L23-Genomics Personalised Medicine PDF

Figure 21.1 in Campbell Biology by Reece et al., (10th edition) The International Human Genome Sequencing Celera Genomics, Science, Vol 291(5507): 1304- Consortium Nature, 409 (6822): 860-921, 2001 1351, 2001 § Fundamentals of genomics and mapping of genomes. § Basics of DNA sequencing, inc...

Figure 21.1 in Campbell Biology by Reece et al., (10th edition) The International Human Genome Sequencing Celera Genomics, Science, Vol 291(5507): 1304- Consortium Nature, 409 (6822): 860-921, 2001 1351, 2001 § Fundamentals of genomics and mapping of genomes. § Basics of DNA sequencing, including key methods and the step-by-step process involved. § Techniques for studying gene expression, focusing on when and how genes are activated in different contexts. § Role of genomics and multi-omics in advancing personalized medicine, illustrating its impact on tailored healthcare solutions. § The complete collection of genetic instructions inherited by an organism is known as its genome. § A typical human cell contains two similar sets of chromosomes. § Each set includes approximately 3 billion nucleotide pairs of DNA. § Estimated 20,000–25,000 genes are encoded in the human genome. § Less than 0.1% difference in DNA sequence among individuals, yet accounts for all human diversity. § Researchers analyze “entire sets of genes” or other DNA segments across one or more species, this comprehensive approach is known as genomics. § Aims to understand the structure, function, evolution, and mapping of genes within and across species. § Relies on high-throughput technologies (DNA sequencing, bioinformatics, and computational biology) to analyze large volumes of genetic data rapidly. https://www.genome.gov/about-genomics/educational-resources/fact-sheets/human-genome-project  Determine sequence of 3 Map genomes of billion chemical bases that selected non- make up human DNA human organisms Identify all estimated genes in human & other model organism DNA Store information in databases, and make it freely available Address ethical, legal, and social issues that may arise from project https://www.genome.gov/about-genomics/educational-resources/fact-sheets/human-genome-project https://www.genome.gov/about-genomics/educational-resources/fact-sheets/human-genome-project Figure 21.2 in Campbell Biology by Reece et al. Page 437, (10th edition) https://www.genome.gov/human-genome-project/timeline  Craig Venter Founded Celera Genomics Pioneered Shotgun Sequencing Private-Public Race Draft Genome Publication https://www.genome.gov/human-genome-project/timeline  Molecular Medicine Environment Agriculture and Livestock Microbial Genetics Risk assessment of individuals to specific allergens Anthropology, Evolution, and Human Migration First genome-wide look at the similarities and differences of the closest evolutionary relative to humans Science, 2010 § DNA Sequencing involves determining the exact order of the bases in DNA — the As, Cs, Gs and Ts that make up segments of DNA. § Human Genome Project aimed to sequence all the DNA (i.e., the genome) so significant effort was made to improve the methods for DNA sequencing. § Human Genome Project used one particular method for DNA sequencing, called Sanger DNA sequencing § This basic method was greatly advanced through a series of major technical innovations. Standard sequencing machine Figure 20.2 in Campbell Biology by Reece et al. Page 409, (10th edition) § Awarded the 1958 Nobel Prize in Chemistry for determining the amino acid sequence of insulin and other proteins. § Received the 1980 Nobel Prize in Chemistry, shared with Walter Gilbert and Paul Berg, for pioneering the first DNA sequencing technique. § Nobel Prize in Chemistry (1958, 1980) From Wikipedia  DNA denatured into single strands. Incubated with a primer designed to base-pair with known 3’ end of the template strand. Added DNA polymerase, 4 dNTPs & 4 ddNTPs, tagged with specific fluorescent molecule. Dideoxyribonucleotide (ddNTP), a modified deoxyribonucleotide (dNTP), its incorporation terminates a growing DNA strand because it lacks a 3’OH group Figure 20.3 in Campbell Biology by Reece et al. Page 410 (10th edition)  Synthesis of each new strand starts at the 3’ end of the primer. The incorporated ddNTP prevents further elongation of the strand. Figure 20.3 in Campbell Biology by Reece et al. Page 410 (10th edition)  The labeled strands in mixture are separated by passage through a gel. A fluorescence detector senses the color of each fluorescent tag as the strands come through. Figure 20.3 in Campbell Biology by Reece et al. Page 410 (10th edition) 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 Haemophilus influenzae sequencing S. cerevisiae sequencing D. melanogaster sequencing A. thaliana sequencing Mouse genome sequencing Human genome sequencing  NGS techniques bypass chain termination (Sanger’s method). Next-generation sequencing machines Figure 20.2 in Campbell Biology by Reece et al. Page 409 (10th edition) 1 Genomic DNA is fragmented. 