GENE TECHNOLOGY Flashcards
What is a genome
the entire set of DNA, including all the genes in an organism
what parts of DNA does gene sequencing work on
fragments of DNA
what is a proteome of an organism
all of the proteins that are made by it
why is it easy to determine the proteome of simple organisms
eg a bacteria
doesn’t have much non coding DNA
therefore relatively easy to determine the proteome from the DNA sequence
why is it hard to determine the proteome of a complex organism
Contain larger sections of non coding DNA
Also have regulatory genes which determine when some genes coding for some proteins are switched on and off
Makes it hard to translate the genome to proteome
what does recombinant DNA technology involve
transferring a fragment of DNA from one organism to another
What are the 3 ways DNA fragments can be made
Reverse transcriptase
Restriction endonuclease enzymes
Using a gene machine
How do you make DNA fragments using reverse transcriptase
mRNA complementary to the gene and easier to obtain as more molecules
It is obtained by isolating it from cells, its then mixed with free DNA nucleotides
mRNA used as template to make lots of DNA
Reverse transcriptase makes DNA from an RNA template
DNA produced is called cDNA (complementary DNA)
How do you make DNA fragments using restriction endonuclease enzymes
Some sections of DNA have palindromic sequences
Restriction endonucleases recognise specific palindromic sequences and cut the DNA at these places
DNA sample is incubated with the specific restriction endonuclease which cuts the DNA fragment out via a hydrolysis reaction
Sometimes the cut leaves sticky ends (unpaired bases at the end of the fragment)
They can be used to bind/ anneal the DNA fragment to another piece of DNA that has complementary sticky ends
How do you make DNA fragments using a gene machine
a database containing the necessary info to produce the DNA fragment is used
The sequence that is required is designed
The first nucleotide in the sequence is fixed to a support
Nucleotides are added step by step in correct order
Protective groups are added (make sure nucleotides join at correct point and prevents unwanted branching)
Short sections of DNA called oglionucleotides (roughly 20 nucleotides long)
How do you make DNA fragments using a gene machine
a database containing the necessary info to produce the DNA fragment is used
The sequence that is required is designed
The first nucleotide in the sequence is fixed to a support
Nucleotides are added step by step in correct order
Protective groups are added (make sure nucleotides join at correct point and prevents unwanted branching)
Short sections of DNA called oglionucleotides (roughly 20 nucleotides long)
They are then broken off from the support and protecting groups are removed
Oglionucleotides can then be joined together to make longer DNA fragments
How do you amplify the DNA fragments you’ve just obtained
DNA fragments inserted into a vector
The vector transfers the DNA fragment into host cells
Identifying transformed host cells
To produce proteins you need a promotor and a terminator region
How is the DNA fragment inserted into a vector and how is recombinant DNA produced
A vector is something that can be used to transfer DNA into a cell
They can be plasmids or bacteriophages
The vector DNA is cut open using the same restriction enzyme endonuclease that was used to isolate the DNA of the target gene
So sticky ends of the vector are complimentary to the sticky ends of the DNA fragment containing the gene
The vector DNA and DNA fragment are mixed together with DNA ligase
DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA
This process called ligation
The new combination of bases in the DNA (vector DNA and DNA fragment) is called recombinant DNA
what is ligation
DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA
How does the vector transfer the DNA fragment into host cells
The vector with the recombaint DNA is used to transfer the gene into cells
If a plasmid vectors used host cells have to be persuaded to take in the plasmid vector and its DNA
With a bacteriophage vector the bacteriophage will infect the host bacterium by injecting its DNA into it
The phage DNA (with the target DNA in it) then intergrates into the bacterial DNA
Host cells that take up the vectors containing the gene of interest are said to be transformed
How do we identify transformed host cells
Marker genes are used to identify the transformed cells
Marker genes can be inserted into the vector at the same time as the gene to be cloned
Host cells are grown on agar plates
Each cell divides and replicates its DNA creating a colony of cloned cells
The marker gene can code for antibiotic resistance
Or it can code for fluorescence where when placed under UV light the transformed cells glow and are then easily identified
what are promotor regions
promotor regions are DNA sequences that tell the enzyme RNA polymerase when to start producing mRNA
what are terminator regions
Terminator regions tell the enzyme when to stop
Without the right terminator region, it won’t stop in the right place and a different protein will be coded for
what does PCR stand for
polymerase chain reaction
Can be used to make millions of copies of a fragment of DNA in just a few hours
what are the 3 stages of PCR and their temperatures
Denaturation 95 degrees
Annealing 55-70 degrees
Extension 72 degrees
what happens in the denaturation stage of PCR
H bonds of DNA are broken, 2 strands of DNA are separate
what happens in the annealing stage of PCR
Primers bind specifically to their complementary sequence in the single stranded DNA
what happens in the extension stage of PCR
DNA polymerase extends the primers forming new DNA. Uses original DNA as a template, complimentary base pairings with use of covalent bonds to join bases.
