19 Genetic Technology

19.1 Principles of Genetic Technology

Recombinant DNA

• Goal: Transfer genes between organisms to express in new hosts
• Recombinant DNA (rDNA): DNA made from different sources
• Transgenic Organism/GMO: Organism expressing genes from another source via rDNA

Gene Transfer Overview

1. Identify the desired gene
2. Isolate and extract the gene
   • Use restriction endonucleases, reverse transcriptase, or artificial creation
3. Multiply gene via PCR
4. Insert gene into vector (plasmids, viruses, liposomes)
5. Deliver vector to host cells
6. Identify and clone cells expressing the new gene

Polymerase Chain Reaction (PCR)

• Purpose: Rapidly replicate DNA
• Requirements: DNA sample, primers, nucleotides, buffer solutions, DNA polymerase
• Taq Polymerase: Heat-stable, used in PCR
  • Not destroyed in denaturation, high optimum temperature
• Primers: Define region for DNA synthesis
• PCR Steps:
  1. Denaturation (95°C)
  2. Annealing (65°C)
  3. Elongation (72°C)

Gel Electrophoresis

• Purpose: Separate nucleic acids/proteins by size and charge
• Steps:
  1. Place sample in gel well
  2. Apply electric field
  3. Molecules move towards the anode
  4. Short fragments move further
• Electrophoresis of DNA: Used for genetic profiling
  • DNA is cut with restriction enzymes, separated by electrophoresis, and visualized with probes

Plasmids

• Properties:
  • Naturally occur in bacteria, transferable, can be artificially produced
  • Double-stranded, replicate independently
• Transfer to Host Cells:
  • Use calcium chloride and heat-shock to increase membrane permeability

Promoters

• Function: Region of DNA where RNA polymerase binds to initiate transcription
• Role in Gene Expression: Ensure RNA polymerase recognizes the template strand

Gene Markers

• Purpose: Identify successfully altered cells
• Examples: Antibiotic resistance genes, GFP, GUS
• Fluorescent Markers: Easier to identify and more economical

Enzymes in Genetic Engineering

1. Restriction Endonucleases: Cut DNA at specific sites
2. Ligase: Splices genes into vectors
3. Reverse Transcriptase: Forms cDNA from RNA

Microarrays

• Purpose: Analyze gene expression and compare genomes
• Process: DNA fragments hybridize with probes on a microarray, detected by fluorescent tags

19.2 Genetic Technology Applied to Medicine

Bioinformatics

• Role: Analyze biological data post-genome sequencing

Human Protein Production by rDNA

• Examples: Insulin, factor VIII, adenosine deaminase
• Advantages: Large volume, identical to human proteins, ethical concerns mitigated

Genetic Screening

• Purpose: Detect specific alleles in adults, embryos, fetuses
• Applications: BRCA genes, haemophilia, sickle cell anemia, Huntington's disease
• Advantages: Preventative measures, informed decisions, research participation

Gene Therapy

• Methods: Use viruses, liposomes, naked DNA
• Applications: SCID, Leber congenital amaurosis
• Somatic vs Germ Cell Therapy: Somatic affects individual; germ cell affects offspring

PCR and DNA in Forensics

• Applications: Genetic profiling, crime scene investigations

19.3 Genetically Modified Organisms in Agriculture

Benefits

• Increase yield and quality in crops and livestock
• Examples: Herbicide/pest resistance, vitamin enrichment

Bt Maize and Golden Rice

• Bt Maize: Insect resistance via Bt toxin gene
• Golden Rice: Enhanced vitamin A content to prevent deficiency

GM Salmon

• Growth hormone genes enable faster growth
• All produced salmon are female and sterile to prevent wild breeding

Advantages of Genetic Engineering

• Faster, universal desired traits, cross-species gene transfer

Consequences and Ethical Implications

• Resistance development, gene spread to wild relatives, ecological effects
• Social concerns: cost, gene transfer risks, loss of biodiversity

These notes provide an overview of the key concepts in genetic technology, including principles, applications in medicine, and implications in agriculture, ensuring a comprehensive understanding of genetic engineering's role and impact.