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Chapter 1 : Nucleic Acid Amplification Methods Overview

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Nucleic Acid Amplification Methods Overview, Page 1 of 2

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Abstract:

The development of the polymerase chain reaction, or PCR, by Saiki et al. (1) was a milestone in biotechnology and heralded the beginning of the modern era of molecular diagnostics. Although PCR is the most widely used nucleic acid amplification strategy, other strategies have been developed, and several have clinical utility. These strategies are based on either signal or target amplification. Examples of each category will be discussed in the sections that follow. These techniques have sensitivity unparalleled in laboratory medicine, have created new opportunities for the clinical laboratory to impact patient care, and have become the new “gold standards” for laboratory diagnosis of many infectious diseases.

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 1
FIGURE 1

Branched DNA signal amplification. Reprinted with permission from reference .

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 2
FIGURE 2

Hybrid capture signal amplification. Reprinted with permission from reference .

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 3
FIGURE 3

Cleavase invader signal amplification. Reprinted with permission from reference .

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 4
FIGURE 4

The PCR cycle. The initial template DNA is first denatured by heat. The reaction is then cooled to anneal two oligonucleotide primers to opposite strands with their 3′ ends pointed inward. A polymerase then extends each primed template to double the amount of targeted DNA. The cycle is repeated 20 to 40 times through successive steps of denaturation, annealing, and extension, accumulating double-stranded PCR products. Reprinted with permission from reference .

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 5
FIGURE 5

Visualization of PCR kinetics. The three phases of PCR (denaturation, annealing, and extension) occur as the temperature is continuously changing (A). Toward the end of PCR the reaction contains single- and double-stranded PCR products. When different points of the cycle are sampled (by snap-cooling the mixture in ice water) (B) and analyzed, the transition from denatured single-stranded DNA to double-stranded DNA is revealed as a continuum (C). Progression of the extension reaction can be followed by additional bands appearing between the single- and double-stranded DNA (time points 5 to 7). Modified with permission from reference .

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 6
FIGURE 6

Model exponential and logistic curves for bacterial growth and PCR. Doubling times of 20 min and 30 s are assumed for bacteria and PCR, respectively. That is, given the equation , is 0.0347 min for bacteria and 1.386 min for PCR. The carrying capacity for bacteria was set at 10/ml. Assuming that PCR is primer limited at one-third the primer concentration ( Table 1 ), a carrying capacity of 10 copies of PCR product/10 µl was used. The shapes of the curves for bacteria and DNA are identical, with only the axis scales specific to each method. Starting with a single bacterium, growth plateaus after 11 to 12 h, while PCR takes only 23 min (46 cycles) to amplify a single copy to saturation.

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 7
FIGURE 7

Real-time PCR with melting analysis. Detection and quantification are enabled by monitoring fluorescence once each cycle at the end of extension (solid squares). Amplification is immediately followed by melting-curve acquisition. Melting-curve analysis identifies PCR products, microbial strains and sequence alterations by melting temperature. The original melting-curve data (solid line) can also be plotted as a derivative melting curve (dotted line). Reprinted from reference with permission from the American Society of Investigative Pathology and the Association for Molecular Pathology.

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 8
FIGURE 8

Common probes and dyes for real-time PCR. The green lightning bolt is the excitation light. The green circles are fluorophores, the dark red circles are quenchers, and the black circles are dark quenchers. The large hungry gray circle is a polymerase with 5′ to 3′ exonuclease activity. The thin black ovals are blockers, and the orange sausages are minor groove binders. (A) Double-stranded DNA dyes show a significant increase in fluorescence when bound to DNA. (B) Hydrolysis probes are cleaved between a fluorescent reporter and a quencher, resulting in increased fluorescence. (C) Dual hybridization probes change color by resonance energy transfer when hybridized. (D) The molecular beacon hairpin quenches fluorescence until target binding that separates the quencher from the flourophore. (E) Scorpion primers are quenched in the native conformation but increase in fluorescence when the original hairpin loop is hybridized to its extension product. (F) Dark quencher probes are initially quenched by a minor groove binder and the dark quencher. Hybridization to the target releases the fluorescence. (G) The short strand of partially double-stranded probes is displaced in the presence of target, releasing fluorescence from quenching.