What are the reagents needed for PCR
DNA sample (template)
Primers- to direct the synthesis of the specific target region
Buffers
DNA polymerase – catalyst
Deoxynucleotide triphosphates- contain the bases complimentary to the DNA
How does the PCR mechanism work
PCR produces a DNA copy from a DNA template
First identify target region
A specific primer is then designed to bind to this target region via complementary base pairing
Primers are short sequences of nucleotides between 20-30 base pairs long that are chemically synthesised
One PCR reaction involves a primer then the chain then another primer (2 primers per reaction)
The second primer binds to the other complementary strand of DNA, its position of where it binds determines the length of the product of the newly synthesised DNA
The final length includes both primers- e.g. base sequence 100 base pairs and each primer is 20 base pairs in total length is 140 base pairs
What can PCR be used for
Can be used for cystic fibrosis diagnosis- as it is sensitive enough to detect individual base changes (point mutations), this is how cystic fibrosis occurs
Can be used in forensic science- looking at samples of DNA in blood or hair follicles, this can be amplified to help identify the individuals
Can also be used for DNA fingerprinting to identify evolutionary relationships between species
How are the results of PCR analysed
PCR products are visualised by gel electrophoresis, by use of agarose gels
DNA molecules are separated on their size- large ones move through the gel more slowly
It moves by inducing an electric current across the gel submerged in buffer in a gel tank, DNA is negatively charged so moves to the positive electrode
Then it is seen by staining with a dye that binds to it as is fluorescent under UV light (normally ethidum bromide)
Define genetic engineering
When an organism is transformed using recombinant DNA technology
How does genetic engineering work
use insulin as an example
The DNA fragment containing gene for insulin is isolated
The DNA fragment is inserted into a plasmid vector
The plasmid containing the recombinant DNA is transferred into a bacterium
transformed bacteria are identified and grown
The insulin produced from the cloned gene is extracted and purified
How can genetic engineering benefit agriculture
Crops can be modified to produce high yields
Crops can be modified to be pest resistant
How can genetic engineering benefit industry
Enzymes can be produced on mass and for less money
How can genetic engineering benefit medicine
many drugs and vaccines can be made quickly, cheaply and on mass
What are the concerns about the use of genetic engineering on agriculture
Farmers might only plant one type of crop (monoculture)
Could make all the farmers crop vulnerable if a disease arose that killed that type of crop
It would also reduce biodiversity
What are the concerns about the use of genetic engineering on industry
Larger companies with lots of money will get bigger and the smaller ones will be wiped out in the process
Without proper labelling people won’t have a choice about whether to consume food made properly or via genetic modification
Some consumers won’t import genetically modified food, leading to an economic loss and they would have previously bought that nations crops
What are the concerns about the use of genetic engineering on medicine
Genetic technology could be unethical eg designer babies
How does gene therapy work
Involves latering defective genes (mutated alleles) inside cells to treat genetic disorders and cancer
In gene therapy if the mutation is caused by 2 recessive alleles what do you do
you can add a working dominant allele to over power the broken recessive ones
In gene therapy if the mutation is caused by a dominant allele what do you do
You can silence the dominant allele
eg stick a bit of DNA in the middle of the allele so that it doesn’t work anymore
What are the 2 types of gene therapy
Somatic therapy
Germ line therapy
How does somatic gene therapy work
involves altering the alleles in body cells, particularly the cells that are most effected by the disorder
Eg cystic fibrosis, is damaging to the respiratory system so somatic gene therapy targets the epithelial cells
Doesn’t effect the sex cells so any offspring could inherit the mutation
How does germ line gene therapy work
Involves altering the alleles in the sex cells
Means that every cell of the offspring will have been effected by the gene therapy and they won’t suffer from the disease
However it is currently illegal in humans
what are the ethical issues with gene therapy
it could become used in cosmetics eg to stop aging
It could do more harm than good as there is a risk of over expression of genes
How can you look for alleles
Using DNA probes anf hybridisation
How do DNA probes work
Are short strands of DNA
They have a specific base sequence that’s complementary to the base sequence of part of a target allele
This means the DNA probe will bind (hydbridise) to the target allele if present in the sample of DNA
The probe also has a label attached so that it can be detected
The 2 common types of labels are radioactive (can be detected using X-rays) and fluorescent (can be detected using UV)
what are some uses of screening with DNA probes
Help identify an inherited condition (look for the mutated allele)
Help determine how a patient will respond to specific drugs (if doctor knows your DNA it will be easier to give out the drugs that will be most effective for you)
Help identify health risks
What is genetic counselling
advising patients and their relatives about the risks of genetic disorders
eg if a family history of breast cancer they can choose whether they want to be screened or not
what does VNTR stand for and what are they
variable number tandem repeats
base sequences that don’t code for proteins and repeat next to each other
why do 2 people not have the same fingerprint
Each individual has a different number of times these sequences/ VNTRs are repeated and therefore the length of the sequences varies which is why no one has the same fingerprint
describe electrophoresis for fingerprinting
DNA isolated
Sample replicated by PCR
DNA mixture is placed into a well in a slab of gel and covered in a buffer solution that conducts electricity
An electrical current is passed through the gel and due t DNA fragments being negatively charged they move towards the positive electrode at the far end of the gel
smaller DNA fragments move faster and travel further through the gel than the larger ones so the DNA fragments separate int bands according to size
what charge does DNA have
a negative charge
how does electrophoresis allow genetic fingerprinting to occur
The DNA spreads out through he gel according to size
If the fingerprints have a band at the same location on the gel it means they have the same number of nucleotides and VNTRs at that place therefore its a match
(looks a bit like chromatography)
give 2 uses of genetic fingerprinting
forensic science - to find a match to any type of DNA at the crime scene
For biological testing- seeing if you are related to someone- paternity testing
how do parents fingerprints link with the offsprings fingerprints
we inherit the VNTR sequences from our parents
Roughly half the sequence of mum and half the sequence from dad
This means the more bands on the fingerprint that match the more genetically similar they are
