Manipulating Genomes 2 Flashcards
Human genome
All of genetic material one contains that is dna in nucleus and mitochondria
Dna that codes for proteins
20-25000 regions of dna code for proteins
Only 2% - extrons
Introns
Large non coding regions of DNA
Removed from mRNA before translation
Satellite dna
Short sequences of dna that are repeated many times
Minisatellites
Sequence of 20-50 base pairs will be repeated from 50 to several hundred times
Occur at more than 1000 locations in genome
Known as variable number tandem repeats
Micro satellites
2-4 bases repeated only 5-15 times
Known as short tandem repeats
Patterns of dna satellites
Appear in same positions on chromosomes however the number of repeats vary (inherited mix)
Closer relation more similar the patterns
Producing dna profile
Extracting dna
Digesting the sample
Hybridisation
Seeing evidence
Extracting dna
Dna extracted from a tissue sample and use pcr in order to produce enough dna to develop a profile
Digesting the sample
Strands of dna cut into fragments using restriction endonucleases
Separating the dna fragments
Cut fragments of dna separated to form a clear pattern using electrophoresis
Hybridisation
Fluorescent dna probes are added in excess to fragments which are complementary to known dna sequence
Probes identify the micro satellites which are more varied than the minisattelites
Seeing the evidence
Sample placed under uv light so fluorescence glows - gives a pattern of bars which is unique to every individual apart from mz twins -THE DNA PROFILE
Restriction endonucleases
Enzymes which split dna into small fragments at specific sequences known as restriction site( complements active site)
Cut both dna strand
Allows scientist ability to cut at known and defined points in introns
Electrophoresis
Dna fragments put into well in gel strips ( one well contains known dna fragments)
Current passed through fragments move towards cathode due to -ve phosphate groups in dna - smaller frag closer to anode
Placed in alkali to denature
Southern blotting
Strands of dna transferred to nitrocellulose paper
Identical positions to the gel (alkali removed using absorbent paper)
Then hybridised
Pcr
Form of artificial dna replication
Semi conservative multiple complies
Amplifies sample
Pcr components
Pcr vial contains dna sample nucleotides 2x primers Taq DNA polymerase
Denature dna - heat 95 give single strands
Primers - cool mixture to 55 allows primers to anneal to dna
Synthesis - at 72 taq DNA polymerase strat synthesising new dna comp to og strand next to primer
Uses of dna profiling
Forensic science - evidence at scene
Paternity of a child
Identifying those at risk of particular disease
Dna sequencing
Process of determining the precise order of nucleotides within a dna molecule
Whyis dna sequencing used
Identifying genetic disorders - base sequence mutations
Identifying species using small conserved sections of DNA
Identifying evolutionary relationships
Pathogen analysis to develop vaccines
Proteonomics
Dna barcoding ( identify species)
Use short section of DNA from conserved region of genome
648 base sequence of Mito. Dna which codes for enzyme analysed
Small enough to be quick varies enough to give clear differences
Proteonomics
Study amino acids
Complexity of genotype phenotype relationship
Enables to predict the sequence of amino acids in all proteins it produces
Chain termination technique
A method to find a base sequence of unknown genes using terminator bases
Terminator bases
Dideoxyribonucleotides - 1 less oxygen the deoxyribose
Form comp pairs with deoxyribose bases
Given fluorescenct tags to identify
Dna polymerase stops replicating when it encounters the terminator bases
Dna sequencing technique
Pcr - single strand dna
Dna polymerase builds up new strands of terminator base binds polymerase stops
Results in different fragments stopped at every base
Separated using capillary sequencing depending on chain length
Fluorescent markers identify final base on each fragment
Computer reassembles the genome
Synthetic biology
Design and construction of novel artificial biological pathways organisms or the redesign of existing natural biological systems
Synthetic bio techniques
Genetic engineering
Use of biological systems in industry - creating enzymes
Synthesis of new genes to replace faulty
Synthesis of entire new organism
Gene therapy
Treatment of a disease by manipulating gene in a persons genetics
Germ line therapy
Changing genes in gametes to create off spring where all cells carry genetic modification (passed on to offspring)
Somatic cell therapy
Only specific group of body cells genetically modified/ different
No modified genes are in gametes not passed onto offspring
Evaluation of somatic cell
Adv=uccessful treatments of leukaemia for example
Dis=somatic cell has limited life replaced by stem cells that will have faulty allele
Germ line evaluation
Adv = successfully done w animals
Dis= ethical concern desirable offspring illegal
Next generation sequencing
Gel plates and electrophoreses replaced by plastic plate called flow cell with dna fragments attached
Produces stacks of DNA via chain termination
Quicker cheaper and more efficient
GM crops
Developed to benefit grower and retailer
Seeds need to be brought in each yr remain sterile - disadvantage poor
Some modified to benefit poor farmers eg scuba rice resistant to floods
Pharming
Production of human medicine
Mice removed genes develop cancer can study
Bacteria making insulin
Gene to cow egg protein harvested in milk
Ethical issues
Should animals be genetically engineered to act as models of human disease
Is it right to put human genes in animals
Religious beliefs
Disease transfer from animal to human etc
Scid
Severe combined immunodeficiency disease
Caused by faulty allele coding for enzyme ADA
Essential for healthy immune system
Gene therapy for SCID
Removal of T cells and insertion of correct allele via vector
Cells take up and clone allele
Alternative injections of ADA
May develop leukaemia
Cystic fibrosis
Thick mucus
Caused by recessive allele that codes for CFTR Protein
No channel proteins
Gene therapy for cystic fibrosis
Normal allele inserted into liposomes
Lipid soluble can move through cell membrane of respiratory pathway
Only last a week - somatic
Genetic engineering
Use of technology to change genetic material of one organism by taking genetic material from another organism
Isolating the gene gm
Use of restriction endonuclease to break at specific sites cutting dna unevenly leaving sticky ends
This makes it easier to insert gene
Or use reverse transcriptase to produce strand of comp DNA
Advantage of using reverse transcriptase
Makes it easier to identify desired gene due to particular cells making v specific types of mRNA
Formation of recombinant dna
Isolated dna inserted into bacterial plasmids
Once plasmid gets into host cell combine w DNA to form recombinant dna
Plasmids use as vectors contain marker gene eg antibiotic res which helps scientist determine whether taken up
Dna and plasmid fusion
Plasmid is cut open w same restriction endonuclease resulting in compl sticky ends
Dna ligase forms phosphodiester bonds between sugar and phosphate groups on the dna strands
Given second marker gene where dna fragment is placed so secondary marker gene doesn’t work
Transferring vector
Calcium rich sol and high temp - causes membrane to become more permeable
Electroporation- current applied to bacteria make membrane v porous
Alternative gm techniques
Electrofusion- fusing of cells using electric current forming a hybrid polyploid cell (don’t survive in living animal)
Agrobacterium tumefacians - plant produce a callous which can be grown into a new transgenic plant
Genetic engineering uses
Bacterial engineering to form medicines eg hormones antibiotics clotting factors etc
In animals to try and cure human genetic diseases eg huntingtons or cystic fibrosis
Benefits and risks of genetic engineering
Microorg adv=make drugs dis= antibiotic resistance can spread
Plant adv= high crop yield dis= damage environment
Animal adv=improve understanding of genetic dis dis= welfare issues
Humans adv=treat disorders dis= illegal and unknown risk
