Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a popular technique used widely to amplify a single or a few copies of specific DNA sequences present in a heterogeneous population to millions of copies with great accuracy in a short time, making possible to detect even minute concentrations of DNA in clinical samples.

From: Advances in Colloid and Interface Science , 2017

POLYMERASE CHAIN REACTION

J.M. WagesJr., in Encyclopedia of Analytical Science (Second Edition), 2005

Introduction

Polymerase chain reaction (PCR) is a technology for exponential amplification of a fragment of DNA. (The PCR is covered by patents owned by Hoffman-La Roche. A license is required to use the PCR process.) The limit of its sensitivity is a single molecule, making PCR a superb qualitative tool for the specific detection of rare DNA sequences. Under proper conditions, the yield of amplified DNA is proportional to the initial number of target molecules, rendering it a quantitative analytical tool as well. Since its original description in 1985, PCR has evolved into an assemblage of varied methodologies almost universally used in basic biological research, biotechnology, clinical research, clinical diagnostics, forensics, food technology, environmental testing, archaeology and anthropology, and other fields. Even though other nucleic acid amplification technologies have been described, PCR remains by far the most widely used.

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Assays for determining cell differentiation in biomaterials

J.A. CooperJr., ... W-J. Li , in Characterization of Biomaterials, 2013

4.2.4 PCR array

PCR array can be viewed as a small-scaled gene microarray with fewer probes. The PCR array technology takes advantage of high throughput screening of microarray and precision of quantitative real-time PCR to detect expression of a large number of genes simultaneously using the highly sensitive real-time PCR technique (SABiosciences, 2010; Chen et al., 2009). The benefit of using PCR array over gene microarray for gene analysis is that PCR array allows researchers to focus on a manageable number of genes and to save an additional procedure of real-time PCR verification. For example, a PCR array that is available in a 96- or 384-well format which focuses on signaling molecules of growth factor-activated pathways can be used to analyze gene expression of regulator molecules during stem cell differentiation. Various PCR arrays for analysis of different cell activities have been developed. To obtain the best analytical result, PCR arrays also follow the same principles as standard real-time PCR to optimize PCR performance (SABiosciences, 2008). For example, PCR efficiency should be between 90% and 110%. Array data can be analyzed by the ΔΔCT method (see section 'Data analysis') or commercial software provided by array manufacturers.

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DEOXYRIBONUCLEIC ACID PROFILING | Capillary Electrophoresis

M. Chiari , L. Ceriotti , in Encyclopedia of Separation Science, 2000

Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a molecular copying process that allows the amplification of the quantity of DNA available for a given test. Using a three-step temperature cycle, PCR allows specific regions of DNA to be amplified to a detectable level. The analysis of PCR products requires the ability to separate the target sequence from nonspecific products that may result from the use of nonoptimized conditions or from overamplification. CE has been successfully used for quantification and sizing of amplified products. Given its small sample requirements, CE can detect PCR products at a low cycle number using LIF, providing an efficient tool to evaluate PCR reaction parameters. Many of the separations which can be carried out on a slab gel have been successfully performed in CE. Some of the most significant papers on separation of PCR products by CE are reported in Table 2.

Table 2. Application of PCR product analysis by CE

Type System examined PCR product size (bp) (resolution needed)
Diagnostic Androgen insensitivity syndrome 136, 139, 160 (3   bp)
Diagnostic Congenital adrenal hyperplasia 127, 135 (8   bp)
Diagnostic Cystic fibrosis ΔF508 95, 98 (3   bp)
Diagnostic Cystic fibrosis, GATT microsatellites (for linkage) 111, 115 (4   bp)
Diagnostic Down's syndrome, D21S11 220, 224 (4   bp)
Diagnostic Duchenne and Becker muscular dystrophy (18) exon 88, 547 (3   bp)
Diagnostic Dystrophin: DXS 164 locus 740   bp→220, 520   bp
Diagnostic ERBB2 oncogene 1.1 kb   bp→500, 520   bp
Diagnostic Factor V mutation 115, 138, 202
Diagnostic Hepatitis C virus 380, 187, 289
Diagnostic HIV-1 virus (gag gene) 115
gag, pol and env genes 142, 394, 442
Diagnostic Kennedy's disease 480, 540
Diagnostic Medium-chain acyl-coenzyme A dehydrogenase deficiency 175, 202
Diagnostic (SSCP) p53   gene mutation clusters A and B 372
Diagnostic Polio virus 53, 71, 97, 163
Diagnostic VNTR: aboB locus 600–1000 (16   bp)
Diagnostic ZFY gene, Y-chromosome 307
Forensic HLA-DQa 242
Forensic Mitochondrial DNA 402, 437, 1021
Forensic STR: HUMTH01 locus 179–203 (4   bp)
Forensic VNTR: D1S80 401–801 (16   bp)
Genotyping Soybean plant simple sequence repeats (SSRs) 401–801 (16   bp)

