Amplifying DNA: The Polymerase Chain Reaction

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Chapter: Pharmaceutical Microbiology : Recombinant DNA Technology

The polymerase chain reaction (PCR) is an extremely simple and powerful technique that was perfected by Kary Mullis in the mid-1980s and has revolutionized many studies in molecular biology, currently having applications ranging from forensic studies to the development of new recombinant drugs.


AMPLIFYING DNA: THE POLYMERASE CHAIN REACTION

 

The polymerase chain reaction (PCR) is an extremely simple and powerful technique that was perfected by Kary Mullis in the mid-1980s and has revolutionized many studies in molecular biology, currently having applications ranging from forensic studies to the development of new recombinant drugs. This technique allows the generation of large amounts of copies of a specified DNA sequence from a single DNA molecule without the need for cloning.

 

The PCR exploits certain characteristics of DNA replication, as it uses single-stranded DNA (ss DNA) as a template for the synthesis of complementary new strands in a 5′ to 3′ direction. The ss DNA templates can be generated by heating ds DNA above 90 °C. DNA polymerase synthesizes ds DNA by extending the complementary strand of a template. Hence the DNA polymerase can be directed to synthesize a specific region of DNA by using a synthetic, complementary oligonucleotide primer that will anneal to the template when the temperature is lowered. The PCR reaction uses a special DNA polymerase (Taq DNA polymerase from the thermophilic bacterium Thermus aquaticus) that can withstand temperatures above 100 °C and has an optimal activity at 72 °C, which has the advantage of reducing non-specific primer annealing that may occur at lower temperatures.

 

In PCR both strands of a target DNA serve simultaneously as templates upon the addition of a pair of primers, one for each strand of DNA. A typical PCR amplification is shown in Figure 25.7. Every PCR cycle is normally repeated up to 30 times. The net result of a PCR is that, at the end of n cycles, it will generate 2n ds DNA copies of a single DNA fragment located between the two primers.



 


A)    Advantages And Limitations Of PCR

 

There are some obvious advantages of using PCR. The main one is specificity, as it allows, using the appropriate primers, the amplification of specific DNA fragments from a population of different cells. It is also a very rapid technique, as it only takes a few hours to amplify a fragment of DNA compared with days using conventional cloning methods. An important feature of PCR is its versatility, as it allows the incorporation of mismatches on the 5′ end of the primers provided that the 3′ end has perfect complementary with the targeted strand. This can be exploited to add restriction sites to enable subsequent cloning of the amplified DNA, or introducing specific mutations into genes. Furthermore, the equipment used for PCR is relatively inexpensive and allows the analysis of a large number of samples at one time. Finally, PCR does not require purified template DNA and can amplify genes from whole cells or tissue samples.

 

However, there are also a number of limitations to the use of PCR. The designing of primers for this technique requires some knowledge of the DNA sequence to be amplified. Although there are new genetically engineered DNA polymerases that can synthesize large fragments of DNA, there are still some restrictions with regards to the maximum length of DNA that can be amplified. Ideally, fragments of 0.1–3 kb can be easily amplified although this technique, under the appropriate conditions, would amplify larger fragments (up to 20 kb). In addition, the slightest sample contamination can lead to false positive results, which can have detrimental effects when this technique is used in diagnostics. Finally, sometimes there is a risk of non-specific amplification when the primers anneal to sequences similar to the targets, leading to the amplification of the wrong DNA.

 

B)  Clinical Applications Of PCR

 

The development of PCR has revolutionized not only basic research but also different areas of medicine. Table 25.4 lists some of the most important clinical applications of PCR. These types of analysis were practically impossible without PCR owing to the large amount of samples that needed handling, the amount of time required to obtain results or the lack of sensitivity of the available tests.

 

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