🐾General Biology II Unit 9 – DNA Technologies and Genomics

Key Concepts and Terminology

• Genomics studies the structure, function, evolution, and mapping of genomes
• DNA sequencing determines the order of nucleotide bases (adenine, guanine, cytosine, and thymine) in a DNA molecule
• Polymerase chain reaction (PCR) amplifies specific DNA sequences, generating millions of copies from a small sample
• Recombinant DNA technology combines DNA from different sources to create novel genetic sequences
• Bioinformatics applies computational tools to analyze and interpret biological data, particularly genomic data
• Genetic engineering modifies an organism's genome by introducing, removing, or altering specific genes
• Personalized medicine tailors medical treatments to an individual's genetic profile, optimizing efficacy and minimizing side effects
• CRISPR-Cas9 is a powerful gene-editing tool that allows precise modification of DNA sequences in living cells

DNA Structure and Replication Basics

• DNA consists of two complementary strands wound together in a double helix structure
  • Each strand is composed of a sugar-phosphate backbone with attached nitrogenous bases
  • The bases pair through hydrogen bonds: adenine with thymine and guanine with cytosine
• DNA replication is a semiconservative process that occurs before cell division
  • Helicase unwinds the double helix, separating the two strands
  • Primase synthesizes short RNA primers to initiate replication
  • DNA polymerase III adds nucleotides to the growing strand, using the original strand as a template
• The leading strand is synthesized continuously in the 5' to 3' direction
• The lagging strand is synthesized discontinuously as Okazaki fragments, which are later joined by DNA ligase
• DNA polymerase I replaces RNA primers with DNA nucleotides
• Telomerase maintains the length of telomeres, repetitive sequences at the ends of chromosomes, to prevent loss of genetic information during replication

DNA Sequencing Techniques

• Sanger sequencing, also known as the chain-termination method, was the first widely used DNA sequencing technique
  • It uses dideoxynucleotides (ddNTPs) that lack a 3' hydroxyl group, preventing further elongation
  • Fragments of varying lengths are generated, each ending with a fluorescently labeled ddNTP
  • Capillary electrophoresis separates the fragments by size, and the sequence is determined by the order of the labeled ddNTPs
• Next-generation sequencing (NGS) technologies enable high-throughput, parallel sequencing of millions of DNA fragments
  • Illumina sequencing uses bridge amplification and reversible terminator chemistry
  • Ion Torrent sequencing detects hydrogen ions released during nucleotide incorporation
  • Pacific Biosciences' single-molecule real-time (SMRT) sequencing uses zero-mode waveguides and phospholinked nucleotides
• Nanopore sequencing directly reads the DNA sequence as the molecule passes through a protein nanopore, detecting changes in electrical current
• Whole-genome sequencing determines the entire DNA sequence of an organism, while targeted sequencing focuses on specific regions of interest

Polymerase Chain Reaction (PCR)

• PCR is a technique used to amplify a specific DNA sequence, generating millions of copies from a small initial sample
• The process involves three main steps: denaturation, annealing, and extension
  • Denaturation: The double-stranded DNA is heated to around 94-96°C, causing the strands to separate
  • Annealing: The temperature is lowered to 50-65°C, allowing primers to bind to their complementary sequences on the single-stranded DNA
  • Extension: DNA polymerase extends the primers, synthesizing new strands complementary to the template at 72°C
• These steps are repeated for 25-40 cycles, exponentially increasing the number of target DNA copies
• Primers are short, synthetic oligonucleotides that define the start and end of the target sequence
• Taq polymerase, a heat-stable DNA polymerase from the bacterium Thermus aquaticus, is commonly used in PCR
• Reverse transcription PCR (RT-PCR) amplifies RNA by first converting it to complementary DNA (cDNA) using reverse transcriptase
• Quantitative PCR (qPCR) measures the amount of amplified DNA in real-time using fluorescent dyes or probes