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Nucleic Acid Techniques

Carl T. Wittwer , G. Mike Makrigiorgos , in Principles and Applications of Molecular Diagnostics, 2018

Multiplex Polymerase Chain Reaction and Nested Polymerase Chain Reaction

Multiplex PCR refers to amplifying more than one target simultaneously in the same solution. This can be done by using consensus primers that bind to and amplify more than one bacterial species, for example, ribosomal sequences with high variation internal to the primers, but flanking identical sequence that can be used for consensus primer binding. Usually, however, multiple primer sets are designed for multiple targets and amplified in the same solution at the same time. Although potential primer interactions increase exponentially with multiplex PCR, it works surprising well most of the time as long as the number of PCR cycles is kept low. If lower primer concentrations are used, the annealing time may need to be increased to maintain efficient annealing. Multiplex PCR is often called "preamplification" if it is followed by an additional amplification reaction.

If PCR is performed with a pair of outer primers and then that PCR product is amplified yet again with a set of inner primers, this is referred to as nested PCR. Typically, the first round PCR product is diluted 1000 to 1 million times before the inner (nested) primers are added. The advantage of nested PCR is that both sensitivity and specificity increase. The disadvantage is increased potential for contamination, particularly if the first round of PCR products is handled manually for dilution and transfer. Multiplex PCR followed by nested PCR has been used in a closed-tube system for multiplex detection of infectious agents. 41

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DNA and Aspects of Molecular Biology

Dipanakar Sen , in Comprehensive Natural Products Chemistry, 1999

7.17.3.2 The Polymerase Chain Reaction

The polymerase chain reaction (PCR) is a relatively recent technology that has had a wide impact on numerous aspects of molecular biology. PCR is, broadly, a means for the amplification of DNA, and its development is central to the subsequent development of SELEX. There are numerous excellent reviews (as well as an ever-increasing number of specialist books) on the methods and uses of PCR, including recent reviews by Arnheim and Erlich 17 and Mullis and Falona. 18 the technical aspects of PCR are covered thoroughly in Section 15 of Current Protocols of Molecular Biology. 16 PCR, as used in SELEX, not only provides a means for the amplification of DNA, but is also useful for introducing mutations into DNA pools (PCR mutagenesis is discussed in Section 7.17.3.5).

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Scientific Fundamentals of Biotechnology

O.E. Tolmachov , in Comprehensive Biotechnology (Second Edition), 2011

1.06.12 Particular Features of Cloning of PCR Amplicons

PCR and LCR are in vitro methods of DNA amplification. There are two types of DNA polymerases used in these chain reactions. Thermostable Taq polymerase is known to introduce occasional mutations to the amplified DNA; it also transfers an additional A to the 3′-ends of the amplicons. Thermostable proofreading polymerases (e.g., Pfu, Pfx, and Pwo) possess correcting 3′–5′ exonucleolytic activity and, therefore, introduce mutations with a much lower frequency; these polymerases generate amplicons with blunt DNA ends. LCR employs both thermostable DNA polymerases and thermostable DNA ligases. PCR is a standard method of choice for in vitro DNA amplification, whereas LCR is a specialized method used only when extra-high template selectivity is required.

