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Mitochondrial DNAs and Phylogenetic Relationships
Published in S. K. Dutta, DNA Systematics, 2019
The mitochondrial genomes of closely related species of fungi show considerable structural and size variation. Such variation has been investigated most fully in the three genera Saccharomyces, Neurospora, and Aspergillus. In all three cases the greater part of this variation is due to a series of insertions/deletions which have been identified as introns located in certain genes, particularly in those coding for cytochrome oxidase subunit 1 and for apocytochrome b. Other insertion/deletion events have also been detected elsewhere in the genome. As a consequence of this large amount of structural variation a particularly high degree of restriction pattern polymorphism is found within and/or between closely related species or strains in all three genera. In spite of this, heteroduplex analysis and detailed comparison of restriction site maps show there to be a high degree of sequence homology in those regions outside the various insertions/deletions.
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.
Common Inherited Genetic Disorders
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Other mutation detection strategies such as allele-specific oligonucleotide hybridization, DGGE, and SSCP have also been used. However, while they appear to be efficient, they suffer from a lack of sensitivity. A mutation detection frequency of 10 to 14% for the mutation tested was reported for the BRCA1 gene using a dual testing strategy of heteroduplex analysis and PTT (Second Australian Mutation Detection Workshop, 1999). Approximately 2 to 3% of BRCA1 patients were found to carry duplications of exon 13; this 6-Kb duplication appears to be Alu-mediated. The high concentration of Alu sequences in the BRCA1 gene suggests that duplications may be present in significant numbers of BRCA1 families with breast cancer.
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
Peripheral blood was collected into EDTA tubes whilst fresh tumor was harvested by pathologists and frozen for storage immediately after enucleation. DNA was extracted as previously described (4,11). RB1 screening covered all 27 exons plus 50 bp upstream and 30 bp downstream to cover associated splice sites, as well as the promoter. Conformation analysis of transitions/transversions and small insertions/deletions was performed by single stranded conformational polymorphism and heteroduplex analysis (GE Biotech ALFexpress), and/or high resolution melt analysis (Corbett RotorGene 6000). Samples giving abnormal traces were reamplified for Sanger sequencing. Polymorphic markers within and around RB1 on chromosome 13 were used to determine tumor LOH. Large exonic deletions were detected by in-house Quantitative Fluorescent PCR (QF-PCR) and Multiplex Ligation-dependent Probe Amplification (SALSA MLPA RB1 probe mix P047, MRC-Holland) (11). Methylation specific PCR (MS-PCR) of bisulphite-modified DNA was performed to detect promoter hypermethylation (4). Around 97% of expected pathogenic variants (including MYCNA cases) were routinely detected over the last fifteen years (April 2005-March 2020).
A novel nonsense variation in the albumin gene (c.1309 A>T) causing analbuminaemia
Published in British Journal of Biomedical Science, 2021
G Caridi, A Farokhnia, F Lugani, AM de Luca, M Campagnoli, M Galliano, D Schröpfer, L Minchiotti
Having obtained written informed consent for genetic testing from both brothers, molecular analysis of the albumin gene (gene accession number: NG_009291.1) [12] was performed based on PCR amplification [5], followed by heteroduplex and single-strand conformational polymorphism analyses. Heteroduplex analysis revealed the presence of a molecular defect in the DNA fragment encompassing exon 11 and flanking intronic junctions [11], indicating that the case was homozygous and his brother heterozygous for the same mutation (Figure 2(a)). Automated DNA sequence analysis of this region in an Applied Biosystems 3100xl capillary DNA sequencer allowed us to identify an A > T transversion at nucleotide position c.1309, the first base of codon AAG for p.437Lys, giving rise to a stop codon TAG (Figure 1). This result confirmed that the case is homozygous and that his brother is heterozygous for this nonsense variant (Figure 2(b)). The premature termination codon would encode a truncated protein consisting of only 412 amino acids (p.Lys437Ter according to the Human Genome Variation Society rules) but, as with other variants resulting in congenital analbuminaemia, it is probably not present in the circulation, since an intact C-terminal end of the molecule is required for its long plasma half-life [13,14].
Copy-number variations in adult patients with chronic immune thrombocytopenia
Published in Expert Review of Hematology, 2020
Emrah Yucesan, Ozden Hatirnaz Ng, Fevzi Firat Yalniz, Hulya Yilmaz, Ayse Salihoglu, Tugce Sudutan, Ahmet Emre Eskazan, Seniz Ongoren, Zafer Baslar, Teoman Soysal, Ugur Ozbek, Muge Sayitoglu, M. Cem Ar
All the cases that were evaluated as mosaic were also evaluated for TCRG (T-cell receptor gamma) and TCRD (T-cell receptor delta) clonality. PCR amplifications were performed using BIOMED-2 protocol; for TCRG (TCRGA:VγI/γ10-Jγ and TCRGB:Vγ9/γ11-Jγ) and for TCRD (VD1-6/JD1-3) regions [11]. For heteroduplex analysis, PCR products were denatured at 95°C for 10 min and renaturated at 4°C for 1 hour. Samples were loaded on 8% non-denaturing polyacrylamide (acrylamide:bisacrylamide = 29:1) gel and run at 100 V for 50 minutes. Gel images were examined for clonality analysis. One or more discrete bands that are identified within the expected ranges (TCRGA:145bp-255bp, TCRGB:80bp-220bp, and TCRD:12bp-280bp) were interpreted as clonal.