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PCR

PCR stands for "polymerase chain reaction." Polymerase means that it makes copies of a target sequence; chain reaction means that it makes millions of copies. Since its development in 1985, the PCR has revolutionized basic and applied research. Introduction of the heat stable DNA polymerase named "Taq" made routine use of this procedure possible.(1)

In a typical PCR reaction, researchers mix target DNA, forward and reverse primers, building blocks, and Taq DNA polymerase. Primers are simply oligomers which are complementary to the target DNA, and thus hybridize to a specific sequence (see the FISH discussion for a discussion complementary/hybridization concepts). The forward and reverse primers are chosen so that they are not too far apart when they anneal to the DNA. They let the mixture react for a while, then heat it to denature the DNA, cool the mixture so that it re-anneals, and allow it to make more DNA. The entire process is repeated 25-40 times, and because the products are also used as template in subsequent reactions, there is an exponential increase in the number of copies of the target sequence.

There are several good web sites that illustrate this process:

As discussed in the clonality section, leukemic B-cells have a specific V-D-J segment encoding the Ig protein. To use PCR to take advantage of this trait to detect leukemia clones, scientists use "forward" and "reverse" primers that flank the region, copying ds DNA from two directions.

In order to amplify the DNA by PCR successfully, the primers must recognize DNA sequences within a short segment of DNA. In the germline configuration, the V and J segments are widely separated, and the primers will be so far apart that the enzyme cannot synthesize new DNA and no PCR product is seen. In a polyclonal B cell population, you would see a smear, since each B-cell would have a different V-D-J length (and sequence). However, if the B-cells are from one clone, they will all have the same V-D-J size segment and therefore only one band.

The choice of primers is critical to the success of the procedure. Each patient's leukemia clone likely has a different V-D-J segment than every other patient's leukemia clone. However, most leukemic clones will have a common sequence found in the J (joining) and another in the V (variable) region; sequences of such primers (oligomers) have been reported in the scientific journals. These short, common sequences of DNA are called consensus V region and consensus J region primers. Consensus primers for IgH (the H stands for heavy-chain) are called JH and VH. (1) These primers work for most but not all leukemias.

Drawbacks of PCR

Due to the variety of rearrangements possible in the regions targeted for PCR, some patients do not have a positive PCR test. This can happen if their PCR amplified region is either too big or too small, or if the areas the primers would hybridize to are themselves deleted. Other problems with PCR are inherent in the reaction itself. The technique is very sensitive, and poor attention to detail or sloppy technique can lead to contamination by DNA from a source other than the patient's sample. (Keep in mind, PCR was used in testing the blood samples for OJ Simpson's trial.)

PCR as a tool for recognition of MRD (8,9)

The proof that a patient's bone marrow contains a clonal population of B-cells indicates that the patient has B-cell leukemia. Therefore, the PCR 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.

In patients with known PCR regions at diagnosis, the PCR test does lend itself to MRD testing. (8, 9) PCR amplifies a small amount of DNA into a large amount of the same sequence DNA, so it can amplify the small amount of leukemic DNA to an amount large enough to detect. Still, there is sometimes not enough leukemic DNA in minimal residual disease situation to visibly see a band on a gel. The amplification process also amplifies normal DNA, which can overshadow any leukemia-specific PCR DNA. Therefore, most strategies for the use of PCR in detection of MRD use a combination of Southerns and PCR.

One of the most critical parts of the assay is the choice of primers, and the groups publishing on this topic almost all use different ones; to cover all kid's leukemias, they have to try first one and then another, and still the researchers are not always successful. (6,20) The initial search for a PCR band is done on the bone marrow aspirate taken at diagnosis, when the clone is plentiful and obvious. When they find the patient's PCR band, they sequence the DNA. Often the researchers must try a few different sets of primers to get the assay to work on all cases of ALL. Some groups use Southern blot techniques to pick out the patient's unique DNA in the regions of interest; once they isolate the novel band on a gel, they cut it out and sequence it. Once they know the sequence of the patient's unique DNA, they can synthesis forward and reverse primers for PCR that are patient-specific. They then use these patient-specific primers to direct PCR synthesis on bone marrow aspirates taken at several times during and after treatment. In this way, they are able to amplify a small amount of residual leukemic DNA. Some groups use patient-specific oligomer probes to look for tiny amounts of leukemic DNA in a procedure called "dot blot", in which labeled probes hybridize with PCR amplified DNA. Some groups sequence the DNA obtained at the later time points to absolutely prove that the DNA is the same as that obtained from the leukemic clone at diagnosis.

A problem with PCR is that it requires stringent quantization protocols because the PCR product is produced exponentially. In other words, the amount of PCR product obtained during an assay does not directly and linearly correlate with the amount of leukemic DNA in the sample. Therefore, they must do a "limit dilution" assay, where there do serial dilutions of each sample (and they usually do each of these in 10 duplicates each) until the math tells them they are in a linear portion of the equation. Or, they might do internal standards. All of these requirements further complicate an already laborious procedure, and the suitability of PCR for routine clinical studies of MRD may be limited.

Another problem is false negatives. One cause of false negatives is that a sub-clone of the original leukemia forms. Thus, when patient-specific probes are used on samples taken late in treatment, it cannot pick up the leukemic DNA because the leukemic DNA is no longer the same sequence as it was at diagnosis.

Specific strategies for the use of PCR to detect MRD are discussed in the sections that follow on individual papers.

The techniques of FISH, PCR, and Southern blots are used in other cancers to detect low levels of cancer cells, or to diagnose cancer. In most cancers, the gene being searched for is the oncogene, the cancer gene - the mutation that actually causes the cancer. In ALL, the gene used to diagnose and detect the cancer is not the cancer gene. The DNA sequence unique to each child’s cancer usually has nothing to do with the mutation responsible for the malignancy.

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