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Chapter 11 : Nucleic Acid Amplification by Polymerase Chain Reaction

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Nucleic Acid Amplification by Polymerase Chain Reaction, Page 1 of 2

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Abstract:

Nucleic acid detection methods have been rapidly evolving and play an important role in viral detection and quantification. One technology which emerged in the 1990s, polymerase chain reaction, better known as PCR, has established itself as a primary tool for molecular biology. In fact, this technology has been so widely adopted in the clinical virology laboratory it has, in many cases, completely replaced culture. At its core, PCR is a straightforward chemical reaction whereby one strand of template DNA is exponentially amplified. This chapter will give an overview of different PCR technologies available, as well as the strength and weaknesses of each. For a more in-depth review of their applications in the clinical laboratory, the reader is directed to the appropriate chapter(s) elsewhere in this text.

Citation: Cárdenas A, Alby K. 2016. Nucleic Acid Amplification by Polymerase Chain Reaction, p 129-136. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch11
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Figures

Image of FIGURE 1
FIGURE 1

Conventional PCR. Start at top, the dsDNA strands are heated causing them to denature. As the solution cools, the two primers anneal to opposite strands of the target DNA (right). The DNA polymerase then extends the primers creating new DNA strands complementary to the target sequence (bottom). The old and new strands serve as templates for further DNA synthesis during the next cycle. dNTPs, deoxynucleoside triphosphates.

Citation: Cárdenas A, Alby K. 2016. Nucleic Acid Amplification by Polymerase Chain Reaction, p 129-136. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch11
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Image of FIGURE 2
FIGURE 2

Nested PCR. The first primer set is used to amplify the target DNA. The product of this first PCR is diluted and serves as the template for a second PCR reaction. The primers of the second reaction are “nested” more centrally than those of the first reaction. If the first reaction amplified the specific target of interest, the second set of primers anneal and generate a new product (A). If the first reaction generated a nonspecific amplicon, the second set of primers will not anneal and no product will be formed (B).

Citation: Cárdenas A, Alby K. 2016. Nucleic Acid Amplification by Polymerase Chain Reaction, p 129-136. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch11
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Image of FIGURE 3
FIGURE 3

TaqMan probes. As the upstream primer is extended, the DNA polymerase (black oval) reaches the probe, which is displaced and digested by the 5′ to 3′ nuclease activity of the polymerase. Once the reporter and quencher molecules are separated an increase in fluorescence is observed from the reporter molecule.

Citation: Cárdenas A, Alby K. 2016. Nucleic Acid Amplification by Polymerase Chain Reaction, p 129-136. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch11
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Image of FIGURE 4
FIGURE 4

Molecular beacon and Scorpion molecules. Molecular beacons are stem cell loop structures where the ends are self-complementary and the center portion is complementary to the target sequence. In the presence of a target sequence, the reporter and quencher molecules are separated and fluorescence is detected (A). Scorpion molecules are also stem-cell loop structures but have a primer added to their 5′ end. After initial extension of the target sequence, the loop unfolds and the loop-region of the probe hybridizes to the newly synthesized target sequence. Since the reporter is no longer in close proximity to the quencher, fluorescence emission takes place (B).

Citation: Cárdenas A, Alby K. 2016. Nucleic Acid Amplification by Polymerase Chain Reaction, p 129-136. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch11
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Image of FIGURE 5
FIGURE 5

Digital PCR. A sample of nucleic acid containing the target sequence (red) is partitioned into many individual cells. The partitions containing the target sequence are amplified causing signal to accumulate. The fraction of positive partitions is determined and the target concentration estimated.

Citation: Cárdenas A, Alby K. 2016. Nucleic Acid Amplification by Polymerase Chain Reaction, p 129-136. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch11
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Tables

Generic image for table
TABLE 1

Comparison of different PCR techniques

Citation: Cárdenas A, Alby K. 2016. Nucleic Acid Amplification by Polymerase Chain Reaction, p 129-136. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch11

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