Читать книгу PCR – diagnostics - Aizhan Zhussupova - Страница 2

Before PCR, molecular biologists utilized nucleic acid sequence data (sequence motifs) to design «hybridization» probes for use in assays for the detection and identification of specific RNA and DNA fragments (moieties). These «hybridization» assays were used to determine the presence/absence of specific RNA or DNA sequences within complex mixtures of nucleic acids, via the use of specifically designed (semisynthetic) complementary DNA or RNA molecules (nucleic acid probes) which had been equipped with radioactive labels for detection purposes. The target nucleic acid population was initially attached to a solid carrier phase and then hybridized with specific labeled probe. After stringent hybridization and extensive washing procedures, the presence of the target DNA fragment could be determined by the presence/absence of the radioactively labeled probe on solid carrier phase.

Alternatively, direct visualization of the probe and target molecule was achieved via electron microscopy, with hybridization being quantified on the basis of the different widths of double stranded (hybridized) versus single stranded (non-hybridized) nucleic acids. The most convenient of these hybridization test systems utilized filter hybridization (where the target DNA extract was first immobilized on nitrocellulose or nylon filters), in combination with post-hybridization autoradiography or scintillography. This format greatly increased test sensitivity and vastly improved the technical reliability and speed of the hybridization procedure, allowing the detection of picogram quantities of target material. However, one major disadvantage of these hybridization systems was the requirement for radioactively labeled probes, not least because working with radioactive materials is hazardous for your health, requires legal permits, correct disposal systems, and is relatively expensive to use. For these reasons, radioactive labels have been largely replaced by various (non-radioactive) chemical labels, facilitating the development of colorimetric, chemoluminescent and chemo-fluorescent hybridization detection methods. However, these «second generation)) chemical-labeling and detection systems do not generally yield as high a degree of sensitivity as the original radio-labeling and detection systems, though both systems are amenable to automation and high throughput applications. To date, a variety of elegant techniques based upon the basic hybridization principle have been developed (e.g. sandwich hybridization, Southern- and Northern-blot hybridizations, etc.) and these are frequently applied in both fundamental research and clinical diagnostics. The need to detect very small numbers of clinically relevant molecules was high. For instance, the detection of low-titer viral infections, minimal residual disease in leukemia patients, point mutations in genes or genetic aberrations in tumors etc., all require highly sensitive methodologies. This has led to the development of novel approaches specifically aimed at the amplification of target (gene) sequences prior to detection, such that sensitivity issues related to hybridization/probe detection protocols would no longer be the limiting step of DNA and RNA detection protocols. As with some of the greatest discoveries in science, from penicillin to microwave ovens, PCR was discovered serendipitously. Thanks to the work of many scientists, including Watson and Crick, Kornberg, Khorana, Klenow, Kleppe and Sanger, all the main ingredients for PCR had been described by 1980. Like butter, flour, eggs, and sugar lined up on a kitchen table, the ingredients of PCR were waiting for someone to scream out «Cake!» and open up the scientific community to a technique with a myriad of applications.

Kary B. Mullis who worked for Cetus Corporation perfecting oligonucleotide synthesis received the Nobel Prize in Chemistry along with Michael Smith in 1993 for his work on PCR and is accredited with its invention (see Fig. 1.1). Like many great inventions and discoveries that later prove immensely important, it took time for the scientific community to become interested in PCR. When Mullis presented an early PCR recipe and the thought process behind it at a conference, no one could conceive the final product.

Figure 1.1. Kary Mullis’ Polymerase Chain Reaction – The PCR method – a copying machine for DNA molecules; http://nobelprize.org/nobel_prizes/chemistry/ laureates/1993/illpres/pcr. html

At his lowest point, Mullis’ career was nearly derailed as his defense of his idea led to an altercation at the conference and he was removed as head of the oligonucleotide synthesis lab. Science would reject his paper on PCR only to name it and thermostable Taq polymerase isolated from Thermus aquaticus «Molecule of the Year» three years later. Thanks to a collaboration with the Erlich lab, the PCR project was back in the oven and over the next few years it was perfected and various applications developed, including DNA fingerprinting (1986), gene amplification systems (1988), real-time PCR with ethidium bromide (1992) and genome sequencing (2001).

