletions. The HindIII digest does not show a novel band (lane 6); it is likely that the rearrangement deleted the recognition site for this restriction enzyme.

Note: If the population were polyclonal, new fragments in the V-D-J region would appear on the restriction enzyme digests, but they would each be of a different size, and there would not be enough of any one of them to show up on a gel. (This case is not illustrated on the gel above.) To be considered positive, a Southern searching for clonal B-cell populations needs to show two novel bands present in two separate enzyme digests.(1)

Drawbacks of Southern blots

This technique requires fresh or frozen specimens, unlike the PCR technique, which can be done on almost any stored bone marrow sample. Selection of probes is also critical, and false negatives are possible. Southerns are not automated they must be done by hand by skilled technician.

Southerns in searching for MRD

The Southern test was developed as a diagnostic tool, and was designed for use on bone marrow samples taken at diagnosis, where there is a large number of clonal B cells. After induction, there are orders of magnitude fewer cells and hence, less DNA.

Southerns alone are not used in any labs to search for MRD, as per the editor's search through MedLine and journal articles in 1999. It is however used in conjunction with PCR studies of MRD.

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