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Genomic Informatics in the Healthcare System
Published in Salvatore Volpe, Health Informatics, 2022
DNA is the code of all biological life on earth. Humans have sought to unravel its mysteries so that the origins of life itself may be revealed. The first sequencing methodology, known as Sanger sequencing, uses specifically manipulated nucleotides to read through a DNA template during DNA synthesis. This sequencing technology requires a specific primer to start the read at a specific location along the DNA template and record the different labels for each nucleotide within the sequence up to 1000–1200 base pairs (bps). Subsequently, an approach called shotgun sequencing was developed for longer read of sequences. In this approach, genomic DNA is enzymatically or mechanically broken down into smaller fragments and cloned into sequencing vectors in which cloned DNA fragments can be sequenced individually. The complete sequence of a long DNA fragment can be eventually generated by these methods by alignment and reassembly of sequence fragments based on partial sequence overlaps.
Basic genetics and patterns of inheritance
Published in Hung N. Winn, Frank A. Chervenak, Roberto Romero, Clinical Maternal-Fetal Medicine Online, 2021
Diagnosis is possible for many genetic disorders using molecular genetic techniques. Genomic DNA can be obtained from peripheral blood leukocytes, solid tissues, or cultured cells. Testing can be accomplished by either direct mutation analysis or by linkage analysis (Fig. 21). In linkage analysis, information about nondisease producing variation, or polymorphisms, such as restriction fragment-length polymorphisms, variable number of tandem repeats, or microsatellite repeat polymorphisms, can trace inheritance of a chromosome containing a disease-producing gene through a family. This method of testing has disadvantages. First, multiple family members are needed to establish the phase of linkage. Second, some families may not be informative for some linked markers. Third, recombination between the marker and the disease gene may occur, causing inaccuracies in the test results.
Investigation of DNA Methylation in Autosomal Dominant Polycystic Kidney Disease
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Unlike working with blood and urine samples, using kidney tissue is advantageous in that a larger yield of genomic DNA can be obtained. However, sample collection is invasive and not easy to get. In mouse studies, an appropriate method is used to euthanize the mouse, followed by perfusion of the kidney to get rid of excess blood. Once the kidneys are harvested, the tissue sample should either be processed fresh or flash frozen and stored at −80°C until needed. When working with frozen tissue, tissue should be removed from the freezer and allowed to thaw on ice if necessary. With an appropriate method, such as the phenol/chloroform extraction technique, genomic DNA is extracted from the tissue, quantified, and either used immediately or stored at −20°C.
Epigenetic control of skin immunity
Published in Immunological Medicine, 2023
Human cells contain two meters of genomic DNA that is tightly folded and packed within the nucleus. Genomic DNA forms a secondary structure referred to as chromatin that fits into a limited space [7]. The basic unit of chromatin, the nucleosome, is consisted of 147 bp genomic DNA and a core histone octamer. DNA is negatively charged and histones are positively charged, and the opposing charges allow DNA to wrap itself tightly around the histone octamer to form a nucleosome. Initiation of transcription requires the binding of RNA polymerase II and several basic transcription factors, called TFIIA and TFIIB, bind to promoters located near the transcription start sites [8]. Sequence-specific DNA-binding transcription factors (TFs) are involved in the enhancement of transcription. TFs bind to enhancers and cause genomic DNA to form looped structures that shorten the distance between enhancers and promoters, thereby promoting the transcription of the target genes. Transcriptional activity is also closely related to the degree of DNA condensation associated with chromatin structure [6,8]. Tightly packed chromatin, called closed chromatin or heterochromatin, restricts the access of RNA polymerase II and the transcription factors to the regulatory sites, and consequently, suppresses the expression of target genes. Open chromatin or euchromatin that is less condensed allows easier access of the transcriptional machinery to DNA, thus setting target genes to be more actively transcribed.
Screening for PIK3CA mutations among Saudi women with ovarian cancer
Published in Journal of Obstetrics and Gynaecology, 2021
Wedad Saeed Al-Qahtani, Manal Abduallah Alduwish, Ebtesam M Al-Olayan, Nada Hamad Aljarba, Al-Humaidhi Em, Fatimah Gh. Albani, Dalia Mostafa Domiaty, Aljohara M. Al-Otaibi, Somaya M. Al Qattan, Alanood S. Almurshedi, Abdelbaset Mohamed Elasbali, Hussain Gadelkarim Ahmed, Bassam Ahmed Almutlaq
Genomic DNA was extracted from FFPW. DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Genomic DNA from adjacent non-cancerous tissues served as control. PCR amplification was carried out targeting exon 9 and 20 of the PIK3CA gene. The primer used for exon 9 was 5′-CATCTGTGAATCCAGAGGGGA-3′ as forwarding primer and 5′- AGCACTTACCTGTGACTCCA-3′ as reverse primer. For exon 20, 5′-CTCTGGAATGCCAGAACTAC-3′ was used as forwarding primer and 5′-ATGCTGTTTAATTGTGTGGAAG-3′ as reverse primer. The PCR mixture contained 50 ng of genomic DNA in a reaction volume of 20 µl containing specific oligonucleotide primers at a final concentration of 5 µM and HotStarTaq Master Mix (Qiagen, Valencia, CA). The cycling conditions include initial denaturation at 95 °C for 10 min followed by 32 cycles of reaction at 95 °C for 30 s, 56 °C annealing temperature for 30 s and 72 °C for 45 s. This was followed by a single extension step at 72 °C for 10 min.
Diagnostic yield of targeted next-generation sequencing in infantile nystagmus syndrome
Published in Ophthalmic Genetics, 2021
Jae-Hwan Choi, Su-Jin Kim, Mervyn G. Thomas, Jae-Ho Jung, Eun Hye Oh, Jin-Hong Shin, Jae Wook Cho, Hyang-Sook Kim, Ji-Yun Park, Seo Young Choi, Hee Young Choi, Kwang-Dong Choi
Genomic DNA was extracted from blood samples of all patients. Standard exome capture libraries were generated using the Agilent SureSelect Target Enrichment protocol for Illumina paired-end sequencing library (version B.3, June 2015) with 1 μg of input DNA. The DNA was quantified and its quality was assessed using PicoGreen and NanoDrop. Each qualified genomic DNA sample was randomly fragmented using the Covaris, followed by adapter ligation, purification, hybridization, and PCR. Captured libraries were subjected to Agilent 2100 Bioanalyzer to estimate the quality and were loaded onto the Illumina HiSeq2500 (San Diego, CA, USA) in accordance with the manufacturer’s instructions. Raw image files were processed for base-calling using HCS software (version 1.4.8) with default parameters, and the sequences for each individual were generated as 100 bp paired-end reads. Sequence reads were aligned to the human reference genome sequence (GRCh37.3, hg19) using the Burrows-Wheeler Aligner (version 0.7.12). PCR duplicate reads were marked and removed using Picard tools (version 1.92). The Genome Analysis Toolkit (version 2.3–9) was used for indel realignment and base recalibration. Variation annotation and interpretation analysis were performed using SnpEff (version 4.2).