3 Fragment is repeatedly copied using PCR. Fragments in range of 400 to All 5ʹ ends of one strand are specifically 1,000 base pairs are selected. captured by the bead. 10⁶ identical copies of the same single strand are attached to the bead. 1 2 3 Each fragment is isolated on a bead. 2 Bead is placed in a droplet of aqueous solution. Figure 20.4 in Campbell Biology by Reece et al. Page 411 (10th edition)  Bead is placed in a small well. DNA polymerases and primers, which can hybridize to the 3ʹ end of single Each well contains a unique DNA fragment. template strand, are added to well. A solution of a single nucleotide is added to all wells and then removed (sequential for each nucleotide) 4 5 Figure 20.4 in Campbell Biology by Reece et al. Page 411 (10th edition)  In each well, if the next base on the template strand (e.g., T) is complementary to added nucleotide (e.g., A), nucleotide is incorporated into the growing strand. This incorporation releases PPi, producing a flash of light. Figure 20.4 in Campbell Biology by Reece et al. Page 411 (10th edition) PPi, or pyrophosphate, is a byproduct released during DNA synthesis when a nucleotide, added as a deoxynucleoside triphosphate (dNTP), incorporates into the DNA strand and releases two of its three phosphate groups.  Nucleotide is removed by washing & a different nucleotide (e.g., dTTP) is introduced. If new nucleotide is not complementary to the next base on template strand (e.g., G), it will not be incorporated into the strand & no flash of light recorded. Figure 20.4 in Campbell Biology by Reece et al. Page 411 (10th edition)  Imaging contribution! The procedure of adding and washing off the four nucleotides is repeated until a complete complementary strand is formed for each fragment. Figure 20.4 in Campbell Biology by Reece et al. Page 411 (10th edition)  DNA is not fragmented or amplified; instead, a single, long DNA molecule is sequenced directly. In one model, nanopore sequencing a single DNA strand passes through a tiny pore (nanopore) in a membrane. Bases are detected individually by measuring how each one interrupts an electrical current as it moves through the nanopore.  nanoporetech.com The pore is a protein channel embedded in a lipid membrane or others use artificial membranes and nanopores. Each base type is expected to interrupt the current for a distinct duration. Figure 20.1 in Campbell Biology by Reece et al. Page 408 (10th edition) Massively parallel sequencing of millions of fragments of DNA simultaneously (Illumina sequencing and Ion Torrent). § HGP announcement by Bill Clinton marked a milestone in genomics. § Genomics: Shotgun sequencing revolutionized DNA analysis. § DNA Sequencing based on chain termination: Sanger method, awarded Nobel Prize, set the foundation. § Next-Generation Sequencing (NGS): Bypasses chain termination, enabling faster and high-throughput sequencing. § Nanopore Sequencing: Single DNA strand passes through a nanopore in a membrane, detecting bases in real-time. § Lab Demo: Overview of Ion Torrent technology. Figure 20.12 in Campbell Biology by Reece et al. Page 419 (10th edition)  In situ hybridization allows us to see the mRNA in place (or in situ) in intact organism. mRNA could be detected by nucleic acid hybridization using complementary sequence Probe is a short, single-stranded nucleic acid that is designed to be complementary to the target mRNA sequence of interest. Wingless (wg) gene encodes a secreted signaling protein; engrailed (en) gene encodes a transcription factor. Figure 20.10 in Campbell Biology by Reece et al. Page 418 (10th edition)  Determining where genes are expressed by in situ hybridization analysis? Yellow probe hybridizes with mRNAs in cells that are expressing the wingless (wg) gene. Blue probe hybridizes with mRNAs in cells that are expressing the engrailed (en) gene. Figure 20.10 in Campbell Biology by Reece et al. Page 418 (10th edition)  One aim is to identify networks of gene expression across an entire