Biotechnology and DNA Technology Study Notes

Chapter 09: Biotechnology and DNA Technology

Human Immunodeficiency Virus (HIV)

Introduction to Biotechnology

  • Learning Objectives

    • 9-1 Compare and contrast biotechnology, genetic modification, and recombinant DNA technology.

    • 9-2 Identify the roles of a clone and a vector in making recombinant DNA.

Overview of Biotechnology

  • Biotechnology:

    • The use of microorganisms, cells, or cell components to make a product.

    • Applications include foods, antibiotics, vitamins, and enzymes.

  • Recombinant DNA (rDNA) Technology:

    • The insertion or modification of genes to produce desired proteins.

Overview of Recombinant DNA Procedures

  • Vector:

    • Self-replicating DNA molecule used to transport foreign DNA into a cell.

  • Clone:

    • Population of genetically identical cells arising from one cell; each carries the vector.

Tools of Biotechnology

  • Learning Objectives

    • 9-3 Compare selection and mutation.

    • 9-4 Define restriction enzymes and outline their use in making rDNA.

    • 9-5 List the four properties of vectors.

    • 9-6 Describe the use of plasmid and viral vectors.

    • 9-7 Outline the steps in PCR (Polymerase Chain Reaction) and provide an example of its use.

Selection and Mutation

  • Selection:

    • Selecting for a naturally occurring microbe that produces a desired product.

  • Mutation:

    • Mutagens cause mutations that might result in a microbe with a desirable trait.

  • Site-directed Mutagenesis:

    • A molecular biology method used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products.

Restriction Enzymes (DNA Cutting Enzymes)

  • Function:

    • Cut specific sequences of DNA; destroy bacteriophage DNA in bacterial cells.

    • Methylated cytosines in bacteria protect their own DNA from digestion.

  • Cut Types:

    • Create blunt ends or staggered cuts known as sticky ends.

    • Blunt Ends:

    • Less efficient; require higher concentrations of enzymes.

    • Sticky Ends:

    • Highly efficient due to complementary overhangs that anneal (hydrogen bond) before ligation.

Animation: Recombinant DNA Technology

  • Process Overview:

    1. Restriction Enzyme Activity:

    • Cuts double-stranded DNA at its particular recognition sites.

    • Example recognition site: GAATTC / CTTAAG.

    • Cuts generate fragments with sticky ends.

    1. Joining DNA Fragments:

    • Two fragments cut by the same restriction enzyme can join by base pairing.

    1. Ligation:

    • The enzyme DNA ligase unites the backbones of the two DNA fragments, producing a molecule of rDNA.

Vectors in Recombinant DNA

  • Functions of Vectors:

    • Carry new DNA to desired cells.

    • Must be able to self-replicate.

    • Examples include plasmids and viruses.

    • Shuttle Vectors:

    • Exist in several different species and can move cloned sequences among various organisms.

  • Plasmid Structure:

    • Contains segments: ori (origin of replication), amp R (ampicillin resistance), lac Z (B-galactosidase), restriction enzyme sites (HindIII, BamHI, EcoRI).

Technique: Polymerase Chain Reaction (PCR)

  • Purpose:

    • Process of increasing small quantities (amplifying) of DNA for analysis.

    • Used for diagnostic tests for genetic diseases and detecting pathogens.

  • Reverse-transcription PCR:

    • Uses mRNA as a template to synthesize complementary DNA (cDNA).

Steps of PCR

  • Thermocycler Stages:

    • Denaturation: 94 °C

    • Priming: 60 °C

    • Extension: 72 °C

  • Components of PCR:

    • DNA polymerase, DNA primers, dATP, dCTP, dTTP, dGTP, and target DNA.

  • Outcome:

    • After each round of PCR, the amount of target DNA doubles.

Inserting Foreign DNA into Cells

  • Methods of Insertion:

    • Transformation:

    • Cells take up DNA from the surrounding environment.

    • Electroporation:

    • Electrical current forms pores in cell membranes to allow DNA entry.

    • Protoplast Fusion:

    • Removing cell walls from two bacteria allows them to fuse.

    • Gene Gun:

    • Inserts DNA-coated “bullets” into a cell.

    • Microinjection:

    • Direct injection of DNA into cells.

Genomic Libraries

  • Definition:

    • A collection of overlapping DNA fragments that together represent the total genomic DNA of a single organism.

  • Construction:

    • DNA extracted from cells and digested with a restriction enzyme to create fragments of specific size.

    • Cloned into plasmid or phage vectors.

  • Purpose:

    • At least one clone exists for every gene in the organism.

