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Preimplantation Genetic Diagnosis for Single Gene Disorders
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Ana Cervero, Jose Antonio Martínez-Conejero, Lucía Sanz-Salvador, Claudia Gil-Sanchís, Maribel Sánchez-Piris, Laura Iñiguez Quiles
Genotyping of the amplified products can be performed by means of different strategies, with minisequencing the most frequently used method for the detection of point mutations (23). In the minisequencing technique, a primer extension reaction is performed, allowing rapid and accurate detection of point mutations. The minisequencing primer is designed to anneal one base before the target site, and it can only be elongated with one specific dideoxynucleotide. The four different dideoxynucleotides are labeled with different fluorochromes, and the products can be analyzed on an automated DNA sequencing system. Other strategies such as amplification refractory mutation system (24), restriction enzyme digestion (25), and real-time PCR (26) have been also applied in PGT-M. Small deletions and duplications can also be detected by sizing of PCR products from specific regions containing the mutation under study.
A Survey of Newer Gene Probing Techniques
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
TAS is primary designed to produce RNA copies of a target DNA or RNA sequence. First, short nucleotide sequences — the polymerasebinding sequences (PBSs) — are recognized and positioned on the 3′ side of the target sequence by a DNA-dependent RNA polymerase. To achieve this, an oligonucleotide primer containing two domains is used. Depending on the target sequences to be amplified either a reverse transcriptase or a DNA polymerase is employed in a primer-extension reaction. A second oligonucleotide primer following a primer extension reaction complements the product of the first primer-extension reaction to form a double-stranded PBS-containing cDNA copy of the target sequence. A number of RNA polymerases (T7, T3, or SP6) can be used to recognize the PBS domains.
Genetic analysis of the embryo
Published in David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham, Textbook of Assisted Reproductive Techniques, 2017
Yuval Yaron, Liran Hiersch, Veronica Gold, Sagit Peleg-Schalka, Mira Malcov
Primer extension preamplification (PEP) is a WGA method designed mainly for use in single cells. Using ran- dom sequence primers of 15 bp, PEP has been claimed to amplify at least 70% of the genome in more than 30 cop- ies (32). This, however, is likely to be a rather conservative estimate, since Paunio et al. (33) reported that PEP yields at least 1000 copies of the genome, and Wells and Sherlock (21) have suggested that more than of 90% of genomic sequences are represented in PEP amplification products. One of the drawbacks of PEP is the time required, which is usually more than 12 hours. Sermon et al. (34) have successfully adopted a modified protocol that requires less than six hours, and Tsai (35) has improved the effi- ciency through further technical modifications. Several autosomal recessive, dominant, and X-linked disorders have been successfully detected in single cells using PEP, including Tay-Sachs disease, cystic fibrosis, hemophilia A, DMD, and FAP (14, 36, 37).
Strategies for targeting RNA with small molecule drugs
Published in Expert Opinion on Drug Discovery, 2023
Christopher L. Haga, Donald G. Phinney
To overcome limitations of enzyme-based RNA footprinting, chemical modifiers of solvent-accessible nucleobases were adopted to probe RNA secondary structures. Perhaps the most well-established reagent is dimethyl sulfate (DMS), a compound that alkylates the N-7 cyclic amine of unpaired guanosine residues, which following reduction and cleavage by analine treatment may be detected by PAGE analysis [21]. DMS has also been shown to alkylate the N1 and N3 positions of unpaired adenine and cytosine, respectively [22]. Since these modifications result in early termination of cDNA elongation, their location can be probed by primer extension using reverse transcriptase. Combining this approach with RNA sequencing permits massively multiplexed RNA secondary structural determination on the level of the entire RNA transcriptome (DMS-Seq and DMS-MaPseq) [23,24]. Because DMS is a small molecule capable of permeating cell membranes, probing RNA secondary structures in cellulo is also possible.
Conjugation of phosphonoacetic acid to nucleobase promotes a mechanism-based inhibition
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2018
Algirdas Mikalkėnas, Bazilė Ravoitytė, Daiva Tauraitė, Elena Servienė, Rolandas Meškys, Saulius Serva
The observed differences in the ability to incorporate PAT among selected DNA polymerases were investigated in further detail. The time course experiments of primer extension were performed under varying concentrations of PAT. Based on our results, HIV-1 reverse transcriptase had the highest preference for PAT (Figure 3(a)). The other three enzymes tested exhibited lower ability to incorporate the aforementioned compound (Figure 3(b)). The intermediate products of primer extension involving one or two PAT moieties were well-preserved during the course of DNA biosynthesis. While the progressive formation of reaction intermediates suggested a distributive mode of DNA synthesis, the enzymes employed in this study showed much higher processivity with dTTP as a substrate for primer extension (Figure 2(b)).
Peptidylarginine deiminase-4 gene polymorphisms are associated with systemic lupus erythematosus and lupus nephritis
Published in Scandinavian Journal of Rheumatology, 2019
L Massarenti, C Enevold, D Damgaard, N Ødum, CH Nielsen, S Jacobsen
Samples were genotyped for the selected SNPs based on a previously developed in-house multiplex SNP assay protocol (36). In brief, target sequences including the selected SNP sites were amplified through polymerase chain reaction (PCR) and annealed to allele-specific oligonucleotides in an allele-specific primer extension (ASPE) reaction. The annealed ASPE oligonucleotides were subsequently hybridized to MagPlex-TAG™ bead sets (Luminex Corporation, Austin, TX, USA) for analysis on the Luminex platform (Luminex Corporation, Austin, TX, USA).