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Describe the process of “sequencing by synthesis” that Illumina uses to determine the
base pair sequence of a single DNA template cluster.Illumina Bridge Amplification/Sequencing
Illumina technology made in California can determine hundreds of bacterial identities
and quantities simultaneously and easily for about $40 per sample.
After the amplification of a template into a cluster by PCR, Illumina Bridge amplification
products are sequenced. This is tedious and can take up to 11 days.
Describe the process of “sequencing by synthesis” that Illumina uses to determine the
base pair sequence of a single DNA template cluster.

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Keith Leannon
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28 Jan 2018
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Read the summary and answer questions in bold below:

In this post, we’ll be focusing on Tyler Jacks and his graduate student Francisco J. Sánchez-Rivera’s review, “Applications of the CRISPR-Cas9 system in cancer biology,” circa 2015 in Nature Reviews. So we understand the context of the authors, Jacks is the director of the Koch Institute for Integrative Cancer Research at MIT. His lab has a large mouse experimental component (MIT 2017). This sets the stage for the angle we’ll be reading from. In all, it’s a nice roundup of his and colleagues' cancer applications of CRISPR-Cas9 with great graphics, but leaves a bit desired in crossing the bridge from mouse to human.

Briefly, the CRISPR-Cas9 system stems from the prokaryotic immune system, where the RNA-guided DNA endonuclease Cas9 uses a single guide RNA (sgRNA) containing a CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) that binds Cas9 and directs it to the genomic sequence of interest, adjacent to a photospacer adjacent motif (PAM). Cas9 causes a precisely-directed double-stranded break (DSB) in the DNA, that gets repaired via non-homologous end joining (NHEJ) or homology-directed repair (HDR).

With NHEJ, this can disrupt the gene by creating Indels: frameshifts and stop codons. In cancer, individual oncogenes or tumor suppressors can be targeted, or drug response modulators, or synthetic lethality genes. With a multiplexed knockout approach, combinatorial vulnerabilities and epistatic relationships can be assessed.

For gene modification via HDR, target single or double-stranded DNA can be introduced, resulting in precise gene knock-ins, with potential effects of introducing point mutations to mimic cancers, fluorescent reporters/synthetic tags can be created, conditional (Cre-lox/Flp-FRT) constructs can used to turn off/on a mutation, non-coding elements like enhancers/insulators/promoters can turn on proto-oncogenes, and ultimately, HDR can be used for gene correction.

With Cas9 and 2 sgRNAs, there can be genomic rearrangements, leading to deletions, inversions (like EML4-ALK), and translocations (like BCR-ABL).

Dead Cas9 (dCas), which lacks its endonuclease activity via mutation to its HNH and RuvC domains, can be used for reversible, transcriptional repression or activation of coding and non-coding genes, or chromatin modification, via an effector.

Much of the practical applications of CRISPR-Cas9 is in ES cells and mouse models, both germinal and somatic (GEMMs and nGEMMs). With this technology, a plethora of GEMM/nGEMM models of cancer can be created, some with the ability to turn on/off the Cas9 system, and multiplexed sgRNAs can help gain further understanding of multi-hit cancers. Patient derived xenografts (PDX) mice can be assayed with CRISPR-Cas9 to determine different treatment efficacies. Entire libraries of sgRNA can be screened in cancer cell lines for enrichment/depletion with Cas9, and dCas9-activators/repressors can be used for complementarity, both of which providing insights into drug targets, synthetic lethality, and chemotherapy modulators.

The delivery mechanisms for the system are diverse, and include plasmids, lentivirus, adenovirus, nanoparticles, hydrodynamic gene transfer, and protein-based. The authors also cite integrating Cas9 into the GEMMs, so it’s one less thing needed in the delivery vehicle. This of course would not be applicable to human.

Ultimately, human cancer treatment is the goal. Yes, we know the boundaries between ES/mice experimental models and human embryos have already been broken, with an international team targeting the autosomal dominant mutation in MYBPC3, which causes cardiac myopathy (Ledford 2017). But the current compromise position of CRISPR-Cas9 technology toward therapeutics and treatment, beyond basic science understanding, is using primary cells, organoids, PDX, or personalized GEMMs to create genetic and drug screens.

In theory, what would it take to bring CRISPR-Cas9 to the ultimate, targeted gene therapy via either NHEJ or HDR, in human cancer? Are there cancers that are better bets for CRISPR-Cas9 than others, delivery vehicles that are safer or better optimized? Is CRISPR-Cas9 really “on target” enough to introduce into a person, and how do we best assess?

