8.3 + 8.4 Flashcards
what are genome projects used for
determining the complete sequence of bases that make up the DNA of an organism
how to gene sequencing work
by cutting the DNA molecule into fragments which are sequenced and then they are put back into correct order to give the sequence of the whole genome
what can sequencing be used for
comparing genomes with species highlighting disease risk, improve understanding of inherited disorders and track early migration patterns with evolutionary relationships between species
what is the proteome
the sequence of proteins coded for by the DNA base sequence of the genome
what can finding the genome of simple organisms help with
intensifying antigens of viruses monitoring mutations as the pathogen evolves allowing us to produce vaccines faster controlling the spread of infection
why is it hard to determine the human proteome
as over 98% are introns (non-coding) so hard to determine which are exons
what are the three methods of making DNA fragments
- using reverse transcriptase to make cDNA
- genes can be found and ‘cut out’ using restriction enzymes
- Using a gene machine to build fragments
what can reverse transcriptase do
an enzyme that can convert RNA back into DNA
what does reverse transcriptase do in making DNA fragments
is is added using free DNA nucleotides to make DNA from the mRNA template which is called cDNA as it is complementary DNA matching the RNA
what do restriction endonuclease do
enzymes that can recognise and hydrolyse hydrogen bonds at specific sequences of DNA
what do restriction endonuclease do in making DNA fragments
they cut at a specific recognition sequence in the DNA which is complementary to their active site
what do restriction enzymes do
form sticky ends (pieces of DNA that have exposed nucleotides on a single strand at each end) allowing different fragments to be joined together
4 steps of using a gene machine to build DNA fragments
- the sequence is designed on a computer
- the first nucleotide in the sequence is attached to a physical support
- nucleotides are added step by step in the right order and joined in the correct place to prevent branching
- short sections of DNA called oligonucleotides are produced (20 nucleotides long). once these are complete they are broken off from the support and can be joined together to make longer fragments
what is the difference between in vivo and in vitro
vivo takes place in living organsims
vitro is not in living organisms translating to in glass
first step of in vivo
- desired gene is isolated using restriction endonuclease enzymess
second step of in vivo
promoter genes and terminator regions are added to the gene to make sure the gene can be correctly transcribed once in the host
- promotor tells RNA polymerase where to start and terminator tel RNA polymerase where to stop
third step of in vivo
the gene is inserted into a vector
cut the plasmid and the gene with same restriction endonuclease enzyme to leave ‘sticky ends’ allowing the target gene to fuse with the plasmid
ligase enzymes can join these two DNA fragments together by catalysing condensation reactions that join the sugar groups and the phosphate groups to the DNA backbone
when DNA fragments and plasmids are mixed not all plasmids will take up the gene correctly so DNA fragments that dont get inserted can also join its own sticky ends to create a loop
fourth step of in vivo
the recombinant plasmid is now transferred to host cells
a solution such as sodium chloride can be used to make cell walls more permeable, heat shock can be used or electroporation to make holes in membrane which DNA can pass through
step five in in vivo
the host cells are allowed to multiply and those that have successfully taken up the gene are identified using a marker gene
marker genes are inserted into vectors at the same time the gene is, these can code for antibiotics resistance and only transformed bacteria will survive.
as the genetic code is universal bacteria can transcribe DNA and translate the mRNA to produce the protein
what is PCR
the artificial replication of short sequences of DNA using artificial DNA primers to amplify a specific strand of DNA
what are the 4 steps of PCR (in vitro)
- the mixture is heated to around 95C to break the hydrogen bond between the complementary nucleotide base pairs and separate the double-stranded DNA into two single strands
- the mixture is cooled to around 55-60C so that primers can bind forming hydrogen bonds to one end of each single strand of DNA which identifies where tax polymerase should bind
- the temperature is raised to around 72C which is the optimum temperature for Taq DNA polymerase catalysing the addition of DNA nucleotides by complimentary base pairing to create two complete double strands of DNA
- repeat
what is the amount of DNA increase
exponential
why does the number of fragments eventually plateau
as primers are the only thing used up in the reaction limiting the number of fragments that can be made. replication stops as they run out
what are the three steps of genetic fingerprinting
- restriction enzymes are used to made DNA fragments that contain VNTRs these are amplified using PCR
- DNA fragments are separated by size using gel electrophoresis
- compare DNA fragments/proteins next to known samples
what are VNTRs
variable number tandem repeats: repeating sequences that occur in different places in the genome of eukaryotes. the probability of two individuals having the same VNTR in the same place is very low meaning the length of VNTR between every person is unique
how can genetic finger printing be used in medical diagnosis
testing to see if causes are genetic or identifying cancer
how can genetic finger printing be used in forensic investigation
to establish w person present at a crime scene with PCR used to amplify trace amounts of DNA
how can genetic finger printing be used in determining genetic relationships
paternity testing
how can genetic finger printing be used in genetic variability
populations with similar genetic fingerprints have little diversity
how can genetic finger printing be used in animal and plant breeding
identifying parents or genetic similarities can help selective breeding however increase genetic disorders
What is a DNA probe
Single stranded pieces of DNA which are complementary to and therefore bind to known base sequences.
