China
Africa
Explore chapters and articles related to this topic
Liquid Biopsies for Pancreatic Cancer: A Step Towards Early Detection
Published in Surinder K. Batra, Moorthy P. Ponnusamy, Gene Regulation and Therapeutics for Cancer, 2021
Joseph Carmicheal, Rahat Jahan, Koelina Ganguly, Ashu Shah, Sukhwinder Kaur
BEAMing is another form of sensitive and specific digital PCR, also based on the emulsification of the PCR mixture. This method, however, requires the added step of attaching streptavidin-coated magnetic beads to each strand of DNA prior to the PCR analysis. Fluorescently tagged complementary primers are next added which can bind with the DNA strands of interest attached to the beads, thus allowing for facile and specific isolation of our DNA targets of interest from the sample. These beads are analyzed using flow cytometry and the presence of DNA is validated by Sanger sequencing. These new advanced digital PCR methods can simultaneously track the number of normal and mutant DNA molecules in a given sample, thus providing another metric of quantification and blood-based characterization [13, 14].
Cardiovascular PET-CT
Published in Yi-Hwa Liu, Albert J. Sinusas, Hybrid Imaging in Cardiovascular Medicine, 2017
Etienne Croteau, Ran Klein, Jennifer M. Renaud, Manuja Premaratne, Robert A. Dekemp
Retrospectively gated acquisitions consist of continuous x-ray acquisition (and beaming) with simultaneous ECG signal recording. After acquisition is complete, any cardiac phase may be reconstructed, affording the ability to monitor heart wall motion, but at the cost of greater radiation dose to the patient. Retrospective gating can be performed with spiral CT and a low pitch, to avoid large gaps in the projection data of each cardiac phase (Mark et al. 2010).
Applications of Circulating DNA Analysis in Personalized Medicine
Published in II-Jin Kim, Cancer Genetics and Genomics for Personalized Medicine, 2017
Dana W.Y. Tsui, Muhammed Murtaza
Digital PCR relies on segregation of template DNA into many hundreds or thousands of PCR reactions with one or no copies of the targeted fragment (Vogelstein and Kinzler, 1999). The presence of DNA template, usually detected by fluorescence, is measured for each reaction, either in real-time or at endpoint, to count the number of positive reactions. Unlike qPCR that requires comparison with a standard input, digital PCR allows direct counting of the absolute number of input molecules. As each positive PCR reaction amplifies one template molecule, digital PCR using mutation-specific fluorescent probes is particularly useful for quantification of low-abundance alleles in a sample. It allows absolute sensitivity for detection of mutant DNA fragments, limited only by number of digital replicates and background noise for each PCR assay. In practice, digital PCR is highly sensitive for point mutations with allele fractions as low as 0.01%. Although digital PCR can be performed by diluting input DNA material and setting up digital reactions in 96- or 384-well plates, several high-throughput methods have been described, including microfluidic devices (Yung et al., 2009), droplet-based platforms (Pinheiro et al., 2012; Taly et al., 2013), and BEAMing (beads, emulsion, amplification, and magnetics) (Diehl et al., 2008). The number of genomic loci that can be assayed in multiplex remains limited to a few.
dPCR application in liquid biopsies: divide and conquer
Published in Expert Review of Molecular Diagnostics, 2021
Andrea Moreno-Manuel, Silvia Calabuig-Fariñas, Antonia Obrador-Hevia, Ana Blasco, Amaya Fernández-Díaz, Rafael Sirera, Carlos Camps, Eloisa Jantus-Lewintre
The main advantages of BEAMing rely on the fact that its protocol was optimized with rigorous quality parameters to minimize false-positive and false-negative results by analyzing many beads to obtain a high signal-to-noise ratio and reaching high sensitivity levels. Therefore, BEAMing can reliably quantify DNA molecules, facilitating calculation of the mutant fraction with a specific LOD [28]. BEAMing can detect mutant alleles with a frequency of 1 in 10,000 among wild-type alleles, although the sensitivity cutoff is usually set at 0.02–0.04% to reduce the possibility of false-positives because mutations found at rates below this baseline are considered random. BEAMing also precisely measures ctDNA even at low concentrations and so it avoids false-negative results caused by low DNA input. These workflow improvements have allowed developers to reduce errors to those caused by the error rate of the polymerase [28,30,31].
Technical considerations for circulating tumor DNA detection in oncology
Published in Expert Review of Molecular Diagnostics, 2019
Claire Franczak, Pierre Filhine-Tresarrieu, Pauline Gilson, Jean-Louis Merlin, Lewis Au, Alexandre Harlé
BEAMing is a four steps digital-PCR method [91–93]. After extraction, purification and pre-amplification, water-in-oil emulsions are prepared [91–93]. Emulsions contain one amplicon, one complementary biotinylated-oligonucleotide sequence, one set of primers and one magnetic bead coated with streptavidin. Increasing copy number per beads is produced by an emulsion-PCR. Finally, specific fluorescent probes for mutations or wild-type sequences are added and hybridized with bead-coated amplicons. The final step consists of flow cytometry analysis, performed in order to quantify mutant allele frequencies (Figure 2). The OncoBEAMTM RAS CRC Kit (Sysmex inostics, Hamburg, Germany) detects 34 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS and NRAS oncogenes [94]. Sensitivity of this method is about 1 mutant allele per 10,000 wild type and is limited by error rate of DNA polymerase [93] (Table 2). In plasma of patients with stage IV CRC, somatic mutations can be detected with near 100% sensitivity [5]. In a prospective study including metastatic breast cancer patients, plasma PIK3CA mutations scanning yielded a 72.5% concordance with tissue analysis [95]. EGFR activating mutations, like L858R, can be assessed in lung cancer patients. Taniguchi et al. reported a 72.5% detection rate of activating EGFR mutations in plasma in 72.5% in a cohort of 44 patients with lung cancers [96]. Emergence of RAS mutations is a frequent mechanism of resistance in metastatic CRC patients treated with-anti-EGFR therapy. BEAMing was used for acquired KRAS mutations detection in several studies, and BEAMing appears as an effective method to monitor acquired molecular alterations involved in acquired resistance to targeted therapies [97–99]. A study evaluated the potential of BEAMing for detection of residual disease and early detection of relapse, and showed that if mutant DNA was undetectable, no recurrence occurred, whereas mutant DNA was detectable in relapsed patients [5].