DNA Fingerprinting of Mycobacterium bovis by Restriction Fragment Length Polymorphism (RFLP)

What is RFLP?
RFLP stands for "Restriction Fragment Length Polymorphism." It is a molecular biology technique that produces a pattern of bands that resembles a bar code. For any isolate of Mycobacterium bovis, this pattern is a characteristic "fingerprint" which serves as an indicator of whether or not there is a relationship with other strains of M. bovis.
Figure 1 shows a DNA "fingerprint" of M. bovis. Note its resemblance to a barcode.

 How is DNA "fingerprinting" by RFLP performed?

Figure 2 provides a schematic diagram of the steps involved in producing a RFLP "fingerprint."
Step 1. In addition to regions that are used specifically to code for the production of various proteins, the genetic material of all M. bovis isolates contain pieces of DNA referred to as "insertion sequences" (open boxes). Isolates of M. bovis may be distinguished by the number of copies of these insertion sequences and their location within the chromosome. Two insertion sequences are detected by this procedure: IS6110 and PGRS. The DNA of the bacteria is cut at specific places by an enzyme, including once within these "insertion sequences".
Step 2. Cutting with the enzyme produces pieces of DNA of a variety of sizes (numbered in this illustration by the size of the piece). Some fragments will contain parts of the insertion sequence.
Step 3. The pieces are separated by a kind of a sieve, made of jelly-like substance (an agarose gel). The DNA that has been cut in pieces is placed at one end of the gel. An electric current applied across the gel will drive the DNA pieces through the gel. The pieces are separated by size, with the smallest pieces moving furthest from the starting point.

Step 4. The separated pieces are then transferred to a nylon membrane. A small piece of DNA (a probe) which matches one side of the insertion sequence is added (black boxes). A substance that emits light is added to the probe so that the presence of the insertion sequence can be determined.

Step 5. When X-ray film is exposed to the nylon membrane, light produced from the probe will produce a banding pattern on the film. The number and location of bands will correspond to the original number and position of the insertion sequence in the bacteria.

How is RFLP used?

M. bovis reproduces by dividing itself in two. The genetic makeup of the new cells are identical to the original parent cell. Even after many generations, bacteria that have been produced from the original parent will share virtually all of the same genetic material (DNA) of the parent. The "fingerprints" of genetically related strains of M. bovis will be identical. Unrelated strains can be easily distinguished since their "fingerprints" will be different.

RFLP analysis is used by laboratory scientists and epidemiologists to evaluate the genetic relationship between isolates of M. bovis.

An example of PGRS patterns comparing M. bovis isolates is shown in figure 3.

The caption for figure 3: RFLP results when DNA is digested with AluI and probed with the probe PGRS. Lane 1, deer isolate; lane 2, raccoon isolate; lane 3, raccoon isolate; lane 4, coyote isolate; lane 5, coyote isolate; lane 6, coyote isolate; lane 7, coyote isolate; and lane 8, MI cow isolate.

The patterns of the deer and cow isolates, which resemble bar codes, are identical to each other. This indicates that the M. bovis isolates from the cow and the deer are related to each other.

Figure 4. RFLP results when DNA is digested with PvuII and probed with IS6110-445 base probe. Lane 1, deer isolate; lane 2, raccoon isolate; lane 3, raccoon isolate; lane 4, coyote isolate; lane 5, coyote isolate; lane 6, coyote isolate; lane 8, MI cow isolate.

The MDCH Molecular Biology Section performs RFLP analysis of both M. tuberculosis. and M. bovis.