👩‍🔬Intro to Biotechnology Unit 3 – DNA: Structure, Function & Manipulation

DNA Basics: The Building Blocks

• DNA (deoxyribonucleic acid) is the hereditary material in humans and almost all other organisms
• Consists of four types of nucleotide bases: adenine (A), thymine (T), guanine (G), and cytosine (C)
• Nucleotides are composed of a sugar (deoxyribose), a phosphate group, and a nitrogenous base
• Bases pair up with each other, A with T and C with G, to form units called base pairs
• DNA typically exists as a double-stranded molecule, with the two strands running in opposite directions (antiparallel)
  • The sugar-phosphate backbones are on the outside of the double helix
  • The nitrogenous bases are on the inside, pairing with their complementary base on the opposite strand
• In human cells, DNA is organized into 23 pairs of chromosomes, with one chromosome in each pair inherited from each parent

Double Helix Structure: Cracking the Code

• In 1953, James Watson and Francis Crick proposed the double helix model of DNA structure
  • Based on X-ray crystallography data collected by Rosalind Franklin and Maurice Wilkins
• The double helix structure resembles a twisted ladder
  • The sugar-phosphate backbones form the "sides" of the ladder
  • The base pairs form the "rungs" of the ladder
• The two strands of DNA run in opposite directions (5' to 3' and 3' to 5')
• The double helix is stabilized by hydrogen bonds between the base pairs and base-stacking interactions
• The width of the double helix is uniform throughout, at about 2 nanometers
• The double helix makes a complete turn every 10 base pairs, or about every 3.4 nanometers
• The complementary base pairing (A-T and G-C) provides a built-in mechanism for DNA replication and helps maintain the genetic information

DNA Replication: Making Copies

• DNA replication is the process by which a cell makes an exact copy of its DNA before cell division
• Replication ensures that each daughter cell receives a complete set of genetic instructions
• The process is semi-conservative, meaning each strand of the original DNA molecule serves as a template for the production of a new, complementary strand
• Replication begins at specific sites along the DNA molecule called origins of replication
• The enzyme helicase unwinds and separates the two strands of the double helix
  • Single-stranded binding proteins stabilize the separated strands
• DNA primase synthesizes short RNA primers, which provide a starting point for DNA synthesis
• DNA polymerase III extends the new DNA strands by adding nucleotides complementary to the template strand
  • DNA polymerase can only add nucleotides to the 3' end of a growing strand
• 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 the RNA primers with DNA nucleotides
• Telomerase, a specialized DNA polymerase, maintains the telomeres (repetitive sequences at the ends of chromosomes) during replication

Genes and Gene Expression: From DNA to Proteins

• Genes are segments of DNA that encode instructions for making specific proteins
• The process of gene expression involves two main steps: transcription and translation
• During transcription, the enzyme RNA polymerase uses one strand of the DNA as a template to synthesize a complementary RNA strand
  • The resulting RNA molecule is called messenger RNA (mRNA)
  • In eukaryotes, the primary mRNA transcript undergoes processing (capping, splicing, and polyadenylation) to produce a mature mRNA
• The mature mRNA moves from the nucleus to the cytoplasm, where translation occurs
• During translation, the mRNA is read by ribosomes, which use the genetic code to translate the nucleotide sequence into an amino acid sequence
  • The genetic code is a set of three-nucleotide sequences called codons, each of which specifies a particular amino acid or a stop signal
  • Transfer RNA (tRNA) molecules, each with a specific anticodon, deliver the appropriate amino acids to the ribosome
• The resulting polypeptide chain folds into a specific three-dimensional structure, forming a functional protein
• Gene expression is tightly regulated at multiple levels (transcriptional, post-transcriptional, translational, and post-translational) to ensure that the right proteins are produced at the right times and in the right amounts

Mutations: When DNA Goes Off-Script

• Mutations are changes in the DNA sequence that can alter gene function and, consequently, the traits of an organism
• Point mutations involve changes to a single nucleotide
  • Substitutions replace one nucleotide with another (transitions and transversions)
  • Insertions add one or more nucleotides to the sequence
  • Deletions remove one or more nucleotides from the sequence
• Frameshift mutations (insertions or deletions that are not a multiple of three nucleotides) alter the reading frame, often resulting in a completely different amino acid sequence
• Chromosomal mutations involve larger-scale changes to the structure or number of chromosomes
  • Deletions, duplications, inversions, and translocations are examples of structural changes
  • Aneuploidy (an abnormal number of chromosomes) and polyploidy (extra sets of chromosomes) are examples of numerical changes
• Mutations can be spontaneous (due to errors in DNA replication or repair) or induced by environmental factors (such as UV radiation, chemicals, or viruses)
• While many mutations are neutral or harmful, some can be beneficial and contribute to genetic diversity and evolution
• Mutations in somatic cells can lead to cancer and other diseases, while mutations in germ cells can be passed on to offspring