genome. Genome-wide expression studies can be carried out using DNA microarray assays. Wikipedia  Tiny DNA spots arranged in a grid on a silicon wafer represent nearly all human genes. This chip allows researchers to analyze expression patterns of all these genes simultaneously. By examining which genes are over- or under- expressed in a specific cancer, physicians may tailor treatments to each patient's unique genetic profile. Figure 21.5 in Campbell Biology by Reece et al. Page 437, (10th edition)  mRNA Isolation: Extract the mRNAs synthesized in the target cell, & generate the corresponding cDNAs through reverse transcription. cDNA Labeling: In microarray assays, label the synthesized cDNAs with fluorescent dyes to enable visualization. Hybridization: Allow the labeled cDNAs to hybridize to a DNA microarray, where they will bind to complementary DNA sequences. Control and Test Comparison: cDNA from control and test samples are labeled with distinct fluorescent colors, allowing simultaneous analysis on the same microarray.  Cancer Cell Normal Cell RNA DNA Microarray Chip Reverse Transcription & Label Cy5 - Red cDNA Cy3 - Green Combine equal Principle - ability of amounts complementary sequence to bind Hybridize to the immobilized DNA probes mixture to chip Figure 20.13 in Campbell Biology by Reece et al. Page 421, (10th edition) Campbell Biology by Reece et al. Page 387, (10th edition) Therese et al. PNAS 2003;100:8418-8423  RNA-Seq is a powerful technique for analyzing the transcriptome, providing insights into gene expression, RNA structure, and alternative splicing. Key Steps: Extraction of total RNA from cells or tissues. Library Preparation: Conversion of RNA to cDNA using reverse transcription. Fragmentation of cDNA and addition of sequencing adapters. Sequencing: High-throughput sequencing of cDNA fragments using platforms such as Illumina or Oxford Nanopore. § Studying Gene Expression: Key techniques for analyzing gene activity discussed. § RT-PCR: Focuses on expression of individual genes. § In situ Hybridization: Visualizes mRNA within intact tissues. § RNA Sequencing (RNA-Seq): Comprehensive approach to study transcriptomes. § DNA Microarray Chip: Enables genome-wide expression studies. § Breast Tumor Analysis: Examined gene expression patterns in tumor subtypes. § Personalized medicine tailors medical treatment to the individual characteristics of each patient, including their genetic profile. § It aims to improve treatment efficacy, minimize adverse effects, and enhance patient outcomes. § It is envisioned that future is “personalized medicine” where each person’s genetic profile could predict diseases or risk conditions and help them with therapeutic modalities. § Genomics enables identification of specific genetic mutations associated with diseases, particularly cancer, leading to the development of targeted therapies that specifically attack those mutations. § HER2-positive Breast Cancer: Women with breast cancer that overexpresses HER2 protein can be treated with trastuzumab (Herceptin) § a targeted therapy that inhibits HER2 signaling, significantly improving treatment outcomes. § Dr. Lukas Wartman, a physician and Dr. Lukas Wartman Washington University cancer researcher, diagnosed with School of Medicine acute lymphoblastic leukemia (ALL) § Genomic information helped identify specific genetic targets for treatment. § Examples: § FLT3 (FMS-like tyrosine kinase 3) over- expression § FLT3 inhibitor sunitinib (FDA - renal cell cancer) New York Times 7/7/12 issue  § Genomic and multi-omics data provide insights into disease progression and treatment response, enabling personalized management strategies. § Chronic Myeloid Leukemia (CML): Patients are monitored for the BCR-ABL fusion gene through quantitative PCR. If levels rise, indicating disease progression, treatment with imatinib (Gleevec) can be adjusted accordingly, enhancing patient outcomes. BCR (Breakpoint Cluster Region) and ABL (Abelson) BCR-ABL fusion gene produces an overactive enzyme (tyrosine kinase) that drives the excessive production of white blood cells in CML. § Multi-omics approaches integrate various biological data (genomics, proteomics, metabolomics) for holistic view of health and disease. § How multi-omics integration can lead to more precise therapeutic targets and personalized treatment plans for cancer patients? § Highlighted the potential for improved patient stratification and tailored therapeutic approaches based on individual molecular profiles. Michigan