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Enzymes
Published in Stephen W. Carmichael, Susan L. Stoddard, The Adrenal Medulla 1986 - 1988, 2017
Stephen W. Carmichael, Susan L. Stoddard
Similar results were reported by O’Malley, Anhalt, Martin et al. (1987). They isolated a full-length genomic clone for human TH. A human genomic library was constructed and screened by using a rat cDNA for TH as a probe. With various techniques, they found that, in contrast to the rat gene, the human gene for TH undergoes alternative RNA processing to generate at least three distinct mRNAs. They also compared the human TH and phenylalanine hydroxylase genes and suggested that both evolved from a common ancestral gene and that major changes in the size of introns have occurred since their divergence.
DNA TECHNIQUES FOR THE AUTHENTICATION OF CHINESE MEDICINAL MATERIALS
Published in Kevin Chan, Henry Lee, The Way Forward for Chinese Medicine, 2001
Pang-Chui Shaw, Fai-Ngor Ngan, Paul Pui-Hay But, Jun Wang
Microsatellites or simple sequence repeats (SSRs) such as (AC)n, (TCT)n, (TCTG)n are tandem repeats of less than six base pairs and are ubiquitous in plants (Saghai-Maroof et al. 1994; Roder et al. 1995) and mammals (Vergnaud 1989). Sequence information can be searched from DNA databases or by screening genomic libraries with oligodeoxyribonucleotides (Cornell et al. 1991). They can also be obtained by screening a genomic library enriched with repetitive sequences. The regions flanking the microsatellite loci can be amplified by PCR, thus providing co-dominant sequence-tagged sites (STS) or repetitive sequence to act as a probe to generate DNA fingerprints such as for the differentiation between Panax ginseng and P. quinquefolius (Leung and Ho 1998). However, the development of such markers may be laborious and expensive as it involves library construction, screening and sequencing of clones.
The Gene for t-PA
Published in Cornelis Kluft, Tissue-Type Plasminogen Activator (t-PA): Physiological and Clinical Aspects, 1988
Tor Ny, Monica Ohlsson, Leif Strandberg
The general strategy for the construction of a genomic library first requires the isolation of DNA fragments from a donor organism. This is done by either shearing or enzymatically cleaving the chromosomal DNA. Using the enzyme DNA ligase, genomic DNA fragments are joined to a suitable cloning vector in vitro. The recombinant DNA molecules are then introduced into E. coli cells where they multiply (Figure 1). When a specific gene is isolated from a genomic library, it will be in the form found on the chromosome. It will therefore contain exons, introns, and regulatory regions, such as promotor and enhancer sequences. Since a gene isolated from a genomic library contains regulatory sequences, it can be reintroduced into eucaryotic cells, and its expression studied.
Comparison of Intraocular Antibody Measurement, Quantitative Pathogen PCR, and Metagenomic Deep Sequencing of Aqueous Humor in Secondary Glaucoma Associated with Anterior Segment Uveitis
Published in Ocular Immunology and Inflammation, 2022
Li Wang, Zhujian Wang, Jinmin Ma, Qiongfang Li, Xueli Chen, Yuhong Chen, Xinghuai Sun
Total RNA nucleic acids were extracted from 50 μl aqueous humor by phenol-chloroform method with TRIZOL. Briefly, nucleic acids (RNA) were reversely transcribed to complementary DNA (cDNA) using random primers and then amplified using a multiple displacement amplification (MDA) random-amplification approach (REPLI-g Single Cell WTA kit, Qiagen, Germany).21,22 Genomic library construction and next-generation sequencing (NGS) were conducted according to the BGI-Seq500 sequencing protocol. Amplified cDNA was purified using AmPure beads (Qiagen, Germany). The products were then quantitated by Qubit Fluorometer 3.0 (Life Technologies, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies Inc. USA). A total of about 1 μg of amplified products was fragmented into 250 bps by Covaris E210 (Covaris, USA) and then was constructed into the library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle amplification. The DNBs were loaded on the patterned nanoarrays and sequenced on BGI-Seq500 platform (BGI, China) using a single-end 100 bp sequencing.
Inulin-grown Faecalibacterium prausnitzii cross-feeds fructose to the human intestinal epithelium
Published in Gut Microbes, 2021
Raphael R. Fagundes, Arno R. Bourgonje, Ali Saeed, Arnau Vich Vila, Niels Plomp, Tjasso Blokzijl, Mehdi Sadaghian Sadabad, Julius Z. H. von Martels, Sander S. van Leeuwen, Rinse K. Weersma, Gerard Dijkstra, Hermie J. M. Harmsen, Klaas Nico Faber
Taxonomy of the fecal microbiome was characterized in a high-resolution fashion using whole-genome metagenomic shotgun sequencing (MGS). Microbial DNA extraction from frozen fecal samples and MGS sequencing using the Illumina HiSeq platform was performed as described previously.55 Genomic library preparation was performed using the Nextera XT Library preparation kit. Trimmomatic (v.0.32) was used to remove adapters and trim the ends of metagenomic reads.49,56 Cleaned metagenomic reads were processed through a previously published bioinformatics pipeline.49 Taxonomic compositions were profiled using the software tool MetaPhlAn2 and were expressed as relative abundances in the microbiome samples.57
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
Targeted error correction sequencing (TEC-Seq) is a barcode-based technique. cfDNA is extracted from plasma and converted to a genomic library through ligation of a pool containing a small number of dual-index barcode adapters. The resulting DNA library is captured and redundantly sequenced to produce multiple duplicates of each DNA fragment. Sequence reconciliation among duplicate fragments identifies alterations present in identical DNA molecules with the same start and end position and barcodes. This technique allows to eliminate error sequencing [123,126].