6.1.3 Manipulating Genomes Flashcards
(26 cards)
PCR (Polymerase Chain Reaction)
Used in almost every application of gene technology at some stage
Rapid generation of multiple copies of DNA samples
Sanger/chain termination technique
- Extract DNA, cut into fragments to various lengths + amplify using PCR
- Prepare four reaction mixtures, include in each a different replication-stopping nucleotide
- Each solution contains:
- DNA nucleotides
- DNA polymerase
- Primers
- Terminator base (A,G,C or T) - Electrophoresis
- Read sequence as xomplement of bands containing labelled strands
- The base sequence is then stored in a database to allow quick comparisons
- Amino acid sequence can also be stored and proteins modified
Faster techniques for DNA sequencing
- Massive parallel sequencing
- High throughput sequencing
- Next generation sequencing
Electrophoresis
- Pipette the 4 solutions containing the 4 different terminator bases into the different wells
- Pass a current through the electrophoresis plate from - to +
- (DNA has a slightly negative charge so it will be repelled by the cathode (-) and attracted to the anode (+))
- (Smaller fragments,ents will travel further as they have less mass and less resistance in the gel)
2 ways to view the DNA
- Southern blotting:
- Using radioactive DNA probes and x rays - Using a GFP (green fluorescent protein):
- DNA probe and UV light
Bioinformatics
- Software is developed to process and understand large complex data (DNA sequences) using computational biology
- Allows to access a large amount of data
- Information is universal
- Allows rapid comparison of sequences with newly sequences alleles
- Amino acid/sequence/protein structures held in database
- Computer modelling of new protein structure from base sequence
Sequencing allowed for: Synthetic biology
- Fieeld of science that involves redesigning organisms for useful purposes by engineering them to have new abilities
- To solve problems in medicine, manufacturing and agriculture
- biofabrication
Bioinformatics used in: Epidemiology
- Study of how often diseases occur in fifferent groups of people and why
- Idemtify the source of the outbreak, identify vulnerable populations, design vaccination programs to target certain individuals
Sequencing allows for: proteomics
- Large-scale study of a set of proteins produced in an organism, system or biological context
Why compare genomes?
- Evolutionary relationships (phylogeny)
- Comoare base sequence to predict function of an unknown protein
- Universal
- Compare between and within species
Uses of DNA profiling
- Paternity tests
- Crime scene DNA comparisons - DNA fingerprints
- Searching for genes that could trigger diseases
Introns
- In most people, the genome is very similar
- Using coding sequences of DNA would not provide unique profiles
- Non-coding DNA contains VNTR/STR/repeating sequences
-STR - short tender repeats, small region 2-4bp repeated 5-15 times - VNTR -variable number tandem repeats, 20-50bp will be repeated 50 - several 100 times
Stages of DNA profiling
- Extract DNA from+ amplify using PCR
- Cut DNA using restriction enzymes
- Put DNA fragments on gel electrophoresis + separate
- use radioactive probe - X-rays/GFP - UV light
Genetic engineering
- Restriction endonucleases (enzymes) are used to cut desired gene from DNA
- Creates sticky ends (exposed bases on DNA) which make it easier to insert desired genes
- The plasmid (vector taken from bacterium) cut using the same restriction enzymes to produce complementary sticky ends
- Desired gene (or cDNA) inserted (annealing) into vector/plasmid using enzyme DNA ligase.
- Desired enzyme inserted with marker - fluorescent marker or gene for antibiotics - Recombinant DNA (plasmid carrying desired gene) inserted into host cell using electroporation.
- Electric shock makes the membrane porous and plasmids can pass through membrane of host cell - Host cell undergoes mitosis, reprocessing desired gene.
- Tested to ensure desired gene been taken up by host cell by looking at fluorescence or applying antibiotic (dependant on marker used)
Alternative method to extract desired gene: Reverse Transcriptase
- Makes DNA from an RNA template- does opposite of transcription
Recombinant DNA
- DNA combined from 2 sources, often from different organisms
Different vectors used from different organisms
Plant- Agrobacterium timefaciens, plasmids, virus
Bacteria - BAC (bacteria artificial chromosome), bacteriophage, virus
Animal - Virus, BAC, plasmid
PCR
- Amplifies DN
1. Denaturation - Heat DNA to high temp to break H bonds between bases
2. Annealing - Cool solution + add primer to base sequence wanted to replicate - allow Taq polymerase to join
3. Heat to optimum temp for enzyme - joins free nucleotides on complementary base pair to form new DNA
Amplifying DNA
- Each round the number of DNA doubles
- Formula 2^x used to calculate number of strands
- x is number of cycles
Insect resistance in genetically modified soya
- Contain genes that make the abt proteins
- Plants produce isecticidal abt proteins - toxic to insects and resistant to weed killer
- Provides and effective environmentally safe pest control
For:
Widely used in organic farming- no chemicals sprayed onto plants
Higher yield crops
Less expense
Less labour
Against:
Some pest insects + predators might be damaged by the toxins made from abt protein
Insects might become resistant foot toxic Bt protein
Genes might spread to wild populations - ‘superweeds’
Reduce biodiversity
Ethical - Patenting issues
Genetically modified pathogens for research
- Scientists can genetically modify pathogens to develop medical treatments
For:
Used for medical + epidemiological research to find treatments
Against:
Health and safety risks of the researcher and the wider public
Ethics - used in biological warfare
Pharming
Genetic modification of animals creating transgenic (contains 2 different types of genetic info) organisms
- some pigs been genetically engineered to express a fatty acid for higher levels of omega-3
For:
Animal has desired gene, leads to:
- Decrease disease risk
- Fastee growth rate
- Production of a human protein in milk that we can harvest
Against:
Unknown risks of GM
Ethics- is it right to put human genes into animals
- creating transgenic animals might cause the animal harm
- welfare of animal might be at risk
Patenting
Where a company that creates a GM seed legally prevents others from using it without payment
For:
Allows company who created GM seed to make profits on their design
Against:
Patented seed can only be used for the year then new seed bought
Ethics- poor farmers who need drought or flood resistant crops with high yield can’t afford to buy GM seeds
