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The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation; Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
Helicase-dependent amplification (HDA) is a method for in vitro DNA amplification that takes place at a constant temperature.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively.
Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses also replicate their DNA or RNA via the rolling circle mechanism.
The overlap extension polymerase chain reaction is a variant of PCR. It is also referred to as Splicing by overlap extension / Splicing by overhang extension (SOE) PCR. It is used assemble multiple smaller double stranded DNA fragments into a larger DNA sequence. OE-PCR is widely used to insert mutations at specific points in a sequence or to assemble custom DNA sequence from smaller DNA fragments into a larger polynucleotide.
Systematic evolution of ligands by exponential enrichment (SELEX), also referred to as in vitro selection or in vitro evolution, is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. These single-stranded DNA or RNA are commonly referred to as aptamers. Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB and CASTing SELEX was first introduced in 1990. In 2015, a special issue was published in the Journal of Molecular Evolution in the honor of quarter century of the discovery of SELEX.
SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. An SNP is a single base pair mutation at a specific locus, usually consisting of two alleles. SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.
Bisulfitesequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity.
The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization which have been developed by molecular biologists to improve PCR performance and minimize failure.
The versatility of polymerase chain reaction (PCR) has led to modifications of the basic protocol being used in a large number of variant techniques designed for various purposes. This article summarizes many of the most common variations currently or formerly used in molecular biology laboratories; familiarity with the fundamental premise by which PCR works and corresponding terms and concepts is necessary for understanding these variant techniques.
The ligase chain reaction (LCR) is a method of DNA amplification. The ligase chain reaction (LCR) is an amplification process that differs from PCR in that it involves a thermostable ligase to join two probes or other molecules together which can then be amplified by standard polymerase chain reaction (PCR) cycling. Each cycle results in a doubling of the target nucleic acid molecule. A key advantage of LCR is greater specificity as compared to PCR. Thus, LCR requires two completely different enzymes to operate properly: ligase, to join probe molecules together, and a thermostable polymerase to amplify those molecules involved in successful ligation. The probes involved in the ligation are designed such that the 5′ end of one probe is directly adjacent to the 3′ end of the other probe, thereby providing the requisite 3′-OH and 5′-PO4 group substrates for the ligase.
High Resolution Melt (HRM) analysis is a powerful technique in molecular biology for the detection of mutations, polymorphisms and epigenetic differences in double-stranded DNA samples. It was discovered and developed by Idaho Technology and the University of Utah. It has advantages over other genotyping technologies, namely:
Multiple displacement amplification (MDA) is a DNA amplification technique. This method can rapidly amplify minute amounts of DNA samples to a reasonable quantity for genomic analysis. The reaction starts by annealing random hexamer primers to the template: DNA synthesis is carried out by a high fidelity enzyme, preferentially Φ29 DNA polymerase. Compared with conventional PCR amplification techniques, MDA does not employ sequence-specific primers but amplifies all DNA, generates larger-sized products with a lower error frequency, and works at a constant temperature. MDA has been actively used in whole genome amplification (WGA) and is a promising method for application to single cell genome sequencing and sequencing-based genetic studies.
A primer dimer (PD) is a potential by-product in the polymerase chain reaction (PCR), a common biotechnological method. As its name implies, a PD consists of two primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PDs may interfere with accurate quantification.
Hot start PCR is a modified form of conventional polymerase chain reaction (PCR) that reduces the presence of undesired products and primer dimers due to non-specific DNA amplification at room temperatures. Many variations and modifications of the PCR procedure have been developed in order to achieve higher yields; hot start PCR is one of them. Hot start PCR follows the same principles as the conventional PCR - in that it uses DNA polymerase to synthesise DNA from a single stranded template. However, it utilizes additional heating and separation methods, such as inactivating or inhibiting the binding of Taq polymerase and late addition of Taq polymerase, to increase product yield as well as provide a higher specificity and sensitivity. Non-specific binding and priming or formation of primer dimers are minimized by completing the reaction mix after denaturation. Some ways to complete reaction mixes at high temperatures involve modifications that block DNA polymerase activity in low temperatures, use of modified deoxyribonucleotide triphosphates (dNTPs), and the physical addition of one of the essential reagents after denaturation.
Massive parallel sequencing or massively parallel sequencing is any of several high-throughput approaches to DNA sequencing using the concept of massively parallel processing; it is also called next-generation sequencing (NGS) or second-generation sequencing. Some of these technologies emerged between 1993 and 1998 and have been commercially available since 2005. These technologies use miniaturized and parallelized platforms for sequencing of 1 million to 43 billion short reads per instrument run.
Multiple Annealing and Looping Based Amplification Cycles (MALBAC) is a quasilinear whole genome amplification method. Unlike conventional DNA amplification methods that are non-linear or exponential, MALBAC utilizes special primers that allow amplicons to have complementary ends and therefore to loop, preventing DNA from being copied exponentially. This results in amplification of only the original genomic DNA and therefore reduces amplification bias. MALBAC is “used to create overlapped shotgun amplicons covering most of the genome”. For next generation sequencing, MALBAC is followed by regular PCR which is used to further amplify amplicons.
Recombinase polymerase amplification (RPA) is a single tube, isothermal alternative to the polymerase chain reaction (PCR). By adding a reverse transcriptase enzyme to an RPA reaction it can detect RNA as well as DNA, without the need for a separate step to produce cDNA,. Because it is isothermal, RPA can use much simpler equipment than PCR, which requires a thermal cycler. Operating best at temperatures of 37–42 °C and still working, albeit more slowly, at room temperature means RPA reactions can in theory be run quickly simply by holding a tube. This makes RPA an excellent candidate for developing low-cost, rapid, point-of-care molecular tests. An international quality assessment of molecular detection of Rift Valley fever virus performed as well as the best RT-PCR tests, detecting less concentrated samples missed by some PCR tests and an RT-LAMP test. RPA was developed and launched by TwistDx Ltd., a biotechnology company based in Cambridge, UK.
Duplex sequencing is a library preparation and analysis method for next-generation sequencing (NGS) platforms that employs random tagging of double-stranded DNA to detect mutations with higher accuracy and lower error rates.
References
- 1 2 Citartan, Marimuthu; et al. (December 2011). "Asymmetric PCR for good quality ssDNA generation towards DNA aptamer production" (PDF). Songklanakarin J. Sci. Technol. 34 (2): 125–131. Archived from the original (PDF) on 2020-01-11. Retrieved 2017-11-18.
- ↑ Wooddell, C I; Burgess, R R (1996). "Use of Asymmetric PCR to Generate Long Primers and Single-stranded DNA for Incorporating Cross-linking Analogs into Specific Sites in a DNA Probe". Genome Res. 6 (9): 886–892. doi: 10.1101/gr.6.9.886 . PMID 8889557.
- ↑ Heiat, Mohammad; et al. (14 January 2017). "Essential strategies to optimize asymmetric PCR conditions as a reliable method to generate large amount of ssDNA aptamers". Biotechnology and Applied Biochemistry. 64 (4): 541–548. doi:10.1002/bab.1507. PMID 27222205. S2CID 21792777.
- ↑ Sanchez, J. Aquiles; et al. (4 December 2003). "Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis". Proceedings of the National Academy of Sciences. 101 (7): 1933–1938. doi: 10.1073/pnas.0305476101 . PMC 357030 . PMID 14769930.