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Preimplantation Genetic Testing of Aneuploidies (PGT-A)
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Daniela N. Bakalova, Darren K. Griffin, Maria E. Póo, Alan R. Thornhill
Drawbacks of the technique are the limit of resolution and the requirement for parental reference samples. There are many possible abnormalities associated with miscarriage and implantation failure that could arise from de novo mutations, which are below the resolution of SNP arrays typically used; this could lead to missed diagnoses and adverse pregnancy outcomes. Additionally, it is inherent to the principle of SNP-based analysis that maternal and paternal DNAs are required that can serve as a reference for the tested embryos; this makes the process more time-consuming and expensive. However, SNP arrays have been successfully applied for aneuploidy screening by several groups [51–53] and the technique allows simultaneous PGT-A and PGT-M (preimplantation genetic testing for monogenic disorders) detection.
Models, techniques, and approaches for change management
Published in Robert Jones, Fiona Jenkins, Penny Humphris, Jim Easton, Key Tools and Techniques in management and leadership of the Allied Health Professions, 2021
DNAs were reduced from 11.3% per annum to less than 1% within the first three months, saving capacity of 11.3%—time wasting is eliminated—with the result that staff are able to spend more time with patients and effectively plan clinical-related work such as record keeping, team meetings, and in-service training. Importantly, capacity for taking more patients is increased.
HLA-DR Typing by Polymerase Chain Reaction Amplification with Sequence-Specific Primers (PCR-SSP)
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
Prepare primer mixes, containing all the components of the PCR reaction mixtures, except Taq polymerase and DNA, in 1-ml batches sufficient for 100 typings. Aliquot the primer mixes in 250 μl batches. Primer mixes can be stored for months at -20°C and can without harm be repeatedly thawed. Between typings the primer mixes should be kept at +4°C. It is recommended that each prepared primer mix batch is tested against two positive and two negative control DNAs.
New αIIbβ3 variants in 28 Turkish Glanzmann patients; structural hypothesis for complex activation by residues variations in I-EGF domains
Published in Platelets, 2022
M Y Koker, N Sarper, C Albayrak, B Zulfikar, E Zengin, B Saraymen, D Albayrak, B Koc, H Avcilar, M Karakükcü, C Chenet, F Bianchi, A G de Brevern, R Petermann, V Jallu
Genomic DNA was isolated from peripheral blood. Promoter and exon sequences of αIIb and β3 genes were sequenced as previously described [7,8]. Sanger sequencing was done by Genoscreen (Lille, France). Nucleotide numbering was according to the coding sequences of the ITGA2B and ITGB3 genes (GenBank accession numbers NM_000419.5 and NM_000212.2, respectively,), the adenine of the ATG start designated +1. The first amino acids of the mature αIIb and β3 proteins sequences (accession numbers NP_000410.2 and NP_000203.2, respectively) were designated +1. Residue numbering according to the Human Genome Variation Society (HGVS) nomenclature (http://www.hgvs.org/mutnomen) designing as +1 the translation initiating methionine is also shown in Tables I and II. Both numberings differ by 31 and 26 residues that correspond to the signaling peptides of αIIb and β3, respectively. To simplify, αIIb and β3 notations were used instead of the coding sequences or proteins accession numbers but full nomenclature is shown in Tables I and II. All identified variations were confirmed by a second Sanger sequencing analysis of DNAs from patients and their relatives (mothers, fathers, and siblings).
The current and future applications of in situ hybridization technologies in anatomical pathology
Published in Expert Review of Molecular Diagnostics, 2022
Hoi Yi Leung, Martin Ho Yin Yeung, Wai Tung Leung, King Hin Wong, Wai Yan Tang, William Chi Shing Cho, Heong Ting Wong, Hin Fung Tsang, Yin Kwan Evelyn Wong, Xiao Meng Pei, Hennie Yuk Lin Cheng, Amanda Kit Ching Chan, Sze Chuen Cesar Wong
Lin et al. [67] developed amplification-based single-molecule FISH (asmFISH) which is the combination of a modified smFISH with RCA. First, a pair of DNA probes is used to target the RNA molecule of interest. The probe is then ligated to the RNA by enzymatic reaction, forming a circular DNA for signal amplification via RCA. Lastly, fluorescently labeled probes are hybridized to the amplified DNAs and detected by microscopy. To test the applicability of asmFISH to detect gene expression, HER2 expression in two breast cancer cell lines, MCF-7 and SK-BR-3, were measured. The results matched with the human protein atlas database, therefore, asmFISH could show the difference in levels of gene expression. It also had a higher capacity in discriminating SNPs compared to in situ padlock probe assay. The author demonstrated that asmFISH is applicable in both FFPE and fresh frozen tissue samples, showing the potential in clinical application.
MYCN amplification levels in primary retinoblastoma tumors analyzed by Multiple Ligation-dependent Probe Amplification
Published in Ophthalmic Genetics, 2021
Elizabeth A. Price, Roopal Patel, Irene Scheimberg, Esin Kotiloglu Karaa, Mandeep S. Sagoo, M. Ashwin Reddy, Zerrin Onadim
This audit was approved by the Barts Health Clinical Effectiveness Unit (audit no. 10839). It covered a sub-set of samples collected during the period 1993–March 2019. Patients were referred to the Retinoblastoma Genetic Screening Unit (RGSU; Barts Health NHS Trust) for Rb genetic analysis by clinical geneticists, genetic counsellors or ophthalmologists, and consent for screening was obtained from parents/guardians. We looked at MYCNA results from 149 Rb DNAs (132 fresh frozen; 17 formalin fixed, paraffin embedded [FFPE]). The patients presented as 21 sporadic bilateral and 128 sporadic unilateral (13 germline, 115 somatic). Table 1 gives an overview of patients and tumor samples. All tumors where an RB1 pathogenic variant was missing (n = 50) were included. 29 had one RB1 change (RB1±) while 21 had no identified RB1 change (RB1+/+) but not all were fully screened (‘Not Determined’ in Table 1). Also included sporadic unilateral cases with especially early age of diagnosis (8 months or less, n = 23). MYCNA testing performed on all 65 fresh frozen tumors received from April 2014-March 2019 provides an unbiased sample set. 114 blood DNAs were tested including samples matched to MYCNA tumors where available.