manipulating genomes Flashcards
the genome
all the genetic material an organism contains . 2 percent exons, large non coding reigons (introns) removed from mrna before translation into polypeptide chains
minisatelite vs microsatellite
miisatelite- 20-50 base pairs repeated 100 times
microsatellite- 2-4 bases repeated 5-15 times
identical twins = identical satellite pattern
electrophesis extra points
dna attaches to the anode due to negatively charged phosphate groups, when smallest fragments reach the end, the electric current is switched off
gel placed in alkaline buffer solution - denatures dna fragements to expose the bases
southern blottoing technique used, strands transferred into nitrocellulose paper/ nylon membrane placed over gel - membrane covered w sheets of dry absorbent paper to draw alkaline solution containing dna through membrane by capillary action .
the process of DNA sequencing
- dna sequencing mixed with primer, polymerase and terminator bases
- mixture placed in thermal cycler- goes through speerating strands and annealing primers by sswithcing from 96 to 50 degrees @ repeated cycles
- 60 degrees, dna poylymerase builds new strands by adding nucleotides to single strand dna
- every time terminator base incoroporated ( terminator bases were added in lower amounts) , results in dna framgents of diff lengths depending on where in chain it got incorporated.
- eventually, all possible dna chains will be produced w reaction being stopped at every base
- dna fragment seperated by length according to capillary sequencing, woks like gel electrophesis
- flourescent markers identify final base of each fragment and laers detect diff colours and order of sequence
- order of bases in capillary tuebs show seq of new, complementary strand of dna which is made. used to build up sequence of original dna strand
- data from sequencing process fed int ocomputer to assemble genome . scientists use it to find bits that code for specific characteristics linked to diseases etc..
uses of dna profiling
performed on traces of dna left at scrime scene
assists in identification of individuals or familial relationships
. identifying ppl at risk of developing diseases
.allows for risk asessments
dna sequencing
first: sanger developed technqiues for sequencing nucleic acids from viruses and then bacteria- involved radioactive labelling of bases and gel electrophesis (flourescent tags use as ALTERNATIVE!)
hgp
scientists all over the world worked together to map genome .
developing tech and faster sequencing techniques= allowed for faster mapping of human genome
next gen sequencing
high throughput sequencing
.eg sequencing reaction takes place of plastic slide (flow cell) instead of gel . allows large clusters to be sequenced and imaged at the same time (with terminator bases ) - “massively paralell sequencing “
.reduced costs, and more efficient ( Sequencing is much faster)
bio informatics etc..
computers than help anayse raw biologica data eg development of algorithms, mathematical models etc ( makes sense of enormous data )
. important in working out 3d structures of proteins and understanding gene regulation - tldr helps use info from dna sequencing eg identifying genes linked to diseases
genomics
analysing structure and function of genomes
.realisation that genes worked with environment to affect physiology and likelihood of disease development
sequencing pathogen genomes allows doctors and scientists to….
find source of infection
.identify antibiotic resistant bacteria so antibiotics used
only when effective+ tracking trnasmissions of TB so it can be treated as it spreads quickly around the world
.scientists: track outbreak of dsiease
.scientsits: identify reigons of pathogen genome to develop new drugs
dna barcoding ( an example of genome sequencing )
identifies sections of genomes that are common to all species but vary between them.
ibol- identfiying species using short sections of dna from genome- chosen section is 648 base pair of cytochrome c oxidase - small enough to be sequenced quickly + cheaply and varies anough to give clear differences
. suitable reigons for barcoding in fungi and bacteria nto identified yet so yo ucant barcode them
use of genome seqwuencing
evolutionary relatin ship- calculating basic mutation rate of dna allows to calculate how long ago sepcies diversion occured from common ancestor
genomics and proteomics
proteomics, study of amino acid sequences
. dna sequence allows prediction of sequence of amino acids in proteins produced . some genes code for many proteins
spliceosomes
exons that are removed adn to be translated from dna during transcirption joined together by enzyme complexes (Specilosomes) to give mature functional mrna thus one gene= produce any versions of functional mrna- thus code for diff amino aicds= thus phenotypes
protein modifiation
some proteins modified - shortened or lengthened to give variety - proteonomics give increasing knowledge of relationship between genotype and phenotype
synthetic biology technqiues
genetic engineering- genetic modifcation of an organism
.using biological systems in industry - eg enzymes and production of drugs from micro organsims
.synthesis of new genes- replace faulty genes- cystic fibrosis
.syntehsis of entire new oganisms
green box: dna sequencing and clinical intervention
mrsa was linked to one hospital worker after dna sequencing thus preventing spread and deaths
genetic engineering
isolting gene and transferring using vector
.restriction endonculeases unevely cut the dna strands - unpaired exposed bases= sticky ends= easier to insert gene
.another technique- reverse transcirptase on mrna to produce complementary dna - easier to idnetify desired gene
formation of recombinant dna: making hte vector
bacterial plasmid- has marker gene ( eg antiiotic resistance) - enable to determine that hte bacteria has taken up plasmid by growing bacteria in antibiotic
. insert dna fragment into plasmid by cutting open using same reaction endonuclease to isolate dna fragment - causes complementeary sticky ends which are now joined by dna ligase
.plasmids given second marker gene-> show plasmid contaisn recombinant gene
.plasmid tehn cut4 by restriction enzyme within marker gene to insert desired gene .