Genetic Engineering and Recombinant DNA

• Recombinant DNA technology involves combining DNA from different sources to create novel genetic sequences
• Restriction enzymes, also known as restriction endonucleases, cut DNA at specific recognition sites
  • They are used to create compatible ends for joining DNA fragments
  • Examples include EcoRI, BamHI, and HindIII
• DNA ligase seals the nicks between the joined DNA fragments, creating a continuous strand
• Vectors, such as plasmids or viral vectors, are used to introduce recombinant DNA into host cells
  • Plasmids are circular, double-stranded DNA molecules that can replicate independently of the host genome
  • Viral vectors, such as adenoviruses or lentiviruses, can efficiently deliver genetic material into cells
• Transformation introduces recombinant DNA into bacterial cells, while transfection is used for eukaryotic cells
• Selectable markers, such as antibiotic resistance genes, allow for the identification and isolation of cells containing the recombinant DNA
• Genetically modified organisms (GMOs) are created by inserting foreign DNA into their genomes
  • Examples include Bt crops (insect-resistant) and Golden Rice (vitamin A-enriched)

Genomics and Bioinformatics

• Genomics is the study of the structure, function, evolution, and mapping of genomes
• Bioinformatics applies computational tools to analyze and interpret biological data, particularly genomic data
• Genome annotation involves identifying and labeling functional elements within a genome, such as genes, regulatory sequences, and non-coding RNAs
• Comparative genomics compares the genomes of different species to identify conserved and divergent regions, providing insights into evolutionary relationships and gene function
• Functional genomics studies the roles and interactions of genes and their products (RNA and proteins) in biological processes
• Transcriptomics analyzes the complete set of RNA transcripts (transcriptome) in a cell or tissue under specific conditions
• Proteomics investigates the structure, function, and interactions of proteins on a large scale
• Metabolomics studies the collection of small molecules (metabolites) in a biological system, providing a snapshot of its metabolic state
• Systems biology integrates data from various -omics fields to understand complex biological systems and their emergent properties

Applications in Medicine and Biotechnology

• Personalized medicine tailors medical treatments to an individual's genetic profile, optimizing efficacy and minimizing side effects
  • Pharmacogenomics studies how genetic variations influence drug response, guiding the development of targeted therapies
  • Genetic testing can identify predispositions to certain diseases, allowing for early intervention and preventive measures
• Gene therapy introduces functional genes into cells to replace or correct defective genes, potentially treating genetic disorders
  • Ex vivo gene therapy involves modifying cells outside the body and then reintroducing them
  • In vivo gene therapy directly delivers the therapeutic gene into the patient's cells
• Recombinant proteins, such as insulin and human growth hormone, are produced using genetically engineered microorganisms or cell lines
• Transgenic animals serve as disease models, bioreactors for producing human proteins, or sources of organs for xenotransplantation
• DNA fingerprinting uses genetic markers to identify individuals, with applications in forensics, paternity testing, and wildlife conservation
• Synthetic biology designs and constructs novel biological systems or organisms with desired functions, such as biosensors or biofuel-producing microbes

Ethical Considerations and Future Directions

• Genetic privacy and confidentiality are major concerns, as genetic information can be misused for discrimination or stigmatization
• Informed consent is crucial when collecting and using genetic data, ensuring that individuals understand the risks and benefits
• The ownership and patenting of genetic information raise questions about intellectual property rights and access to genetic resources
• Genetically modified organisms (GMOs) have sparked debates about their safety, environmental impact, and labeling
• Human germline editing, which introduces heritable changes to the genome, raises ethical concerns about altering the human gene pool
• The unequal access to genetic technologies and personalized medicine may exacerbate health disparities
• Future advancements in DNA sequencing technologies aim to increase speed, accuracy, and affordability
• The integration of genomics with other -omics fields and artificial intelligence will provide a more comprehensive understanding of biological systems
• Precision medicine initiatives, such as the All of Us Research Program, seek to collect and analyze genetic and health data from diverse populations to improve healthcare outcomes