Genetic Engineering

Genetic Engineering and Biotechnology

Understanding the Genome

  • Definition of Genome:

    • A genome is the complete set of genes or genetic material present in a cell or organism.

  • Human Genome Project (HGP):

    • An international research project aimed at sequencing the entire human genome (3.1 billion base pairs) from 1990 to 2003.

    • Cost: Approximately $2.7 billion.

    • Goals of the HGP:

    • Sequence the DNA.

    • Identify genes associated with diseases.

    • Map the location of every gene on chromosomes.

    • Majority of DNA was donated by an anonymous male from Buffalo, NY.

Findings of the Human Genome Project

  • Key Outcomes:

    • Human DNA consists of approximately 3 billion base pairs.

    • There are about 35,000 genes in human DNA.

    • Many diseases have a genetic basis, leading to the idea that knowledge gained can lead to disease cures.

  • Availability of Data:

    • All genomic data is free and accessible online for public use.

  • Implications for Biotechnology:

    • Companies are focused on developing improved drugs and methods for testing genetic disorders based on HGP findings.

Definitions of Key Concepts

  • Biotechnology:

    • The application of cellular and biological processes to develop technologies and products aimed at improving human life and the health of the planet.

  • Genetic Engineering:

    • The modification or alteration of an organism's genes through biotechnological methods.

Historical Context of Genomic Alteration

  • Selective Breeding (Artificial Selection):

    • A process humans have employed for centuries to choose organisms with desirable traits for reproduction, evident in domestic animals and crops.

    • Methods include:

    • Hybridization:

      • Creating hybrids by mating dissimilar individuals to achieve offspring with traits from both parents.

    • Inbreeding:

      • Breeding similar organisms, often referred to as pure breeds.

Technological Approaches to Genomic Modification

  • Methods to Change Genomes:

    • Genetic material can be altered through techniques such as cutting, adding, extracting, or copying segments of DNA.

  • Restriction Enzymes:

    • Enzymes that cut DNA at specific nucleotide sequences, facilitating easier manipulation of DNA fragments.

    • Example of cutting: Restriction enzyme identifies the sequence AGCT and cleaves the DNA between specific nucleotides.

Types of Restriction Enzymes

  • Blunt Ends:

    • Result from a clean cut of DNA without overhangs.

  • Sticky Ends:

    • Created by staggered cuts, allowing for easier DNA annealing through complementary overhangs.

  • Restriction Enzyme Example - SAL1:

    • Cuts between G and T wherever the sequence GTCGAC appears in DNA.

Amplification Techniques

  • Polymerase Chain Reaction (PCR):

    • A technique that rapidly amplifies specific DNA sequences, producing millions of copies of a gene from a small sample.

    • Components of PCR:

    • Sample DNA

    • Free nucleotides (A, T, G, C)

    • Taq Polymerase (heat-resistant DNA polymerase)

    • Specific primers to initiate the replication process.

    • Thermocycler Process:

    • Cycles through three temperatures:

      • 98°C (denaturation),

      • 55°C (primer annealing),

      • 70°C (DNA synthesis).

    • Copy Calculation Formula:

    • Total copies made is given by the formula 2^n, where n is the number of cycles.

Applications of PCR

  • DNA Fingerprinting:

    • Each individual's DNA is unique; after amplification, a DNA fingerprint can be created.

    • Restriction enzymes are used to cut the amplified DNA at specific sites, resulting in different fragment patterns across individuals.

Gel Electrophoresis

  • Technique Overview:

    • Common method for separating DNA fragments based on size.

    • DNA is loaded into a gel, and an electric field is applied, causing smaller fragments to move faster towards the positive end.

    • Visualization occurs under X-ray imaging after electrophoresis, producing a distinct banding pattern for each individual’s DNA.

  • Visual Example:

    • The display shows bands separated by size, indicating the lengths of the DNA fragments post-electrophoresis.

Practical Applications of DNA Fingerprinting

  • Crime Scene Investigation:

    • DNA samples collected from a crime scene can be amplified, cut, and analyzed to match against potential suspects.

    • An exact match identifies the perpetrator, while lack of a match excludes suspects.

  • Paternity Testing:

    • DNA fingerprinting can determine parental lineage; a child should match half of the DNA bands from the mother and half from the father.

Introduction to Genetic Engineering

  • Definition:

    • Genetic engineering involves designing and building specific characteristics in organisms by manipulating their genetic material.

  • Gene Functionality:

    • Genes encode traits, such as eye color, hair texture, etc.

Transgenic Organisms

  • Definition:

    • Transgenic organisms contain genes from different species.

    • Involves using restriction enzymes to cut suitable DNA from one species and inserting it into another, a process called transformation.

  • Recombinant DNA:

    • Refers to DNA composed of gene sequences from two or more organisms.

  • Plasmids:

    • Small, circular DNA molecules prevalent in bacteria, often used as vectors during transformation processes.

Steps for Creating Recombinant DNA Plasmids

  • 1. Isolate the gene of interest.

  • 2. Cut both the gene of interest and plasmid DNA with the same restriction enzyme.

  • 3. Mix the DNA fragments and add ligase to fuse them through base pairing.

  • 4. Transform the recombinant plasmids into E. coli.

  • 5. Plate cells on agar with antibiotics to select for transformed cells.

  • 6. Expand positive clones to replicate the desired DNA exponentially.

Applications of Recombinant DNA and Transgenic Organisms

  • Medical Production:

    • Bacteria engineered to produce human hormones and enzymes, e.g., human insulin, avoiding the use of animal sources.

    • Following steps:

    • Identify the gene of interest.

    • Remove and insert it into bacterial DNA.

    • Extract and purify the produced enzyme.

  • Examples of Transgenic Organisms:

    • Bacteria producing human proteins.

    • Tobacco plants with luminescent proteins.

    • Transgenic pigs and sheep that secrete human proteins in their milk.

    • Genetically modified crops (e.g., corn and soybeans with built-in insecticides or rice enriched with vitamin A).

Cloning Techniques

  • Cloning Process:

    • A method for creating a genetically identical organism by doing the following:

    • Remove the haploid nucleus from an egg cell.

    • Fuse a diploid nucleus from a somatic cell with the empty egg cell.

Gene Therapy

  • Definition:

    • Gene therapy involves replacing defective genes with functioning ones to treat disorders.

  • Methodology:

    • Viruses are often utilized as vectors for delivering new DNA; steps include:

    • Removal of viral genes.

    • Insertion of the desired gene into the emptied virus.

    • Injection into target tissue to facilitate gene expression.

Stem Cells

  • Definition:

    • Stem cells are undifferentiated cells capable of developing into various specialized cell types.

  • Sources:

    • Found in embryos, placenta, and bone marrow; also under research for creating stem cells artificially.

  • Potential Uses:

    • Regeneration of new organs through manipulation of stem cells.