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Cancer Informatics
Published in Trevor F. Cox, Medical Statistics for Cancer Studies, 2022
To amplify DNA using PCR, a segment of DNA is placed in solution containing primers (short sequence of single stranded DNA), free DNA nucleotides, DNA polymerase (an enzyme that catalyses the synthesis of DNA molecules) and other ingredients. Figure 10.1 illustrates the process where a target section of DNA is to be amplified. The solution is heated to over 90∘C to separate the DNA strands. The temperature is then lowered and primers attach themselves to the ends of the target section of DNA. The polymerase builds two new strands starting at the primers, but note only one end is extended. Heating again separates the strands into four separate ones. On lowering the temperature, primers again attach themselves and the polymerase extends the strands. There are now four strands of DNA. The next step will produce eight strands of the target DNA. The process continuously repeats, producing strands and so easily making many copies of the target DNA, which can then be used in experimentation.
Constitutional Mismatch Repair Deficiency Syndrome
Published in Dongyou Liu, Handbook of Tumor Syndromes, 2020
Human cells are exposed to both extrinsic (e.g., ultraviolet radiation, carcinogens, and oxidative stress, etc.) and intrinsic (e.g., replication error) factors that can damage DNA, impair gene expression, and induce genomic instability and mutagenesis. To maintain stability and prevent transmission of mutations to progeny, human cells have evolved a number of mechanisms to repair damaged DNA. Of these, DNA mismatch repair genes play a vital role in repairing mistakes that remain after DNA polymerases replicate DNA.
Cytomegalovirus
Published in Avindra Nath, Joseph R. Berger, Clinical Neurovirology, 2020
Foscarnet is a pyrophosphate analogue with antiviral activity against sensitive strains of CMV [109]. The drug inhibits DNA polymerase by directly binding to the enzyme and acting as a reversible competitive inhibitor. Because this mechanism of action does not require the presence of the protein kinase gene to phosphorylate the antiviral drug (as is necessary with ganciclovir), foscarnet is active against many ganciclovir-resistant strains of CMV. However, other mutations, especially of the viral DNA polymerase, can result in foscarnet resistance.
The discovery of novel antivirals for the treatment of mpox: is drug repurposing the answer?
Published in Expert Opinion on Drug Discovery, 2023
Ahmed A. Ezat, Jameel M. Abduljalil, Ahmed M. Elghareib, Ahmed Samir, Abdo A. Elfiky
Other important viral targets are the orthopoxvirus DNA and RNA polymerases. DNA polymerase protein, encoded by the E9L gene, is the target of Brincidofovir (CMX001) [69]. DNA polymerase protein is essential for replicating its double-stranded DNA (dsDNA) genome, and therefore, it is considered a primary target for developing antiviral drugs. However, some conserved mutations between members of the orthopoxviruses in the DNA polymerase genes have produced resistance against treatment with brincidofovir [70]. Tremendous efforts are made to combine multiple antivirals and vaccines, develop new techniques and drugs, and investigate new targets for complete virus defeat. For the targeting of viral RNA, Methisazone (Thiosemicarbazone) is used to inhibit the viral RNA polymerase protein, encoded by several viral genes like J3R, H5R, and A24R, and is believed to interfere with viral maturation in most of the orthopoxviruses [71].
An insight into clinical and laboratory detections for screening and diagnosis of cervical cancer
Published in Expert Review of Molecular Diagnostics, 2023
Shruthi Padavu, Pooja Aichpure, Ballamoole Krishna Kumar, Anoop Kumar, RadhaKanta Ratho, Shipra Sonkusare, Indrani Karunasagar, Iddya Karunasagar, Praveen Rai
The chemistry of PCR is based on a thermostable DNA polymerase that identifies and amplifies two oligonucleotide primers flanking the region of interest. The viral DNA is amplified sufficiently in vitro to yield an acceptable amount of target, which is then observed by agarose gel electrophoresis [65]. The HPV PCR usually targets the L1 gene, the most conserved gene within the HPV genome [66]. Consensus primers such as PGMY-LB, SPF10-LiPA25, GP5+/6+, and MY09/11 primer sets are most commonly used to identify HPV by PCR [67]. PCR is a sensitive assay for detecting HPV types, particularly HR-HPV subtypes. The PCR genotyping is time-consuming, expensive, and requires a sophisticated laboratory and equipment such as a thermocycler, which obviates its use as a point-of-care application [55]. Though inadequate sensitivity is reported during low viral load, HPV DNA PCR has been widely used in diagnosing cervical cancer in most countries [60].
Design, optimization, and application of multiplex rRT-PCR in the detection of respiratory viruses
Published in Critical Reviews in Clinical Laboratory Sciences, 2022
Jing Yang, Dandan Li, Jie Wang, Rui Zhang, Jinming Li
For multiplex rRT-PCR, it is necessary to reduce the secondary structure and interaction among primers, probes and the template. The secondary structure of the target region will markedly damage its affinity in hybridization, thus damaging the detection performance (Figure 4(B-a,b)) [37,38]. Fan et al. designed primers covering different positions for the hairpin structure template containing a 9 bp stem and 15 nt loop, and specifically described the effect of the secondary structure of the template on amplification [39]. The results showed that Ct values from big to small were primer 3 > primer 4 > primer 5 > primer 2 > primer 6 > primer 1 > primer F (Figure 4(B-c)). Compared with primer F, primer 6 was only 5 nt complementary to the stem, but the Ct value increased significantly. Primers 3 and 4 covered a large area of the hairpin structure, and thus the amplification was inhibited. The coverage areas of primers 1 and 2 were close to the 5′ end of the hairpin structure and had little effect on amplification. Additionally, if two primers are complementary at the 3′ end, DNA polymerase can bind to them and extend the primers in both directions to generate primer dimers. However, if two primers are bound at the 5′ end or in the middle, they will not be bound by DNA polymerase and extended [40]. The 3′ end complementarity of primers is an important parameter for evaluating the formation of primer dimers; it is critical to analyze the position and secondary structure of amplification before design (Figure 4(B)) [41].