4 uses of gene probes
- locate specific alleles of genes
- to see if a person carries a mutates allele that causes a genetic disorder such as cystic fibrosis
- help determine how patients will respond to drugs such as breast cancer treatment which only responds to a certain mutation
- identify health risks such as some peoples risk of cancer can be increased if they carry certain mutations
method of gene probes (3 steps)
- DNA sample digested into fragments using restriction enzymes and separated using gel electrophoresis
- Separated DNA fragments transferred to nylon membrane and incubated with the DNA probe. The probes will only bind if the complementary sequence is present
- The membrane is exposed to UV light and if the gene is present it will fluoresce. DNA would be invisible without the probes so only bands which contain the gene will be visible
what is a microarray
used to screen a sample with multiple DNA probes all at once
how does a microarray work
- the sample of fluorescent DNA is washed over the array which has the DNA probes fixed to it
- DNA sequences that match the probe stick to the array
- the array is washed to remove any fluorescent DNA not stuck to a probe
what do genetic counsellors do
guide people to make informed decisions about matters related to genetics that may come up in their or their families health
example of genetic counselling
screening for genetic mutations leading to cancers
what does personalised medicine do
personalised medicine can be used target different dosages or compounds as medicine depending on the genetic make up of the person
example of personalised medicine
some breast cancer treatments are targeted at specific mutations so screening can be used to make sure the right medicine is used
what is gene therapy
the manipulation of the genetics of a human cell.
what is somatic cells gene therapy
only affects certain cell types, the alterations made to the patients genome in those cells are not passed onto the patients offspring.
method of delivering the functional alleles to the patients body cells: (4 steps)
- the liposomes are placed into an aerosol inhaler and sprayed into the noses of patients
- some liposomes will pass through the plasma membrane of the cells lining the respiratory tract
- some liposomes will then pass through the nuclear envelope and insert into the host genome
- the host cell will express the CFTR protein
what are viral vectors
genetically engineered viruses used to deliver the functional gene copy
why are viruses used as vectors
as they are already engineered to encapsulate and deliver genes to human cells to be transcribed and translated into proteins
why is it a risk to used viruses as vectors
could be destroyed by the immune system, triggering an immune response the viral vector has potential to also reactivate its ability to cause disease
what is germline gene therapy
altering the gamete or zygote of the organism changing the genetic make-up of the descendants of the original patient
why is gremline gene therapy a risk
the gene may find their way to a location that could disrupt the expression or regulation of other genes or increase the risk of cancer
ethical praises to do with animals (2)
- genetic manipulation is no different from crossbreeding
- genetic manipulation reduces cost and increase productions of crops
ethical praises to do with plant (3)
- plant pathogen can reduce crop yield so getting rid of them helps famine
- reduces the cost and increase production of plant crops
- there is no evidence of any gene escaping crop plants into the wild
ethical praises to do with bacteria (4)
- bacteria can be seen as little machines rather than parts of a larger ecosystem
- bacteria swap their DNA amongst themselves all the time
- many life saving drugs can be produced from genetically modified bacteria
- these bacteria can be completely isolated to the laboratory
ethical issues to do with animals (3)
- animals are sentient
- genetic manipulation reduced the variation within the populations as clones are used
- there could be health implications for the animals
ethical issues to do with plants (4)
- plants are harder to contain and could cross breed with wild varieties releasing new genes into the environment
- potential harm to human health from food
- negative impact on traditional farming practices
- excessive corporate dominance
ethical issues to do with bacteria
- there is danger that they will escape from the lab
- there could be contamination from the other products of the bacteria
- antibiotic resistance genes are used and these could be create and untreatable pathogen as are carried on a plasmid that can be shared between species of bacteria
