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The Precision Medicine Approach in Oncology
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
The DNA sequencing methods used in the 1970s and 1980s such as Maxam-Gilbert and Sanger sequencing were manual techniques requiring a very large amount of manpower, time, and effort. The shift to more rapid automated sequencing methods in the 1990s finally allowed the sequence of whole genomes to be carried out within a reasonable time period. The first bacterial genomes, including that of Haemophilus influenzae, were sequenced by “Shotgun Sequencing” of shorter DNA fragments. The first eukaryote genomes were also sequenced by this method, but involved larger DNA clones from DNA libraries such as Bacterial Artificial Chromosomes (BACs) and Yeast Artificial Chromosomes (YACs). One of the first automated DNA sequencers was the capillary-based ABI PRISM 3100 Genetic Analyzer which allowed a much more rapid and extensive output of data.
Molecular Genetic Approaches to Obesity
Published in Claude Bouchard, The Genetics of Obesity, 2020
Streamson C. Chua, Rudolph L. Leibel
In positional cloning one starts by identifying genes or DNA markers which flank the mutation. All of the DNA between the two closest flanking markers is then cloned by a chromosome “walk”. Such a walk entails using a cloned segment of DNA containing a marker known by conventional genetic analysis to be near the mutation to identify another genomic clone containing a shared sequence. This process is repeated sequentially until a clone is obtained which contains a sequence known by conventional genetics to be on the other side of (i.e., to flank) the mutation. The enormous amount of labor which was initially required to clone loci by positional information has been reduced to more manageable levels by the cloning of large segments of DNA (0.3 to 1.0 million bp) with yeast artificial chromosome (YAC)41 and PI42 cloning systems.
Preimplantation Genetic Testing for Structural Rearrangements
Published in Darren K. Griffin, Gary L. Harton, Preimplantation Genetic Testing, 2020
Until comprehensive chromosomal analysis techniques such as aCGH and NGS systems came along, researchers tried to improve the informativity of the FISH system both quantitively (adding extra probes for aneuploidy) and qualitatively, such that it could also distinguish a balanced chromosome complement from a normal one. For this reason, carrier-specific probes were developed to be used in interphase cells [53]. This approach was based on hybridization of breakpoint spanning yeast artificial chromosome (YAC) DNA probes; however, it requires a major pre-clinical work up developing those probes for each specific translocation.
John Sulston (1942–2018): a personal perspective
Published in Journal of Neurogenetics, 2020
Robert H. Waterston, Donald G. Moerman
John and Alan (the two were so closely associated in communications about the map that a technician in the Waterston lab thought they were one person – John N. Allen) tried new vectors, modified bacterial hosts and targeted searching in even more complex libraries, all with little improvement in genome coverage. Fortunately, students in Maynard Olson’s laboratory were developing yeast based cloning methods, called Yeast Artificial Chromosomes (YACs), that not only offered the possibility of cloning larger genome fragments (the larger the pieces, the easier the puzzle) but also a eukaryotic host with at least different biases than those of E. coli (Burke, Carle, & Olson, 1987). At the time Bob Waterston had the good fortune to be on sabbatical at the LMB and located in John’s lab, spending at least some of his time trying to find solutions to the gap problem. After some discussion, including sessions in the local pub, they agreed that Bob should pursue making worm genomic libraries in YACs upon returning to St. Louis. That cemented a close collaboration that extended over the next 15 years and the sequencing of two genomes, first the worm and then the human.
Yeast-inspired drug delivery: biotechnology meets bioengineering and synthetic biology
Published in Expert Opinion on Drug Delivery, 2019
Chinnu Sabu, Panakkal Mufeedha, Kannissery Pramod
Few types of yeast cloning vectors are seen, and they are so-called shuttle vectors. Shuttle vectors have the ability to replicate and be selected in both bacteria and yeast. Since plasmid preparation from yeast is ineffective, shuttle vectors are developed. Yeast cloning vectors based on 2-μm plasmid are called yeast episomal plasmids (YEPs). YEPs have the capability to replicate independently or can be integrated into one of the yeast chromosomes. They are considered as high copy number vectors consisting of a selective marker and origin of replication. The major disadvantages associated with their recombinants are they are highly unstable making it difficult and time-consuming to achieve a reliable result. Another type of yeast vectors is yeast integrative plasmids (YIPs). YIPs contain a selective marker and lack origin of replication. They cannot replicate independently and have a low transformational frequency. On the other hand, the recombinants are highly stable making it more beneficial. Yeast replicative plasmids are another type of cloning vectors. Their backbone consists of origin of replication in close proximity to the selective marker. They replicate independently with high transformational frequency. Like YEPs, their recombinants are highly unstable. As time passes, there was a large demand for large pieces of DNA to be manipulated. Yeast artificial chromosome (YAC) was developed at this time to combat the problem. YAC composes three main components; centromeres, the origin of replication and telomeres [43]. Researchers are still continuing to engineer yeast to produce eukaryotic proteins. The expressed foreign proteins can be used to synthesize life-saving drugs for the pharmaceutical industry. Yeast offers the simplicity of microbial growth and ability to perform post-translational modification [47]. In addition, they possess certain demerits such as expression of the heterologous protein and inability to perform certain post-translational modification [48].