Center for Translational Pathology Chinnaiyan et al. Nat Genet, 2013, 45, 180-5; Sci Transl Med. 2011, 3, 111ra121 https://www.cancer.gov/research/key-initiatives/moonshot-cancer-initiative/about § The Clinical Proteomic Tumor Analysis Consortium (CPTAC) is a program under the Cancer Moonshot aimed at advancing the understanding of cancer through proteomics. § To characterize the proteomes of various tumors, enabling the identification of novel biomarkers and therapeutic targets. https://proteomics.cancer.gov/programs/cptac  Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. To identify genetic alterations in GBMs through comprehensive genomic analysis. Next-Generation Sequencing: Conducted gene expression analyses on 22 human tumor samples. Identification of recurrent mutations in the active site of isocitrate dehydrogenase 1 (IDH1) in 12% of GBM patients. Science, 2008 Patient Groups: Wild-Type: 79 patients Mutant IDH1: 11 patients Comparison: Wild-type IDH1 vs. Mutant IDH1 Median Survival: Patients with Mutant IDH1: 3.8 years Patients with Wild-Type IDH1: 1.1 years IDH1 mutations are often associated with a distinct tumor biology, leading to less aggressive tumor behavior and different clinical outcomes Science, 2008 Cancer Cell, 2024 § New human pangenome reference includes genomic data from 47 people (collectively more globally diverse). Expected that number to reach 350 people by 2024. § A human pangenome reference will help ensure that it is beneficial for everyone and that genomics will advance in an equitable way. https://investors.vrtx.com/news-releases/news-release-details/vertex-and-crispr-therapeutics-announce-authorization-first https://www.csrwire.com/press_releases/812736-emerging-population-genomics-revolution https://www.thehindu.com/opinion/editorial/decoding-the-script-on-the-genome-india-project-and-its-sequencing-10000-indian-genomes/article67899979.ece https://www.ncgindia.org/about https://www.newindianexpress.com/states/kerala/2024/Jul/15/kerala-turns-to-genome-sequencing-for-dengue-sickle-cell-anaemia-treatments AI/ML are transforming genomics by enabling precise identification of genetic disorders, detecting cancer types, predicting disease progression, and analyzing genomic variants. Facial analysis AI programs are expected to assist in identifying genetic disorders by examining facial features for unique patterns. https://www.genome.gov/about-genomics/educational-resources/fact-sheets/artificial-intelligence-machine-learning-and-genomics  Machine learning models can identify the primary type of cancer from a liquid biopsy, offering a non-invasive method for cancer detection. AI algorithms also predict how specific cancers are likely to progress in individual patients, providing insights that guide treatment planning. https://www.genome.gov/about-genomics/educational-resources/fact-sheets/artificial-intelligence-machine-learning-and-genomics  Deep learning is enhancing the accuracy of gene- editing tools such as CRISPR, towards precise genomic modifications and therapeutic applications. AI/ML technologies may advance more personalized treatment options and novel therapeutic approaches. https://www.genome.gov/about-genomics/educational-resources/fact-sheets/artificial-intelligence-machine-learning-and-genomics § Personalized Medicine: Tailored treatments based on individual genomics/omics data. § Multi-Omics Integration: Combining genomics, proteomics..for deeper insights. § Personalized Diagnostics: Case examples like Lukas Wartman & Angelina Jolie illustrate impact. § Key Initiatives: § Cancer Moonshot & CPTAC: Advancing cancer research with multi-omics. § Glioblastoma Study: Comprehensive genomic analysis in cancer. § Global Genomic Projects: Human Pangenome & 10,000 Indian Genomes. § AI/ML in Genomics: Enhancing data analysis & personalized applications.  Biology and Chemistry Incorporating ddNTPs in place of nucleotides (dNTPs); Fine-tuning ratio of dNTPs to ddNTPs Biology and Physics Design fluorescent dyes that emit at Biology and Engineering different wavelengths & no interference Automation & Gel Electrophoresis with DNA or each other Detection Systems Specialized optics in sequencing machines Biology and Computer Science Impact on Medicine Bioinformatics emerged Foundation for genomics and Quality control algorithms personalized medicine As engineers, your expertise will be vital for driving advancements in biotechnology and medicine, paving the way for a healthier and more innovative future for India!