DNA Manipulation Techniques: Playing with Genes

• Recombinant DNA technology involves the manipulation and combination of DNA from different sources to create new genetic sequences
• Restriction enzymes (endonucleases) are used to cut DNA at specific recognition sites, generating sticky ends or blunt ends
  • These enzymes are derived from bacteria and serve as a defense mechanism against viral DNA
• DNA ligase is used to join DNA fragments, such as inserting a gene of interest into a vector
• Vectors, such as plasmids or viruses, are used to introduce foreign DNA into host cells
  • Plasmids are small, circular DNA molecules that can replicate independently of the host cell's chromosome
• Transformation is the process of introducing recombinant DNA into bacterial cells, often using heat shock or electroporation
• Polymerase chain reaction (PCR) is a technique used to amplify specific DNA sequences
  • PCR involves three main steps: denaturation, annealing, and extension
  • The process is repeated for multiple cycles, exponentially increasing the number of copies of the target sequence
• DNA sequencing technologies, such as Sanger sequencing and next-generation sequencing (NGS), are used to determine the precise order of nucleotides in a DNA molecule
• CRISPR-Cas9 is a powerful gene-editing tool derived from a bacterial adaptive immune system
  • CRISPR-Cas9 uses guide RNA to target specific DNA sequences and make precise cuts, allowing for the insertion, deletion, or modification of genes

Applications in Biotechnology: DNA in Action

• Genetically modified organisms (GMOs) are created by introducing foreign genes into their genomes
  • GM crops (corn, soybeans, cotton) can have improved traits such as herbicide resistance, pest resistance, or enhanced nutritional content
  • GM animals (salmon, pigs, goats) can be engineered to grow faster, produce human proteins in their milk, or serve as disease models
• DNA fingerprinting uses unique patterns in an individual's DNA to identify them
  • Applications include forensic investigations, paternity testing, and wildlife conservation
• Gene therapy involves the introduction of functional genes into cells to replace or correct defective genes
  • Approaches include ex vivo (cells modified outside the body) and in vivo (direct delivery to target tissues) gene therapy
  • Potential applications include treating genetic disorders, cancer, and infectious diseases
• Personalized medicine uses an individual's genetic information to tailor medical treatments and preventive strategies
  • Pharmacogenomics studies how genes influence drug response, allowing for the optimization of drug therapy based on a patient's genetic profile
• DNA vaccines use DNA encoding viral or bacterial antigens to stimulate an immune response
  • Advantages include ease of production, stability, and the ability to induce both humoral and cellular immunity
• Ancient DNA analysis involves extracting and studying DNA from ancient specimens (fossils, mummies, sediments) to gain insights into evolutionary history, population genetics, and past environments
  • Examples include studying Neanderthal DNA, tracing human migrations, and reconstructing ancient ecosystems

Ethical Considerations: The DNA Debate

• Genetic privacy and discrimination are major concerns as genetic testing becomes more widespread
  • The Genetic Information Nondiscrimination Act (GINA) in the United States prohibits discrimination based on genetic information in health insurance and employment
• Informed consent is crucial when collecting and using individuals' genetic data for research or clinical purposes
  • Participants should be fully informed about the risks, benefits, and potential implications of genetic testing
• The use of gene editing technologies, such as CRISPR-Cas9, raises ethical questions about the potential for designer babies and the modification of the human germline
  • Many countries have regulations or guidelines restricting the use of gene editing on human embryos
• Ownership and patenting of genetic information is a contentious issue
  • The patenting of genes and genetic tests can limit access and hinder research, while also providing incentives for innovation
• The development of genetically modified organisms (GMOs) has led to debates about their safety, environmental impact, and socioeconomic consequences
  • Concerns include the potential for unintended ecological effects, the development of resistant pests, and the control of the food supply by a few corporations
• Genetic engineering of animals raises ethical questions about animal welfare and the creation of new species
  • Considerations include the suffering caused by genetic modifications, the use of animals for human benefit, and the potential ecological impacts of genetically engineered organisms
• The unequal access to genetic technologies and their benefits is a concern, particularly in developing countries
  • Efforts to promote equity and global health should consider the distribution of genetic resources and technologies