The reaction conditions for PCR often require optimization for a particular amplicon and a particular set of primers. The important parameters include Mg2+ concentration and cycling time and temperature regiment. The choice of PCR annealing temperature is particularly essential; close melting temperature for both PCR primers is normally desirable. Taq-polymerase is normally used to catalyze reaction at 72   °C, whereas thermostable proofreading polymerases have lower optimal temperature and are normally used to catalyze reactions at 68   °C. The success of PCR can depend on the primers' concentration and the choice of a particular thermostable polymerase. Specificity of PCR can be increased when thermosensitive polymerase–antibody complexes are used to initiate hot-start PCR. In this scenario, polymerases become active only at the denaturation temperature (about 95   °C) and initial low-temperature mis-priming events are minimized. Sometimes, PCR specificity and amplicon yield can be dramatically improved with PCR enhancer substances such as betaine, DMSO, dithiotreitol, β-mercaptoethanol, formamide, and their cocktails. Longer PCR amplicons require more time to be synthesized and require more rigorous optimization. Cocktails of different proofreading polymerases are known to allow the amplification of the particularly long templates. Additional internal primers can be added to the reaction, increasing reaction specificity and the attainable amplicon size; this version of PCR is called 'assembly PCR'.

Once PCR amplicon is generated, it needs to be purified before the ligation with vector DNA. Contaminants can be removed by purification on a microcolumn. As this step normally results in unacceptable DNA dilution, the PCR amplicon should then be concentrated, for example, through ethanol–ammonium acetate precipitation. Acetate salts are used to create high salt conditions as acetates are well dissolved in ethanol–water mixtures and are not likely to precipitate with DNA.

A single nucleotide overhang generated by Taq polymerase, which transfers an additional A to each of the 3′ ends of its PCR products, can be exploited for a simplified ligation to vector DNA. The procedure is known as 'TA cloning'. The vector fragment is engineered to contain a single nucleotide 3′ overhang T, normally using restriction enzymes AhdI [29] or XcmI [30]. Another approach for the cloning of Taq-polymerase-generated PCR products is blunt ending the product with its subsequent ligation between the blunt ends of the cloning vector, perhaps combined with a restriction digestion of the ligation mixture as described in Section 1.06.10. A similar approach can be used for PCR products generated with proofreading DNA polymerases; in this case, the DNA fragments ends are already blunt. TOPO cloning system, which relies on vaccinia virus DNA-topoisomerase for DNA fragment joining, can be applied to both TA cloning and blunt-end cloning of PCR products. An additional cloning strategy is to include suitable recognition sequences into the PCR primers in order to engineer flanking restriction enzyme sites into the PCR product. When following this strategy, it is important to add some extra nucleotides to the primer sequences at their 5′ ends to ensure the efficient digestion of the flanking restriction sites by restriction endonucleases.

Finally, PCR primers can be designed to supply a PCR product with long single-stranded overhangs matching the vector sequence. In this method, one uracil nucleotide is included in both the forward and reverse PCR primers, so that long single-stranded overhangs can be created in the PCR amplicon through the removal of the uracil residues with a mixture of uracil DNA glycosylase and endonuclease VIII. Complementary long single-stranded overhangs in the vector DNA are obtained via restriction enzyme digestion and site-specific nicking. After annealing the insert and vector DNA, no in vitro ligation is required and the annealing mixture is used to transform bacteria [31].

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Microfluidic devices for DNA amplification

Ali Shahid , ... P. Ravi Selvaganapathy , in Bioelectronics and Medical Devices, 2019

Polymerase chain reaction

PCR was the first DNA amplification method, developed in the early 1980s (Mullis et al., 1986). PCR is a well-known and widely used DNA amplification method due to its simple operation. PCR exponentially amplifies DNA molecules from a single copy to several millions of copies of DNA with a few repetitive temperature cycles. Every temperature cycle of PCR consists of three steps: denaturation, annealing, and extension, as shown in Fig. 28.1.

Figure 28.1. Reaction sequence of polymerase chain reaction. (A) Denaturation (95°C). (B) Annealing (50°C–70°C). (C) Extension (~72°C).

A double strand of DNA is split to form two single-strained DNA molecules at the elevated temperature of ~95°C (Fig. 28.1A) during the denaturation step. Then, the annealing step (Fig. 28.1B) is performed at the lowered temperature (~50°C–70°C). During the annealing process, small complementary synthetic single-stranded DNA molecules known as primers are attached to the specific defined site of the single-stranded DNA molecule. Finally, in the extension step (Fig. 28.1C), which occurs at 72°C, the polymerase enzyme copies the single-stranded DNA in between the two primer locations. DNA molecules exponentially increase when exposed to this temperature cycle repeatedly. Conventional PCR process is straightforward, but it requires significant instrumentation to obtain the different temperatures in each stage and to monitor and control them. Also, it takes longer to complete the amplification reaction. The microfluidic technology was used to establish PCR systems to overcome the limitations of conventional PCR systems.

