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Rapid HLA-DR-Dw and DP Matching by PCR Fingerprinting and Related DNA Heteroduplex Technologies
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
The initial development of DNA heteroduplex matching was in response to an urgent clinical need for a fast screening method for selection of DR-Dw and DP matched unrelated donors for patients awaiting bone marrow transplantation. The simplicity, speed, and potential for simultaneous analysis of large numbers of donors by PCR fingerprinting fulfills this need and allows the same-day elimination of HLA class I matched but DR-Dw and/or DP mismatched candidate donors. The techniques were recently tested in a pilot clinical study of 53 unrelated donor-recipient pairs. Here, PCR fingerprinting and confirmatory DNA cross-matching for HLA-DRB alleles were found to be more informative than DNA-RFLP analysis alone, and were equivalent to DNA-RFLP supplemented with dot-blot PCR-SSO (sequence-specific oligonucleotide) typing of DR4-Dw subtypes.4
Hereditary and Acquired Causes of a Hypercoagulable State
Published in Harold R. Schumacher, William A. Rock, Sanford A. Stass, Handbook of Hematologic Pathology, 2019
The PCR based DNA assay for factor V Leiden relies on relatively straightforward molecular biology techniques (19). Primers flanking the G-to-A mutation at nucleotide 1691 are used to amplify a 267-base pair fragment, which is then digested with the restriction endonuclease Mnll. The wild-type factor V fragment is cleaved at two sites by the restriction enzyme to yield three fragments of 163, 67, and 37 base pairs. The mutation abolishes one of the endonuclease cleavage sites, resulting in a single cleavage to yield fragments of 200 an 67 base pairs. Interpretation of the electrophoresis gels is straightforward and readily allows identification of wild-type and heterozygous and homozygous mutants. Newer, more rapid, and less expensive PCR heteroduplex techniques are now in use to look for point mutations in multiple genes at one time (71).
Molecular Genetics and Diagnostic Testing
Published in Merlin G. Butler, F. John Meaney, Genetics of Developmental Disabilities, 2019
Searching for disease-causing mutations in a gene may require identification of a single nucleotide or a few nucleotides in a coding sequence composed of thousands of nucleotides. To avoid sequencing large amounts of DNA, strategies have been developed to rapidly scan genes for small mutations. The techniques use PCR to amplify the gene or gene segment to be scanned and, ultimately, each method leads to sequencing of the region shown to contain a mutation. Single-strand conformational polymorphism analysis is based on the conformational change in DNA resulting in an alteration in electrophoretic mobility that is produced by some mutations.Mismatch heteroduplex analysis is a method in which the PCR products are denatured and allowed to renature forming heteroduplexes, if a mismatch (mutation) is present. The heteroduplexes can be distinguished because they move more slowly on nondenaturing polyacrylamide gels than do homoduplexes.Denaturing gradient gel electrophoresis and denaturing high-performance liquid chromatography are methods that are based on the differences in mobility of heteroduplex and homoduplex molecules when they are heated to temperatures just below the melting temperature of the homoduplexes.
A profile of the FDA-approved and CE/IVD-marked Aptima Mycoplasma genitalium assay (Hologic) and key priorities in the management of M. genitalium infections
Published in Expert Review of Molecular Diagnostics, 2020
Elena Shipitsyna, Magnus Unemo
The captured RNA target sequence is amplified in an isothermal TMA reaction (Figure 2), using two MG-specific primers and two different enzymes (reverse transcriptase (RT; including RNase H activities), and RNA polymerase). The first primer contains a promoter sequence for the RNA polymerase, and when this primer has hybridized to the target RNA, RT initiates a reverse transcription, creating a complementary DNA (cDNA) copy. The RNA in the resulting RNA:DNA heteroduplex is degraded by the RNase H activities of the RT. This enables the second non-promoter primer to bind to the DNA copy and a new strand of DNA is synthesized by the RT, creating a dsDNA molecule. Both strands of the created dsDNA molecule then contain the promoter sequence for the RNA polymerase, and can be used as a template to initiate transcription. Each of the newly synthesized RNA amplicons reenters as templates in the TMA process, leading to an exponential expansion of the RNA target sequence (Figure 2).
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
PCR protocol is started by a primer annealing step and a hybridization step. When the reference sequence is hybridized with the wild-type sequence a homoduplex is formed. If the sequence is bearing a single nucleotide variation, the duplex formed with the reference sequence is a heteroduplex. Tm is different between homoduplex and heteroduplex. A critical denaturation step is performed at a temperature lower than the usually Tm. Heteroduplexes, which melt at a lower temperature than homoduplexes can be selectively denatured and preferentially amplified throughout the course of PCR. With this method, mutations at any position along the sequence are preferentially enriched during COLD-PCR amplification. For mutations analysis, these methods are then coupled with a mutation detection assay as sequencing, MALDI-TOF genotyping or PCR as TaqMan® real-time PCR [46].
Comparison between flow cytometry and standard PCR in the evaluation of MRD in children with acute lymphoblastic leukemia treated with the GBTLI LLA – 2009 protocol
Published in Pediatric Hematology and Oncology, 2019
Juliana Maria Camargos Rocha, Sandra Guerra Xavier, Marcelo Eduardo de Lima Souza, Mitiko Murao, Benigna Maria de Oliveira
For MRD analysis, at least two clonal rearrangements identified at diagnosis were tested, whenever possible. Sensitivity of 10−3 for IGK (Vk2-Vk3/Kde) and TRD (Vd2-Dd3) and of 10−2 for all other primer combinations were achieved. PCR and heteroduplex analyses were performed as described for diagnostic samples, except 500 ng of DNA were used. Follow-up samples were analyzed in parallel with those from the diagnosis and PBL and were considered positive when the resulting bands had the same migration pattern as those detected at diagnosis.