(marker genes now= floruescene not antibotic )
transferring vector t
process= transformation
methods=, culture bacterial cells in calcium solution and increase temperature. ( makes membrane porous)
electroporation (current makes membranes porous) ( electric urrent has to be carfeully monitored so not to destroy the memebrane
electrofusion (Another way of transferring vector)
currents applied to memb of 2 differetn cells = fuses cell and nuclear memb of both cells to make hybrid cell. used to produce GM plants
used to make monoclonal antibody - (antibody combiens with tumor to divide rapidly)
engineering plants
use agrobacterium tumefaciens - causes tumours in plants. combine desired gene ( eg pesticide protection ) with the bacteria with flourescent amrker gene and carry int plant cell dna. callus is formed (mass of gm plant cells to be grown into plant )
.electrofusion- another method of making transgenic plants- remove cellulases form cell walls and electrofuse them to form polyploid cell. plant hormones stimulate growth of cell wall followed by callus fomration and production of cloned transgenic palnts
engineering animals
membranes harder to manipulate - harder to engineer dna .
animals produce preteins which can be used to cure CF + huntingtons etc
advantages of genetic manipulation
.bactria used to produce insulin + vaccine
.gm microogranisms- store record of dna of another organisms (eg in bacteria) - useful for dna sequencing projects
gm microorganisms used for development of medicine and gene tech (Eg epidemic research ). cons: suspicion of bio warfare using genetically modified pathogens (eg resistance)
gm soya beans have..
insect resistance - produces bt proetin toxic to insects
.resistance to weed killer- allows for higher crop yield
pros/ cons of pest resistance
.reduce amount of pesticied= decrease cost. increase yield
non pest and insect eating preds can be damaged by toxins in gm plants
.pests develop resistance
pros/ cons of disease restance?
.increases crop yield
.transferred genes might spread to wild popualation + cause problems
pros/ cins of herbidcide resistance
reduces competing weeds = inc yield
reduced biodiversity due to overused herbicides
pros / cons of extended shelf life
reduces food waste
reduces commericla value and demand
pros / cons of growing conditions
grow in wider range of conditons and survive droughts
NO CONS
pros. cons of nutritional valuee
enhanced level of vitammins
allergic reaactons to proteins made in gm corps
pros/ cons of medical uses
used for human medicines/ vaccines
NO CONS
Patenting problems
poor countries cant use gm crops because they are patented ( eg soya was apptented so that farmers could only buy them and grow them to use or sell them or porcess them onlt in the year bought , cnat save the seed to grow next year )
.pros L IRI engineered flood resistant rice to support farmers
gm animals
swine fever resisetant pigs
faster growing salmon ( producees growht hormones all year and grows to full adult size in half the tike )
pharming
. production o human medicines from animals
.creating animal models- developing dieasing in animals for development of new therapies. eg mice hace genes deleted so they develop cancer
.creating human proteins - human gene introduced into cow or sheep egg with promoter sequence so gene only expressed in mammary glands. ftransgenic female embryo returned to mother and whne it matures and gives brith it will produce milt that contains desired human protein to be harvested
ethical issues
should animals act as models to human diseasE
is it right to put human genes itno animals??
is it right to put genes from other species into animals wihtout being ertain that it wont harm ?
.does genetically modifying animals reduce them to commodities?
is welfare compromised?
somatic and germ line therapy
used to treat faulty genes that cause CF, haemophilia and severe combinedi mmunodefficiency (SCIDS)