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 9
FIGURE 9

Typical real-time PCR amplifications monitored with SYBR Green I, hydrolysis probes, and hybridization probes. Both once-per-cycle and continuously monitored displays are shown. Note the hybridization information inherent in reactions monitored with SYBR Green I and hybridization probes.

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 10
FIGURE 10

Variant typing by melting analysis. Primer and probe designs are shown on the left with typical data on the right. Dual (A) and single (B) hybridization probes use covalent fluorescent labels (asterisks), and typing is solely derived from the probe signal. Single hybridization probes discussed here include molecular beacons, scorpion primers, dark quencher probes, and partially double-stranded probes. Unlabeled probes (C) and snapback primers (D) require no covalent labels because fluorescence is provided by a dye that binds to dsDNA. With unlabeled probes and snapback primers, both probe and PCR product melting transitions are observed and can contribute to typing. Any free 3′ ends on the probes are terminated with a phosphate (Pi) or other blocker to prevent probe extension by the polymerase. The snapback primer (D) incorporates an unlabeled probe into the 5′ end of one primer, generating a self-probing amplicon that forms a hairpin. In panel E, no probe is present, but typing of the PCR product is still possible by high-resolution melting. High-resolution melting identifies heterozygotes by a change in curve shape and distinguishes homozygotes by .

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 11
FIGURE 11

Transcription-based target amplification. NASBA and TMA are examples of transcription-based amplification systems. Reprinted with permission from reference .

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Image of FIGURE 12
FIGURE 12

Strand displacement target amplification. The process is shown for only one strand of a double-stranded DNA target, but amplification occurs on both strands simultaneously. Reprinted with permission from reference .

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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FIGURE 13

(a) Primer design of the LAMP reaction. For ease of explanation, six distinct regions are designated on the target DNA, labeled F3, F2, F1, B1c, B2c, and B3 from the 5′ end. Because c represents a complementary sequence, the F1c sequence is complementary to the F1 sequence. Two inner primers (FIP and BIP) and outer primers (F3 and B3) are used in the LAMP method. FIP (BIP) is a hybrid primer consisting of the F1c (B1c) sequence and the F2 (B2) sequence. (b) Starting structure producing step. DNA synthesis initiated from FIP proceeds as follows. The F2 region anneals to the F2c region on the target DNA and initiates the elongation. DNA amplification proceeds with BIP in a similar manner. The F3 primer anneals to the F3c region on the target DNA, and strand displacement DNA synthesis takes place. The DNA strand elongated from FIP is replaced and released. The released single strand forms a loop structure at its 3′ end (structure 3). DNA synthesis proceeds with the single-strand DNA as the template, and BIP and B3 primer, in the same manner as described earlier, to generate structure 5, which possesses the loop structure at both ends (dumbbell-like structure). (c) Cycling amplification step. Using self-structure as the template, self-primed DNA synthesis is initiated from the 3′ end F1 region, and the elongation starts from FIP annealing to the single strand of the F2c region in the loop structure. Passing through several steps, structure 7 is generated, which is complementary to structure 5, and structure 5 is produced from structure 8 in a reaction similar to that which led from structures 5 to 7. Structures 9 and 10 are produced from structures 6 and 8, respectively, and more elongated structures ( ) are also produced. Reprinted with permission from reference .

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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FIGURE 14

HDA amplifies target sequences using two sequence-specific primers flanking the fragment to be amplified and a mixture of enzymes for DNA strand separation and polymerization. In the first step of the HDA reaction, the helicase enzyme loads on to the template and traverses along the target DNA, disrupting the hydrogen bonds linking the two strands. Exposure of the single-stranded target region by helicase allows primers to anneal. The DNA polymerase then extends the 3′ ends of each primer using free deoxynucleotides (dNTPs) to produce two DNA replicates. The two replicated DNAs independently enter the next cycle of HDA, resulting in exponential amplification of the target sequence. Reprinted from http://www.biohelix.com/HDA_mechanism.asp.

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1
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Tables

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TABLE 1

Typical reactant amounts in PCR (10-μl reaction mixture)

Citation: Nolte F, Wittwer C. 2016. Nucleic Acid Amplification Methods Overview, p 3-18. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch1

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