Human body can be described by many means – one of them – it is relatively big vessel with complex body fluid. For diagnostic purpose it is very complicated to detect insignificant number of biomarkers on very early stages of pathological process. This article, present pioneering method to initiate and conduct diagnostic by researching body fluids.

**With additional research provide additional examples of scientific way of dealing with solutions to improve functionality of human life. (QUESTION - This is exactly what is witten on my assignment)**

Analyzing human blood for a very low virus concentration or a sample of water for a bioterrorism agent has always been a time-consuming and difficult process. Researchers at the Georgia Institute of Technology and Emory University have developed an easier and faster method to detect these types of target molecules in liquid samples using highly porous, micron-sized, silica beads. The researchers developed a technique to simultaneously or sequentially add optical and magnetic nanoparticles into the beads. Adding magnetic nanoparticles allows the use of a magnetic field to attract and easily remove the beads from a liquid sample. “These nanoparticles enter the pores of the microbeads so quickly and so completely -- essentially more than 99 percent of the nanoparticles go into the pores of the beads,” explained Shuming Nie, the head researcher on the project and the Wallace H. Coulter Distinguished Chair in Biomedical Engineering and director of Emory-Georgia Tech Nanotechnology Center. The beads are mixed in a liquid such as urine. Viruses, proteins or other biomarkers are captured on the bead surface. After the beads are removed from the liquid, optical imaging is used to determine the concentration of a specific protein or virus in the liquid sample based on the number of proteins or viruses attached to the surface of the beads. Tushar Sathe, a graduate student in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, described the process of creating these novel beads and their clinical applications on Jan. 20 at SPIE Photonics West in San Jose, California. The work was also published in the Aug. 15 issue of Analytical Chemistry. The technology involves embedding fluorescent quantum dots and magnetic iron oxide nanoparticles inside the beads to create dual-modality magneto-optical beads. Nie and Sathe synthesize the quantum dots in different colors by varying their size, giving the beads a unique optical signature. Having different color beads allows the researchers to detect several target molecules at the same time in the same liquid sample. “We use the quantum dots to create a set of beads that are unique and can be distinguished from each other. It’s similar to bar-coding -- once you barcode the beads and put them in the urine or blood sample, you can remove them and decode what proteins or viruses have attached to individual beads based on their spectral signature,” explained Sathe. The process of creating these beads is quite simple, according to Sathe. The surface of the beads contains a long-chain carbon molecule that makes the beads hydrophobic, meaning they repel water. The beads are dissolved in butanol and washed several times. Then the beads are counted and optical and magnetic nanocrystals are added to the suspension either simultaneously or sequentially. After 15-20 minutes, the butanol is removed to get rid of any remaining nanoparticles that didn’t get incorporated into the beads and the beads are washed with ethanol. Then the beads are coated with a polymer that creates a hydrophilic surface on the beads. This allows the beads to be functionalized by adding antibodies or DNA molecules to the surface that will capture the target molecules. These beads are dual-function -- both optical and magnetic -- but according to Sathe, more functions can be added to the beads. “Adding them is as easy as adding the nanoparticles into the solution. You just have to make sure the nanoparticle surface is hydrophobic so that it interacts with the beads,” said Sathe. The primary biomedical applications for this new technology will be to detect cancer and neurological diseases by identifying certain molecules present in human blood or urine that indicate specific diseases, according to Nie, who is also professor of biomedical engineering, chemistry, materials science & engineering, and hematology and oncology at Emory University and the Georgia Institute of Technology. “Some of the biomarkers for Alzheimer’s disease have very low concentrations in the blood so you need highly sensitive techniques that can find a specific molecule to diagnose this disease,” explained Nie. “Our technique could also be used to monitor therapeutic response. For example, if the viral level decreases in samples taken at later dates, then we know the drug is probably working.” This new technology allows the researchers to analyze very low concentrations of target molecules. “Instead of analyzing a liter of sample where the concentration could be very dilute and you might not see the target molecule you’re looking for, you can let the beads capture the molecules on their surface, remove them from the liquid, and then just measure the number of molecules attached to the beads,” said Nie. This ongoing research is funded by the National Cancer Institute, the Department of Energy’s Genomes to Life (GTL) Program, the Department of Defense and the Georgia Cancer Coalition, a public-private partnership established by the Georgia General Assembly in 2001.

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