Heaphy 11 Genetic engineering Flashcards
Recombinant DNA
(genetic engineering) extends traditional biotechnology.
• ability to cut & join heterologous DNA. Relies on restriction enzymes.
Recombinant DNA Used for:
- Botox, protein from C. botulinum -> lethal to paralyse, can be clinical use to stop muscle contractions.
- Can be purified from anerobic cultures Or from engineered E.coli
- Often carried out in micro-organisms. Bacteria E. coli, yeast.
- Cheap well understood. Both have autonomously replicating plasmids as cloning vectors
Requires:
RESTRICTION ENZYMES
- Cloning vectors
- Other enzymes used in cloning
- PCR
- Protein production
RECOMBINANT DNA CORE TECHNIQUE
Sticky ends cut, then stuck together
Compatible cohesive ends
T4 DNA Ligase enzyme +ATP ligate the ends together
• Plasmid can be amplified to prepare enormous amounts of cloned DNA if desired
EcoR1:
restrict, sticky ends, compatible, base pair with each other, anneal, covalently linked => cloned, amplified
Can self ligate to create mismatch => titrations to ensure the major product is desired plasmid w/ insert
Vectors
amplify DNA
Have marker gene => resistance to antibiotics
Grow on plates with antibiotics
Enzymes of genetic engineering
ENDOnucleases:
- Most important restriction enzymes- cut DNA molecules at internal sites).
- Restriction enzymes isolated and characterised from hundreds of bacterial species have different specificities i.e. recognition sequences for cutting:
• HindIII
from Haemophilus influenzae A*AGCTT blunt ends
• EcoRI
from Escherichia coli G*AATTC compatible cohesives
BamHI
from Bacillus amyloliquefaciens G*GATCC large fragments
• Sau3A
from Staphylococcus aureus *GATC small fragments
• Sma I
from Serratia marcescens CCC*GGG make blunt ends
• All palendromes *
shows cutting point.
•
DNA Ligase
rejoins the cut DNA
Phage T4. Requires ATP.
Why genetically engineer?
- ‘engineered’ to contain recombinant DNA molecules comprising genes. Usually to express ‘foreign’ proteins, but sometimes RNA.
- Mutations could be ‘corrected’
Uses of genetic engineering:
x7
- Vaccines: surface protein of pathogen used to immunise without symptoms or side-effects.
- human insulin humulin: for diabetics, previously animal (associated allergy problems
- human growth hormone: protropin, humatrope, dwarfism
- interferons: a therapeutic. -> MS, Hep C
- Factor VIII: for haemophiliacs
- Iinsecticidal toxin of Bacillus thuringiensis; kill insect pests but cannot spread to natural insect population.
- botox
CLONING VECTORS
ORIGINS
• Developed from research on genetics, biochemistry and physiology of bacteria and bacterial viruses (phages) i.e. microbiology
pUC 19
- derived from plasmids or phage
CLONING VECTORS
Function:
- replicate stably & independently of the host DNA
- easily separated from host DNA
- easily introduced into a host by transformation or infection
- may carry selectable markers , antibiotic resistance gene
- restriction enzyme cutting sites for cloning
- efficient promoters for expression of cloned genes.
Phage vectors
- λ (lambda).
- M13
- Different vectors used for different purposes.
- very popular for making DNA libraries.
- M13 chromosome is single stranded DNA, used directly for DNA sequencing by Sanger method.
DNA polymerase
(usually E. coli DNA polymerase I) Thermostable DNA polymerase (e.g. Taq from Thermus aquaticus) active at high temperatures, is used in polymerase chain reaction (PCR).
Modifying enzymes
- phosphorylases, phosphatases other nucleases, etc. 5’ – OH grp +/ -
- reverse transcriptase (from retroviruses) copies RNA template into cDNA. So labile mRNA converted to stable DNA.
PCR
Process:
• DS DNA • Add primers • Anneal at 50’C • Polymerase copy DNA 72’C • Raise temp • Repeat = clonal amplification Can introduce unique restriction sites
PCR
Uses:
x4
- Forensics
- Diagnostics
- DNA manipulation for cloning
- RT-PCR. Capture mRNA
Protein production
uses
x3
- Design gene for human growth hormone.
- PCR from existing DNA
- Chemical synthesis from scratch? Codon usage.
