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Genetic Engineering
Published in Shintaro Furusaki, John Garside, L.S. Fan, The Expanding World of Chemical Engineering, 2019
The Sanger method, which is also called the dideoxy-termination method, is now widely used for DNA sequence determination and automated or semi-automated instruments were developed more than ten years ago. In the Sanger method, fluorescent dye labeled primers or terminators (dideoxynucleotides) are used. The terminators are analogs of the normal deoxyribonucleoside triphosphates that lack 3’-hydroxyl group. In a polymerase reaction, template single strand DNA, excess amounts of primers and four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP and dTTP), and fixed amounts of dideoxy-terminators are included. Due to the enzyme reaction, nucleotides are incorporated into the growing strand one by one to complement the template. Chain elongation by DNA polymerase occasionally stops at each step when a dideoxyribonucleotide is incorporated, instead of a corresponding normal deoxyribonucleotide, since the absence of the 3’-hydroxyl group in dideoxynucleotides prevents addition of the next nucleotide. If the mixing ratio of dideoxy-terminators and normal deoxyribonucleotides is suitable, a set of DNAs of different lengths complementary to the template and terminating at each of the different nucleotides of the sequence is produced by the reaction (Figure 12.2).
Basic Molecular Cloning of DNA and RNA
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
DNA sequencing involves the determination of the identity of each of the base pairs in a target region. Sequencing technology has become orders of magnitude faster and cheaper over the past three decades. As of 2016, a large number of full eukaryotic genomes have been sequenced, including those of mammals (human, cow, rat, mouse, cat, dog, tiger, elephant); birds (chicken, duck, eagle, pigeon—a very large number of bird genomes were reported in 2014); other animals (puffer fish, mosquito, alligator); plants (corn, rice, poplar, watermelon, papaya); and many protists. Dozens of bacterial and archaeal genomes have also been sequenced. Sequencing is performed on automatic sequencers that work on essentially the same principle as PCR. The DNA is denatured in the presence of primers, and elongation begins. However, into the mix of abundant free A, C, G, and T are added dideoxynucleotides, each fluorescently labeled with a different color. (This is called dye-terminator sequencing.) The dideoxynucleotides lack a 3′ OH group, so when they are added to a strand, elongation terminates—no new nucleotides can bind. Since most of the nucleotides in the reaction are ordinary, the point at which a deoxynucleotide will be added is random, leading to an eventual series of fragments of all different sizes that terminate in one of four colors. By resolving the size of these fragments with single-base-pair resolution, the position of each A, C, G, or T in the sequence can be known (Figure 2.12). Size resolution used to be done on meter-sized sequencing gels; modern sequencers use capillary electrophoresis.
DNA Structure, Sequencing, Synthesis, and Modification: Making Biology Molecular
Published in Richard J. Sundberg, The Chemical Century, 2017
A second method was developed at about the same time by Frederick Sanger,g working at Cambridge in the United Kingdom. The concept is similar but the method relies on synthesis of a complementary strand of DNA, rather than cleavage of the strand. Each of four tubes is set up with the single-stranded DNA to be sequenced, a primer, DNA polymerase and the four deoxynucleotides. In each tube is placed a small amount of one of the four dideoxy analogs of the nucleotides. Each time a dideoxynucleotide is introduced into the growing strand it terminates, because there is no 3-hydroxy group for continuation. A radioactive label is incorporated into the primer that is used to start the synthesis. The four lanes are subjected to electrophoresis and the sequence can be read. It is the complement of the strand that is being sequenced. This method is several times faster than the Maxam–Gilbert method. Figure 20.5 shows the concept of the Sanger dideoxy method. Gilbert and Sanger shared the 1980 Nobel Prize in Chemistry with Paul Berg, who pioneered the formation of recombinant DNA (see Section 20.4).
Culturing the uncultured microbial majority in activated sludge: A critical review
Published in Critical Reviews in Environmental Science and Technology, 2023
Sanger sequencing developed in 1977 is a method to sequence DNA using electrophoresis based on DNA polymerase's random incorporation of chain-terminating dideoxynucleotides during in vitro DNA replication. Sanger sequencing is still a widely used method now although it has been largely replaced by high-throughput sequencing methods in recent years, particularly for large-scale automated genome analyses. In cultivation studies, Sanger method is used in the final identification of a single colony or isolate as it can produce DNA sequence reads of >500 nucleotides and maintains a very low error rate (>99.99%). The new species is identified if its similarity of 16S rRNA gene with cultured species is below 98.65 (Kim et al., 2014; Yarza et al., 2014), and the current commonly used 16S rRNA databases include NCBI (Sayers et al., 2022), Silva (Quast et al., 2013), Greengene (DeSantis et al., 2006), and EzBioCloud (Yoon et al., 2017). Once the new species are confirmed, the corresponding steps should be followed for new species announcement, such as the brief description of sources and growth conditions of isolates, a panel of experiments to evaluate the morphological and biochemical characteristics, genotypic and phenotypic information for taxonomy classification, and constructions of GenBank 16S rRNA accession numbers and an assembly of strain numbers to promote the propagation of novel species.
Next-generation DNA sequencing of oral microbes at the Sir John Walsh Research Institute: technologies, tools and achievements
Published in Journal of the Royal Society of New Zealand, 2020
Nicholas C. K. Heng, Jo-Ann L. Stanton
Ever since Avery et al. (1944) provided experimental evidence supporting DNA as the ‘transforming principle’ (i.e. genetic material) of cells and the subsequent elucidation of the molecular structure of DNA by Watson and Crick (1953), there has been much research conducted into finding more effective, cost-efficient, and accurate ways to determine the nucleotide sequence of any DNA molecule. It was not until 1977 that two competing sequencing methods were introduced: (i) the dideoxynucleotide chain-termination method of Sanger et al. (1977), and (ii) the chemical modification/cleavage method of Maxam and Gilbert (1977). The ‘Sanger’ sequencing method, which utilised radioactively-labelled synthesis-terminating dideoxynucleotides in combination with DNA polymerase and X-ray films, prevailed as it was more efficient and used less radioactivity. Over the next two decades, Sanger sequencing underwent several key advances including (a) the replacement of radioactive labels with fluorescent dyes, (b) the development of highly-sophisticated fluorescence detection instruments, and (c) the use of thermostable DNA polymerases to facilitate longer sequence read lengths. Current Sanger-based sequencing instruments such as the Applied Biosystems 3730xl Genetic Analyzer can process up to 384 sequencing reactions in a single run, each yielding >900 basepairs (bp) of reliable sequence data (Liu et al. 2012). Despite the long read lengths, Sanger-based sequencing is labour-intensive and relatively expensive (NZ$9.50 per sequence) and is thus largely limited to confirmatory sequencing, e.g. of cloned DNA fragments in recombinant plasmids. Nevertheless, the first bacterial genome to be sequenced completely, that of Haemophilus influenzae, was achieved purely with Sanger-based sequencing (Fleischmann et al. 1995). Even more than 40 years since its invention, Sanger sequencing remains the ‘gold standard’ for nucleotide sequencing with unsurpassed accuracy (Liu et al. 2012). However, significant advancements in miniaturisation technology meant that it was only a matter of time before cost-effective high-throughput DNA sequencing systems would become a reality.