What happens in Leukemias, Lymphomas, and Myelomas?
Tariq I Mughal, John M Goldman, Sabena T Mughal in Understanding Leukemias, Lymphomas, and Myelomas, 2017
In the late 1970s, doctors in Japan noticed an unusual form of lymphoma. Unlike most lymphatic tumors, which are made up of B lymphocytes, the unusual Japanese lymphomas are composed of T lymphocytes. By 1980, Japanese scientists had identified an RNA-containing virus that was present in cells derived from most cases of this T-cell leukemia/lymphoma (Fig. 3.12). The virus was named ATLL virus (ATLV). An important attribute of ATLV was that it could induce cells to produce an enzyme capable of constructing new sequences of DNA from an RNA model or template. DNA normally directs cells to produce (or transcribe) RNA; there is usually no flow of information from RNA to DNA. For this reason, the enzyme that directs the synthesis of DNA from RNA is known as reverse transcriptase.
The Complex Nature of the Epidemic
Jeffrey T. Huber in HIV/AIDS Community Information Services, 2013
The human immunodeficiency virus—HIV-1 and HIV-2 are genetically related, with HIV-2 considered to be less virulent—is a parasite which is dependent upon host cells for survival. This is why it cannot be transmitted via an inanimate object, such as a toilet seat. The human immunodeficiency virus is a retrovirus; and like other retroviruses, it consists of ribonucleic acid (RNA) and lacks deoxyribonucleic acid (DNA), and cannot replicate in the usual way. It carries reverse transcriptase, or an enzyme which catalyzes the process of forming a compound (by combining simpler molecules) in order to synthesize DNA using certain aspects of RNA. The DNA copy is then incorporated into the chromosomes of the host cell, which is thereby programmed with the viral genetic code. When the cell becomes active, it reproduces the viral RNA which encodes viral proteins, and millions of new complete virus particles (virions) are formed to infect other susceptible hosts. It is this retrovirus which is indicative of the acquired immunodeficiency syndrome since “HIV or antibodies to it are found in essentially all patients with AIDS.”29
Dengue Fever: A Viral Hemorrhagic Fever of Global Concern
Jagriti Narang, Manika Khanuja in Small Bite, Big Threat, 2020
Reverse transcriptase-polymerase chain reaction (RT-PCR): RTPCR is a well-exploited technique for the detection of RNA-based viruses. The first step is the isolation of RNA from the DENV by commercially available kits (Qiagen, Invitrogen, Ambion, etc.) or the TRIazol method (Chomczynski-Sacchi) (DePaula et al., 2002; Deubel et al., 1990; Deubel and Pierre, 1994; Figueiredo et al., 1998; Fulop et al., 1993; Lanciotti et al., 1996; Suk-Yin et al., 1994), and then it is amplified using primers. Reverse transcriptase enzyme helps in the conversion of RNA into DNA template. Three authors from the chapter have designed indigenous set of primers for all DENV serotypes (Joshi et al., 2018). DENV can be tested by using different types of PCR such as semi-nested PCR (Gomes et al., 2007), NASBA technique (Usawattankul et al., 2002), and RT-LAMP (Sahni et al., 2013).
A Study on the Effect of Vitamins A and C to Modulate the Expression of NKG2D Ligands in Hepatic and Colon Cancer Cells
Published in Nutrition and Cancer, 2021
Mazin A. Zamzami, Mohammad Nasrullah, Hani Choudhry, Mohammad Imran Khan
High-Capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Lithuania, Catalog no. 4368814) uses the random primer scheme for initiating cDNA synthesis. The reverse transcription process is usually used to synthesize DNA from an RNA template to produce complementary DNA (cDNA). Reverse transcriptase is an RNA-dependent DNA polymerase and is directed the synthesis of the first standard DNA by the RNA template and short primer complementary to the 3-end of the RNA. To synthesize cDNA, the reagents of the kit were combined to form a reverse transcription master mix. RNA samples (300 ng) were added after the necessary normalization step with nuclease-free water. The reactions were prepared and thermocycler reactions were programmed following the manufacturer's protocol. Primers for RT-PCR reaction (Table 1) were designed to measure the expression of NKG2DLs, DNA methyltransferases (DNMTs), and TETs. The primer sequences were obtained using the USCS browser. mRNA sequence (only exons) were taken and by the primer three web tool (http://bioinfo.ut.ee/primer3-0.4.0/), primers were designed after confirming all primer characteristics. Moreover, sequences of primers were confirmed (in silico PCR) by the UCSC browser (https://genome.ucsc.edu/cgi-bin/hgPcr). RPL0 was used as a housekeeping gene to compare the expression of other genes.
Rapid and simple molecular tests for the detection of respiratory syncytial virus: a review
Published in Expert Review of Molecular Diagnostics, 2018
Catherine A. Hogan, Chelsea Caya, Jesse Papenburg
There are four main steps to the reaction: extraction, reverse transcription, amplification, and detection. In the extraction step, an acidic extraction reagent lyses the RSV virions and releases their RNA. A first DNA template anneals to its target sequence in the RSV N gene, forming an RNA/DNA complex. The reverse transcriptase enzyme recognizes this complex, produces a DNA copy from RNA, and digests the RNA strand to reveal a single-stranded DNA copy. The second template then binds to its complementary DNA sequence and is extended by DNA polymerase, producing a partially double-stranded DNA product. The nicking enzyme identifies this specific site, binds to it and cuts the sequence. From this, the DNA polymerase can bind to the sequence and extend it to produce a fully double-stranded DNA product that harbors two nicking enzyme recognition sites. Amplification can thus continue through repeated cycles of this same process. Specific amplification products of the RNA target are identified through fluorescently-labeled molecular beacons.
Critical insight into recombinase polymerase amplification technology
Published in Expert Review of Molecular Diagnostics, 2022
Mustafa Ahmad Munawar
Kary Mullis developed the revolutionary technique of polymerase chain reaction (PCR) in 1983 [1], and its modern version has evolved through several of the improvements in the chemistry and machinery. PCR is a cyclic process of amplifying template DNA through primers, polymerase, deoxynucleoside triphosphates (dNTPs) (nucleotides or building blocks), magnesium ion, buffer, and two to three different incubation temperatures. PCR utilizes (1) a high temperature such as 95°C to convert double-stranded DNA into single-stranded DNA (melting), then (2) a temperature around 55°C to facilitate annealing of a primer pair with target DNA, and (3) a temperature around 70°C to extend annealed primers through polymerase. A typical PCR comprises 30–40 cycles, and it exponentially amplifies target DNA into millions of amplicon copies. PCR is also utilized to amplify RNA. RNA template is first converted into complementary (c)DNA through reverse transcriptase (RT) and then amplified through PCR. In summary, PCR is utilized for qualification, absolute quantitation, and relative quantification of target nucleic acid, and it is used massively in many fields including diagnostics, forensics, and agriculture. PCR applications include detection of pathogens or contaminants, genotyping, relative quantification of gene expression, molecular cloning, sequencing, methylation detection, and site-directed mutagenesis.
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