When Mullis first tried PCR, his hope was that rather than needing temperature cycles, the ingredients would take care of themselves. A few experiments with no apparent product showed him what he dreaded most: that the temperatures would have to be readjusted to cycle the reaction from single to double-stranded DNA temperatures and, since the polymerase used at the time was thermally unstable, fresh enzyme would need to be added for every one of the 30 round required to create an almost pure product. Painstakingly, the temperatures would have to be controled by hand. This meant heating the reaction up to 95 °C, then allowing it to cool, adding DNA polymerase and heating the temperature back up 30 times over. Thus was time-consuming, exhausting, and tedious work. Imagine troubleshooting the optimal temperature for a primer to bind? Rather than popping your plate into a PCR cycler and giving different annealing temperatures to the different rows, one would need to do every single sample by hand, moving from bath to bath every 30-60 seconds. At this time, the samples were not done on convenient 96-well plates, but instead in tubes so you can imagine that hours of work would go into running from waterbath to waterbath to a timer ringing constantly. By 1985 and 1987, thermostable Taq polymerase and the first PCR machine, the PCR-1000 Thermal Cycler, became commercially available following a joint venture by Cetus and Perkin-Elmer. These contributed to reducing the cost and hours spent performing this technique and opened up numerous new applications for its commercial use and use in research. In 1991, Roche bought the rights to PCR from Cetus and invested in refining the science for use in molecular diagnostics to detect diseases. Roche Molecular Diagnostics has not only defined and refined PCR, but it still remains one of the leaders in the industry. Currently, you can run a simple PCR reaction in 2-3 hours, while doing the other lab procedures. That PCR has become one of the most widely used tools in molecular biology is clear (see Fig. 1.2).

Figure 1.2. Results of a PubMed search for articles containing the phrase «Polymerase Chain Reactions. Graph shows number of articles listed in each year expressed as a percentage of the total PubMed citations for each year (Bartlett and Stirling, 2003)

For instance, search on real-time PCR or RT PCR as a common tool for detecting and quantifying expression profiles of selected genes, technology facilitating the detection of PCR products in real-time, i.e., during the reaction yielded 7 publications in 1995, 357 in 2000, and 2291 and 4398 publications in 2003 and 2005, respectively. What is not clear from this simplistic analysis of the literature is the huge range of questions that PCR is being used to answer. Techniques have been developed in areas as diverse as criminal forensic investigations, food science, ecological field studies, and diagnostic medicine; just as diverse are the range of adaptations and variations on the original theme.

In the PCR procedure trace amounts of DNA can be quickly and repeatedly copied to produce a quantity sufficient to investigate using conventional laboratory methods. In this way, for example, it is possible to sequence the DNA, i.e. determine the order of its building blocks. Given these capabilities, Mullis’ method ultimately ushered in the age of genomics. Only with the advent of increasingly sensitive DNA chips in recent years has PCR faced any notable competition. But even then it is often necessary to first copy or amplifies the DNA of interest. For this reason PCR and DNA chips often go hand in hand.

«Polymerase Chain Reaction» is now a word in Merriam Webster’s Collegiate Dictionary and if you put «PCR» into Google searching machine, you get about 59,000,000 hits in 0.35 seconds. As Dr. Kary Banks Mullis says on his personal website (http://www.karymullis. com/pcr.shtml): «Most people in molecular biology today are not old enough to remember pre-PCR. But try to do your job without it, and you will see what a difference that simple little technique has made».

Questions for self-control

1. What do the PCR initials stand for? Who invented it?

2. For what specific purpose PCR is used?

3. The three main stages of the PCR process are usually repeated around 30 times over several hours. Approximately how many copies of the original DNA molecule are made during that time?

4. The development of PCR has had a major impact on a number of different areas. It has led to some major developments in medicine and forensic science, some of which are still in the early stages. What molecular technologies do not rely on the PCR reaction?

5. Ramunas Kondratas, curator at the Smithsonian’s National Museum of American History, documented the discovery, development, commercialization, and applications of PCR technology. Three sessions were recorded May 14 and May 15, 1992 at Emeryville, California; September 25, 1992 at Alameda, California; and February 25, 1993 at Norwalk, Connecticut. What they mainly included?