reombinant DNA technology Flashcards
what is recombinant dna
introduction of a foriegn gene into the dna of another organisum
what are the 5 processes of making a protein using dna technology
isolation of dna fragments containing desired protein
insertion of dna using a vector
transformation inserting vector into a host
identification of host cells that have taken up the gene
growing/cloning of population of host cells
describe isolation of dna fragments using mrna
the mrna has been transcribed from the gene of interest
reverse transcriptase synthesises a single strand of complementray dna
dna polymerase forms another strand using free nucleotides making double stranded dna
what are the advantages of using mrna rather than dna when isolating dna fragments
introns already removed
more mrna compared to dna in a cell
easier to find and quicker to remove
describe isolating dna fragments using restriction endonuclease endonuclease
cuts dna at a spesific recognition site to cut out the desired gene from the rest of the genome
they cut leaving sticky ends with exposed bases or blunt ends
when will a sticky end join to another sticky end
when they have been cut by the same restriction endonuclease
characteristics of a restriction endonuclease
highly spesific active sites that catalyse the hydrolysis of the sugar phosphate backbone of both stands of dna
cut a recognition site between 4-8 base pairs long
recognition sites are palandromic
describe isolating a dna fragment using the gene machine
desired nucleotide sequence is derived from desired protien
they are fed into a computer checked and small overlapping single strands assemble to form the complete gene called oligonucleotides
the gene is replicated many times using pcr making it into double stranded dna
using sticky ends it is inserted into a plasmid to act as a vector
advnantages of the gene machine
only takes 10 days
any sequence can be produced
high accuracy
introns are removed
when sticky ends form recombinant dna enzymes form phosphodiester bonds
dna ligase in a condenstaion reaction
what is a promotor
length of dna added before a dna fragment which transcritpional factors and rna polymerase bind to to initiate transcritption
what is a terminator
length of dna added after the dna fragment to release rna polymerase and stop transcritpion
how do plasmids enter host cells
host cells and plasmids are mixed in ice cold temperatires and calcium ions
heat shock of 42 degrees is applied for 2 minutes to increase bacteria wall permeability allowing plasmids to enter host cell
describe 3 ways to identify if a plasmid entered a host cell
- antibiotic resistance markers - bacteria have two genes to be resitstant to two antibiotics in the same plasmids. they grow on an agar dish and are treated with amplicilin so only the resistant ones grow and it becomes clear which one is not
- florecent markers - insert florecent gene into plasmid protein , insert the gene of interest into the middle of the florecent gene
grow them as normal and any successful bacteria will glow - enzyme marker - there is a substrate that is usually colourless but turns blue in lactate - same process as florecent markers
what is needed for the polymerise chain reaction
the dna fragment to be copied
taq polymerase
primers - short single stranded lengths of dna with complementrray base sequences and prevenets two dna strands rejoinig
free nucleotides
thermocycler - comptuter controlled machine
stages of pcr
1 strands seperation - dna heated to 95 degrees to break hydrogen bonds for 5 minutes
2 mix with primers and make hydrogen bonds with them at 55 degrees
3 mix with free nucleotides and taq polymerase
4 dna synthesis at 70 degrees because it is taqs optimum
why is taq polymerase better
it has a higher optimum so is faster
advantages of in vivo coloning
introduces gene to another organisum
no contamination risk
very accurate
cuts out spesific genes
trandformed bacteria to make large quantities of the gene
advantages of in vitro coloning
rapid
makes more crime scene evidence quickly
not require lvivng cells
advantages of recombinant dna to humans
increase yeild and nutritional value of aniamls and plants
pest and disease resistant plants and aniamls
crops resistant to harsh conditions and pesticides
making medicine , vaccines
examples of genetically modified microorganisums
antibioics
hormones - insulin
enzymes - amalyse and lipase in food production
examples of genetically modified plants
tomatos to keep them fresh for longer in transit
herbicide resistant croops
disease resistant crops
examples of genetically modified aniamls
disease resistant genes
goats with improved milk protein
growth hormones in fish and sheep
what causes cystic fibrosis
cftr - channel protein used to transfor chloride ions across epithlial cell
water leaves by osmosis creating a thin runny mucus
cf - recessive allele coding for cftr is mutated by deletion so water is retained in epithelial cells and mucus is thick so cant be wafted out of the lungs
what is gene therapy
replacement of a defective gene by introducing a healthy one
what is somatic gene therapy
it targets affected tissue but is not passed on to future generations only effective for the life of the individual
what is germ line therapy
replacing the defective gene in the fertilised egg
is passed on to future generations
what is a dna probe and how does it work
short single stranded sectio of dna with bases complementary to the target gene
they are labeled to make them visible
describe hybridisation
dna strands are seperated by heat and mixed with a probe with complementray bases so they bind
dna washed to removed excess
hybridised dna detected using x ray paper
how to test for different disorders at the same time
fix samples of each dna probe in a well on a glass slide
add dna sample
complementry dna bases will bind to one or more probe
what varies about vntrs in different people
length of vntrs
number of vntrs
who has the same vntrs
identical twins
are vntrs coding or non coding
non coding
process of genetic fingerprinitng
extraction of dna from a sample and amplified by pcr
digestion by restriction endonuclease cuting dna
seperation by size in gel electrophoresis
dna is negativly charged so attracted to the possitive end of the gel
hybridisation - dna is transfered onto a nylon sheet which is immersded in alkali to seperated double stranded dna and probes are attached to vntrs
development - nylon placed under xray and radio active probes will be visible
