Chapter: Pharmaceutical Biotechnology: Fundamentals and Applications - Molecular Biotechnology

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PCR Technology

For the detection of DNA or for testing the presence of mutations in DNA the probe method described above is very powerful.

PCR Technology

 

For the detection of DNA or for testing the presence of mutations in DNA the probe method described above is very powerful. Within the current probe techniques, however, a substantial amount of DNA is required to allow the detection of target DNA. The PCR (polymer-ase chain reaction) technology became very popular in recent years to acquire large amounts of DNA.

In the PCR technology target DNA is amplified by in vitro DNA synthesis, occurring in a number of fast repeating steps. The reaction starts with the conversion of the double-stranded target DNA to single-stranded DNA and uses specific oligonucleo-tides as primers to allow DNA polymerase to do its job. The choice of the oligonucleotide primers, hybridizing with each of both target strands, will determine the left and right limits of the DNA to be amplified.

Each PCR cycle (illustrated in Fig. 16) consists of three steps each requiring only 1 to 3 minutes. In the first step the target DNA must be made single stranded and this is done by heating the sample to 92LC. The second step involves the specific hybridiza-tion of the two primers to the complementary single-stranded DNA. The optimal temperature for this process is about 55LC. In the third step DNA polymerase will extend the primer sequence using the single stranded DNA as a template. The optimal extension temperature is about 72LC since the DNA polymerase chosen is derived from a thermophilic bacterium, Thermus aquaticus, which normally grows in hot springs at temperatures above 80LC. This DNA polymerase is extremely resistant against heat dena-turing and survives the 92LC DNA denaturing step. All reagents (target DNA, primers, dNTPs and polymerase) are put in a tube which is sealed and usually 20 to 30 PCR cycles are performed. The procedure can be automated and PCR machines are available which control the temperature for each of the three separate steps of a PCR cycle. Such machines can process hundreds of tubes simultaneously and produce results within 2 to 4 hours.


Ideally each cycle of DNA replication doubles the amount of DNA which is located in-between the chosen primers. Thirty PCR cycles will give an amplification of 230 times. This means that minute quantities of DNA can be amplified with specific primers to easily detectable levels. It should be realized that the specificity of the reaction is fully determined by the PCR primers and these primers will also determine the length of the amplified fragment. The tremendous sensitivity of the technique has sparked the development of a great number of applications where such sensitivity is of paramount importance. Also, compared to many other detection methods, the PCR procedure is very fast.

For example, the presence of microbial patho-gens in raw and processed food products can be unequivocally determined using this technology. DNA is extracted from this material and the PCR reaction is performed using primers which are specific for the suspected pathogen(s). Detailed knowledge of DNA sequences of all sorts of genes in all sorts of organisms allows the development of such specific primers, the main prerequisite for diagnostic PCR technology. If specific amplified DNA products can be detected, this is proof that the pathogen is present in the material. Also in clinical material (blood, urine, etc.) the technique is usedextensively as a rapid and sensitive test for the presence of bacterial and viral pathogens. A third area where PCR has become standard technology is in forensic science. At a crime scene often minute quantities of potentially important evidence is found (single hairs, blood drops, semen stains, etc.) and PCR technology can be used to get enough DNA to show the origin of this material. These are only a few examples of the use of PCR technology. PCR is often an essential step in elaborate diagnostic and detection procedures and novel applications are continuously being developed.

As for the application of the PCR technology for diagnosis of pathogens, one has to realize that for most purposes the intent is to detect viable pathogens. The PCR technology obviously cannot distinguish DNA from vital or dead material and in that respect it is not always an adequate technique. The PCR technique is a very sensitive one since minute amounts of DNA are highly amplified. This high sensitivity may limit the discriminative power of the technique when applied for diagnostic purposes. For example, it may detect minor contaminants in the samples. Moreover, DNA contaminants may be introduced during the perfor-mance of the tests. It is therefore a major concern in the application of PCR to avoid DNA contaminations that could cause false positive reactions.

Modified PCR techniques and related methodol-ogies are discussed.


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