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Global Perspective

J.K. Osbourn , in Comprehensive Medicinal Chemistry II, 2007

1.10.2.6 Sexual Polymerase Chain Reaction

PCR was invented in the 1980s by Kary Mullis and colleagues 28 and represented a major breakthrough in the field of molecular biology. PCR enables large amounts of DNA to be produced from short segments.

It is possible that novel proteins, with different properties and characteristics and potential therapeutic applications, may be constructed from the starting point of naturally occurring DNA. This can be achieved by amplifying segments of DNA using the PCR technique, but with the introduction of deliberate variation in the order of segments, resulting in changes to the sequence of amino acids. This technique was first called 'sexual PCR' by Willem Stemmer. 29 A biological target is identified and its genes and related genes are isolated and 'shuffled' into novel combinations via sexual PCR. The process is repeated until a whole library of novel genes is created with potentially improved characteristics from the parental clone. The novel genes are expressed as proteins, which are then screened for desirable therapeutic properties and functions. In essence, this technique speeds up genetic evolution by testing new combinations of DNA segments. 30

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Mass Spectrometry Applications in Infectious Disease and Pathogens Identification

Phillip Heaton , Robin Patel , in Principles and Applications of Clinical Mass Spectrometry, 2018

Limitations and Future Perspectives

PCR-ESI-MS remains an emerging technology for the clinical microbiology laboratory and, although CE marked for in vitro diagnostic use in Europe, the technology is not currently available in the United States. The platform can be used for detecting bacteria in blood, sterile fluids, BAL fluids, ETA, and soft tissues as well as fungi in BAL fluids and from isolates, and for detecting viruses in plasma. The studies mentioned in this chapter have shown the potential utility of this platform because it may provide direct-from-specimen results with a faster turnaround time than culture combined with subsequent MALDI-TOF-MS. However, it remains to be determined whether the improvements in the platform overcome the problems with ease-of-use and throughput that have been observed with previous renditions of the technology. The PLEX-ID can hold 15 plates, limiting the number of patient specimens that can be tested in a batch. Although PCR-ESI-MS can detect polymicrobial infections, this remains an area where improvement is needed. Also, although the sensitivity of the platform is a strength, it remains an open system, so strict adherence to a unidirectional workflow is imperative to prevent amplified DNA contamination. Finally, this technology will most likely be limited to large laboratories because of the high costs of reagents and instrumentation, although the latest redesign of the platform may lower the costs. Laboratories that adopt this technology must decide which patients will benefit most from this testing. PCR-ESI-MS may have the potential to affect patient care by reducing lengths of stay, and/or time to initiate targeted therapy, thus improving antimicrobial stewardship; however, outcome-based studies are needed.

Points to Remember

PCR-ESI-MS

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PCR-ESI-MS couples PCR to TOF-MS to detect a wide range of pathogens and some antimicrobial resistance genes.

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PCR-ESI-MS allows testing directly from patient samples.

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PCR-ESI-MS is capable of providing semi-quantitative data.

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PCR-ESI-MS is not reliant on viable organisms being present.

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PCR-ESI-MS is an open system requiring strict adherence to unidirectional workflow.

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Multiplexed, lateral flow, polymerase chain reaction (PCR) techniques for biological identification

W.M. Nelson , ... L.M. Cockrell , in Biological Identification, 2014

2.3.3 Reverse transcription real-time PCR

Although PCR requires DNA as a template, expansion of the protocol to include a reverse transcription step allows the user to start with an RNA template that is subsequently converted to a DNA sequence, which then becomes the template for the PCR reaction. In this variation of PCR, the RNA template is converted into a complementary DNA (cDNA) sequence by a reverse transcriptase enzyme. This step typically requires the addition of a long (30–60   min) incubation step at a relatively low (37–55   °C) temperature before the PCR thermal cycling begins. However, newer and more robust enzymatic formulations now offer increased speed, allowing this time to be shortened.

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