Complementary DNA (cDNA)

  • Definition:

    • Made from mRNA by reverse transcriptase, a DNA polymerase enzyme that transcribes single-stranded RNA into DNA.

  • Purpose:

    • Used for obtaining eukaryotic genes due to introns present in eukaryotic DNA, which are removed from mRNA.

Synthetic DNA

  • Definition:

    • Artificially created deoxyribonucleic acid molecules, built using a DNA synthesis machine.

  • Uses:

    • Data storage, product tagging, or signal processing.

Selecting a Clone

  • Techniques:

    • Blue-white screening:

    • Rapid technique to identify recombinant bacteria based on β-galactosidase activity.

    • Process:

      • Cells transformed with recombinant DNA produce white colonies when grown on media containing ampicillin and X-gal; non-recombinant plasmids create blue colonies.

    • Colony Hybridization:

    • Selecting bacterial colonies with desired genes using DNA probes that are complementary to the gene of interest.

Making a Gene Product

E. coli

  • Advantages:

    • Easily grown; complete genomics known.

  • Disadvantages:

    • Produces endotoxins; does not secrete protein products.

Saccharomyces cerevisiae

  • Advantages:

    • Eukaryotic expression; larger genome than bacteria.

Plant Cells and Whole Plants

  • Advantages:

    • Easily grown; large-scale; low-cost.

Mammalian Cells

  • Advantages:

    • Capable of producing complex eukaryotic proteins; used for medical products.

  • Disadvantages:

    • More difficult to culture and grow.

Applications of DNA Technology

  • Learning Objectives

    • 9-13 List at least five applications of DNA technology.

    • 9-14 Define RNA interference (RNAi).

    • 9-15 Discuss the value of genome projects.

    • 9-16 Define terms: random shotgun sequencing, bioinformatics, proteomics.

    • 9-17 Diagram the Southern blotting procedure with examples.

    • 9-18 Diagram DNA fingerprinting and provide examples.

    • 9-19 Outline genetic engineering with Agrobacterium.

Therapeutic Applications (Examples)

  • Products:

    • Human enzymes (e.g., Insulin).

    • Subunit vaccines:

    • Made from pathogen proteins in genetically modified yeasts.

    • Gene therapy:

    • Replace defective genes.

    • CRISPR:

    • Gene editing to correct mutations.

Examples of Pharmaceutical Products of rDNA

  • Cervical Cancer Vaccine:

    • Contains viral proteins; produced by yeast or insect cells.

  • Erythropoietin (EPO):

    • Treatment of anemia; produced by mammalian cell culture.

  • Hepatitis B Vaccine:

    • Produced by yeast carrying hepatitis-virus gene.

  • Human Insulin:

    • Produced by E. coli; better tolerated than animal-derived insulin.

Genome Projects

  • Shotgun sequencing:

    • Sequences small genome pieces, which computers assemble into complete sequences.

  • Metagenomics:

    • Study of genetic material extracted directly from environmental samples.

  • Human Genome Project:

    • Aims to sequence the entire human genome.

  • Human Proteome Project:

    • Maps proteins expressed in human cells.

Scientific Applications

  • Bioinformatics:

    • Understanding gene function through computer-assisted analysis.

  • Proteomics:

    • Identifying all expressed proteins in a cell.

  • Genetic Testing:

    • Screening parental or fetal tissue for various genetic diseases.

Southern Blotting Technique

  • Description:

    • Separates DNA fragments by size via electrophoresis, transfers to a membrane, probes with a labeled sequence, and detects labeled DNA bands.

Forensic Microbiology

  • DNA Fingerprinting:

    • Used to identify pathogens and track infectious diseases.

Nanotechnology in Biotechnology

  • Usage:

    • Bacteria can produce molecule-sized particles; used in drug targeting and delivery.

Agricultural Applications

  • Ti Plasmid (Agrobacterium tumefaciens):

    • Integrates into the plant genome; used to introduce rDNA into plants.

    • Widely employed to create transgenic plants.

  • Examples of Genetically Modified Products:

    • Bt cotton and Bt corn; contain toxin-producing gene from Bacillus thuringiensis.

    • Genes for polyphenyl oxidase deleted in button mushrooms to prevent browning.

  • Animal Products:

    • Genetically modified mosquitoes to control the spread of the Zika virus.

Safety Issues and Ethics of Using DNA Technology

  • Considerations:

    • Prevent accidental release of GMOs into the environment (e.g., suicide genes).

    • Ensure GM crops are safe for consumption.

    • Ethical concerns regarding individual access to genetic information.