Assessing the Microbiome—Current and Future Technologies and Applications
David Perlmutter in The Microbiome and the Brain, 2019
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.
Encephalitozoon
Dongyou Liu in Handbook of Foodborne Diseases, 2018
The report about the implementation of methods of DNA chips (DNA “microarray”) for the parallel detection of several species of microsporidia (E. cuniculi, E. hellem, and E. intestinalis) in clinical samples75 is very interesting. The great advantage of a DNA “microarray” compared to the PCR method is the ability to diagnose a high number of unknown samples. Unlike PCR, DNA is not obtained through laborious processing of spores by preextraction steps, but by using FTA filters, which not only eliminates these labor-intensive procedures, but also helps avoid the significant loss of DNA and effectively removes inhibitors from fecal samples. Compared to the commercial DNA extraction kits, this method results in lower financial costs, requires less technical training, requires less equipment, and can process a larger number of samples simultaneously.76 The disadvantage of DNA “microarray” is not only expensive laboratory instrumentation, but also the synthesis of large amounts of primers. But once the preparation of the DNA microarray is completed, the actual execution of tests is considerably cheaper. The method of the DNA microarray, as described by Wang et al. (2005),75 represents a combination of the PCR method, which is followed by hybridization of the amplicons using more specific probes immobilized on a microchip. The fluorescence intensity correlates with the abundance of DNA in a sample.
Sexually Transmissible Viral Pathogens: Human Papillomaviruses and Herpes Simplex Viruses
Attila Lorincz in Nucleic Acid Testing for Human Disease, 2016
New automated extraction methods currently under development162 are intended to optimize DNA extraction from collected samples and thus decrease overall cost. However, because the cost of HSV DNA testing can vary widely among laboratories and is currently limited to in-house techniques rather than standardized commercial diagnostic kits, cost should be considered in deciding on the use of PCR-based technology in different clinical settings. PCR-based ascertainment methods are also characterized by significant costs for equipment and technician time. Reimbursement systems for the diagnosis of HSV infections also vary and will influence decisions concerning which diagnostic methods are ultimately chosen.
A microbial signature following bariatric surgery is robustly consistent across multiple cohorts
Published in Gut Microbes, 2021
Farnaz Fouladi, Ian M. Carroll, Thomas J. Sharpton, Emily Bulik-Sullivan, Leslie Heinberg, Kristine J. Steffen, Anthony A. Fodor
Fecal samples were collected at baseline and each timepoint after surgery and stored at −80°C until analysis. DNA extraction was performed as previously described.41 Briefly, a phenol/chloroform extraction method combined with physical disruption of bacterial cells and a DNA clean-up kit (Qiagen DNeasy Blood and Tissue extraction kit, Valencia, CA) was used to extract DNA from fecal samples. For 16S rRNA gene sequencing, the V4 variable region was amplified by polymerase chain reaction (PCR) using 16S rRNA gene primers (forward: 5ʹ-CAACGCGARGAACCTTACC-3ʹ; reverse: 5ʹ-CAACACGAGCTGACGAC-3ʹ) and sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA) at the High-Throughput Sequencing Facility in the Carolina Center for Genome Sciences at the UNC School of Medicine as previously described.42 For metagenomics, extracted DNA was subjected to 2 × 150 bp paired-end sequencing on the Illumina HiSeq 4000 platform at the UNC-Chapel Hill high throughput sequencing facility. The microbial profile of BS study was primarily characterized through metagenomic sequencing (n =135 metagenomes) and 16S rRNA gene sequencing was additionally performed for a subset of samples (n = 61) to validate the results from metagenomic sequencing.
Diagnostic markers for glaucoma: a patent and literature review (2013-2019)
Published in Expert Opinion on Therapeutic Patents, 2019
In 2001, Craig J. E. et al. formulated another method for the detection of mutation p. Q368X in the myocilin gene which analyzes single-stranded DNA conformational polymorphisms (SSCP) [65,66]. SSCP analysis detects sequence variations (single-point mutations and other small-scale changes) through electrophoretic mobility differences. DNA with a sequence mutation shows a significant mobility difference compared to wild type DNA when it is subjected to nondenaturing (or partially denaturing) conditions [65,66]. The method consists of 5 steps, where DNA extraction is the critical, first step. The overall quality, accuracy, and length of the read DNA sequence can be significantly affected by the characteristics of the sample itself, and the method chosen for nucleic acid extraction. The choice of the DNA extraction method depends on the source or tissue type, and on how it was obtained, handled or stored prior to extraction [66].
Association of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms with coronary artery disease (CAD) in a North Indian population
Published in Cogent Medicine, 2018
Stephen Butler, Aaron Young, Elizabeth C. Akam, Nakul Sinha, Suraksha Agrawal, Sarabjit Mastana
The study population consisted of 166 healthy controls and 159 patients with diagnosed CAD, from Uttar Pradesh, North India. Patients were classified on the basis of at least 50% or more stenosis in one or more coronary arteries, verified through coronary angiography. The healthy controls had no known history of ischemic heart disease, hypertension, diabetes, endocrine or metabolic disorders and were selected after administering a treadmill test to exclude the possibility of the patients having an underlying CAD (Rai, Sinha, Finn, Agrawal, & Mastana, 2016; Rai et al., 2012). The DNA samples were collected with full written consent and study protocol was approved by SGPGIMS Lucknow ethics committee and Loughborough University. Clinical data was gathered on: age, gender, diet (vegetarian vs. non-vegetarian), smoking-status and lipid profiles (plasma total cholesterol (TC), triglycerides (TG), high-density lipoproteins (HDL), low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL) and apolipoprotein B (ApoB)). DNA extraction was done through the use of organic methods, as detailed in Rai et al. (2012).
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