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An Introduction and Review of DNA Profile Interpretation
Published in Jo-Anne Bright, Michael D. Coble, Forensic DNA Profiling, 2019
Jo-Anne Bright, Michael D. Coble
DNA testing is recognized as the “gold standard” in forensic identification science methods. The techniques used in forensic DNA analysis are based upon the accepted principles of molecular biology, including DNA extraction methods, the polymerase chain reaction (PCR), capillary electrophoresis (CE), and population genetic models including Mendelian inheritance and the Hardy–Weinberg equilibrium. The interpretation of a good-quality DNA profile generated from a crime scene stain from a single donor (termed “single source”) provides an unambiguous result when using the most modern forensic DNA methods.
Assessing the Microbiome—Current and Future Technologies and Applications
Published in David Perlmutter, The Microbiome and the Brain, 2019
Thomas Gurry, Shrish Budree, Alim Ladha, Bharat Ramakrishna, Zain Kassam
DNA extraction is followed by polymerase chain reaction (PCR) amplification. In the case of 16S rRNA sequencing, primers first bind to the constant region of the 16S gene and are subsequently extended into the 16S variable regions using a specifically engineered DNA polymerase enzyme. This process creates amplified sequences of the variable 16S regions, referred to as amplicons. In shotgun metagenomics, PCR is used to amplify the fragmented DNA sequences from the sample. Amplified sequences are then tagged with sample-specific barcodes, which facilitate multiplexing—a process in which multiple samples are run in a single Illumina sequencing lane, significantly increasing the sequencing throughput and reducing the overall cost of analysis. In the final preparatory step, adapters, which are required for binding the Illumina flow cell, are added to the amplicon sequences. Once this is completed, the sample is ready for sequencing. In a process similar to PCR, the Illumina platform is able to identify the exact nucleotide sequence of amplicons and amplified fragments by monitoring fluorescent output. Barcoded and adapter-modified nucleotide sequences are amplified using fluorescently labeled nucleotides, emitting a unique fluorescent pattern that can be directly translated into a sequence readout. The Illumina sequencing platform outputs FASTQ files which contain the “raw” data comprised of both sequence reads and accompanying quality control scores. Finally, using open-source computational pipelines, the raw data can be quality trimmed and filtered.
Microbiome and pregnancy complications
Published in Moshe Hod, Vincenzo Berghella, Mary E. D'Alton, Gian Carlo Di Renzo, Eduard Gratacós, Vassilios Fanos, New Technologies and Perinatal Medicine, 2019
Maria Carmen Collado, Omry Koren
In recent years, the presence of a placental microbiome has been a subject of intensive debate. In 1900 Henry Tissier, a French pediatrician, articulated the sterile-womb dogma, which stated that the fetoplacental unit is germ free, and our first encounter with bacteria occurs upon birth (23). This dogma was subsequently challenged, especially in the last decade, due to the implementation of highly sensitive culture-dependent and culture-independent (next-generation sequencing) approaches to identifying bacteria. In 2014, Aagaard et al. described a unique placental microbiome dominated by the phylum Proteobacteria, and most similar in composition to the oral microbiota, suggesting an oral-placenta transmission route (24). Nevertheless, the descriptions of a true placental microbiome by Aagaard and others have been questioned, especially based on the fact that culture-independent techniques identify DNA and not viable bacterial cells (25). Another problem that has been raised is bacterial contamination of the DNA extraction kits, which is problematic when working with low biomass samples (26). Several consortia are currently engaged in large-scale efforts to determine whether the placental microbiome exists and to ascertain the potential biological role.
Tongue coating microbiome composition reflects disease severity in patients with COVID-19 in Nanjing, China
Published in Journal of Oral Microbiology, 2023
Zongdan Jiang, Lu Yang, Xuetian Qian, Kunhan Su, Yuzhen Huang, Yi Qu, Zhenyu Zhang, Wanli Liu
To ensure the precision of tongue coating collection and minimize the influence of food residues, several measures were implemented. Participants were instructed to observe a fasting period, refrain from smoking and consuming alcohol, and avoid performing any oral hygiene procedures for a duration of 4 hours prior to sample collection. A sterile throat swab was employed, and it was gently rolled along the base of the tongue, ensuring approximately 30 brush strokes to collect the tongue coating. The swab was then immersed in a centrifuge tube containing phosphate buffer solution, while continuous stirring facilitated the transfer of microorganisms from the swab to the buffer. This process was repeated twice, and the collected samples were combined to ensure an adequate quantity of tongue coating samples. Following that, the collected tongue coating samples were subjected to centrifugation at 4°C and 5000 g for 5 minutes, with subsequent removal of the supernatant. The remaining material was stored at −80°C in a refrigerator. All items used were sterile. The DNA extraction process adhered to a previously established protocol [9].
The population incidence of thalassemia gene variants in Baise, Guangxi, P. R. China, based on random samples
Published in Hematology, 2022
Bixiao Wei, Weijie Zhou, Mingkui Peng, Ju Long, Wangrong Wen
The study was approved by the Ethics Committee of the Baise People’s Hospital. Samples were obtained from patients at the same hospital, with a total of 4800 participants. Of these, 2341 were female and 2459 were male. All samples were obtained from healthy individuals participating in annual physical examinations in the medical examination center without any selection for thalassemia-related phenotypes. The individuals from whom the samples were obtained were informed of the study aims. All samples were of venous blood, and EDTA was used as anticoagulant. Blood was stored in a refrigerator at 4–8 °C for backup, and DNA extraction was completed within 3 days of collection. DNA extraction was done by the magnetic bead method, and the DNA concentration was detected using an UV spectrophotometer. Samples were then stored at –20 °C.
The successful strategy of comprehensive pre-implantation genetic testing for beta-thalassaemia–haemoglobin E disease and chromosome balance using karyomapping
Published in Journal of Obstetrics and Gynaecology, 2022
Sirivipa Piyamongkol, Suchada Mongkolchaipak, Pimlak Charoenkwan, Rungthiwa Sirapat, Wanwisa Suriya, Tawiwan Pantasri, Theera Tongsong, Wirawit Piyamongkol
Biopsied cells were washed thoroughly in phosphate-buffered saline (PBS, Cell Signaling Technology, Theera Trading Co. Ltd., Bangkok, Thailand) with 0.1% polyvinyl alcohol (PVA, Sigma-Aldrich, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) before transference to microcentrifuge tubes. DNA extraction was performed using an alkaline lysis buffer protocol (Sermon et al. 1995). 2.5 μL of lysis buffer (0.75 μL of water, 1.25 μL of 0.1 M DTT and 0.5 μL of 1 M NaOH) was added, mixtures were incubated at 60 °C for 10 min. After that, a neutralisation buffer (2.5 μL of 0.4 M tricine) was added. Whole genome amplification with multiple displacement amplification (MDA, REPLI-g® Single Cell Kit, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) was then carried out by manufacturer’s instructions. A mixture of 12.5 μL of water, 29 μL of reaction buffer and 1 μL of DNA polymerase (REPLI-g® Single Cell Kit) was added to extracted DNA, making a total volume of 50 μL. The mixtures were incubated at 30 °C for 2 h then at 65 °C for 5 min to inactivate the reaction.