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

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.

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.

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.

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

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

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.

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 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.

Identification of pathogens, forensic analysis, and specific gene identification.
- Example: Used to identify victims of disasters or exonerate wrongly convicted individuals.

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.

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.

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.

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

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.