8-BIOTECHNOLOGY

Biotechnology Overview

• Definition: Biotechnology is the use of enzymes and DNA from various natural sources to copy, move, and express genetic information in novel systems.

  • Applications include:

    • Development of genetic diagnostic tests

    • Production of pharmacologically useful proteins

    • Evolutionary relationship analyses

    • Genome mapping

Genetic Modification Process

• Steps in Genetic Modification:

  1. Isolation of Vector: Plasmid is isolated from a bacterium.

  2. Cleavage of DNA: DNA containing the gene of interest is cleaved into fragments using enzymes.

  3. Insertion of Gene: Desired gene is selected and inserted into the plasmid.

  4. Transformation: Plasmid is taken up by a bacterial cell.

  5. Cloning: Cells that now have the gene of interest can be cloned with two main goals:

     • Create and harvest copies of the gene.

     • Create and harvest protein products of the gene.

• Example: COVID-19 mRNA vaccines utilize plasmids to produce RNA and proteins needed for immune response.

Enzymes in Biotechnology

Restriction Endonucleases

• Function: Also known as restriction enzymes, these naturally occur in bacteria and serve a defensive function by destroying bacteriophage DNA.

  • They create either blunt ends or staggered cuts, known as sticky ends, by cutting double-stranded DNA at specific recognition sequences that are palindromic.

Examples of Restriction Enzymes

• DNA Palindrome:

  • Example: EcoRI recognition sequence is:

    • 5’...GAATTC...3’

    • 3’...CTTAAG...5’

• Restriction enzymes function like molecular scissors, making the cuts necessary for DNA manipulation.

• New DNA Molecules: Two different DNA pieces cut by the same enzyme can be glued together, resulting in recombinant DNA.

Types of Enzymes

Ligase

• This enzyme links adjacent nucleotides in a DNA strand, used to permanently glue together DNA sticky ends cut by restriction enzymes.

Reverse Transcriptases

• Naturally occur in retroviruses, they read single-stranded RNA to produce double-stranded DNA, allowing for the reverse transcription process.

DNA Polymerase

• Enzymes that replicate DNA by reading double-stranded DNA to produce new strands. Enzymes from extreme thermophiles (e.g., Thermus aquatics) are used due to their ability to withstand high temperatures in PCR.

Polymerase Chain Reaction (PCR)

• Purpose: Amplification of small amounts of DNA.

• Reaction Cycle Stages:

  1. Denaturation: Heating to 95ºC separates DNA strands.

  2. Priming: Lowering the temperature allows RNA primers to bind.

  3. Extension: Temperature raised to 72ºC enables Taq polymerase to elongate, creating copies of target DNA.

• Exponential Amplification: Each cycle doubles the amount of DNA, leading to over 1 billion copies after 30 cycles.

DNA Fingerprinting

• DNA is purified and cut with restriction enzymes, producing unique restriction fragment length polymorphisms (RFLPs) for individuals.

• Visualization: RFLPs are then separated using electrophoresis and can be stained to reveal unique patterns for identification.

Scientific Applications of DNA Fingerprinting

• Identification of pathogens, forensic analysis, and specific gene identification.

  • Example: Used to identify victims of disasters or exonerate wrongly convicted individuals.

Genomic Libraries

• Development: Cloning an organism's DNA to have at least one clone for every gene.

• cDNA Production: Using reverse transcriptase, mRNA is converted into complementary DNA to obtain eukaryotic genes without introns.

Transformation in Biotechnology

• Use of Recombinant Plasmids: Bacteria and yeast can be transformed with recombinant plasmids to produce desired quantities of DNA or proteins.

• Selection methods include enhanced green fluorescent protein (EGFP) and blue/white screening for lacZ activity.

Applications of Recombinant DNA Technology

Therapeutic Applications

• Products: Recombinant proteins such as insulin, human growth hormone, and vaccines are produced using biotechnology.

• Table of Pharmaceutical Products: Includes cervical cancer vaccine, erythropoietin, and interferons among others.

Agricultural Applications

• Transgenic Plants: Produced for pest and herbicide resistance, improved nutritional value, and longer shelf life. Examples include Bt corn and golden rice.

Forensic Science and Large Scale Projects

• Human Genome Project: Sequencing the entire human genome to improve understanding and applications in health.

• Evolutionary Biology: DNA fingerprinting aids in understanding evolutionary relationships and conservation biology.