Manipulating genomes Flashcards
Define the term genome
All the genetic material in an organism (including mitochondrial DNA)
Define the term exon
Small parts of DNA that code for proteins (2%)
Define the term intron
Larger parts of DNA that don’t code for proteins, removed from mRNA before its translated
Define the term micro satellite
small regions of 2-4 intron bases repeated 5-15 times (STRs)
Define the term mini satellite
larger regions of 20-50 bases repeated 50+ times (VNTRs)
Define the term DNA profile
visible picture of a satellite
Define the term PCR
version of natural DNA replication that amplifies DNA
Define the term Restriction endonuclease
special enzymes that cut DNA into small fragments (restriction fragments) at restriction sites
Define the term electrophoresis
a technique to separate fragments based on length
Define the term southern blotting
DNA fragments transferred to nylon membrane
Define the term hybridisation
radioactive/fluorescent DNA probes added in excess to DNA fragments to identify microsatellite regions
Define the term recombinant DNA
DNA formed by joining together DNA from multiple sources
Define the term vector
something used to transfer DNA into a cell e.g. plasmid, bacteriophages (viruses that infect bacteria)
What are the techniques for studying genes
PCR
Cutting out DNA fragments using restriction enzymes
Gel electrophoresis
Describe the process of PCR
• Reaction mixture set up containing DNA sample, free nucleotide primers and DNA polymerase
• DNA mixture heated to 95 to break H bonds between the two strands of DNA.
o DNA polymerase doesn’t denature at this high temp which is important so new enzymes don’t need to be used every time
• Mixture then cooled to 50-65 so primers can bind to strands
• Mix heated to 72so DNA polymerase can work lining up the free DNA nucleotides alongside each template strand
• Two new copies of the fragment of DNA formed and one cycle of PCR complete
Describe the use of restriction enzymes
- Restriction enzymes recognise palindromic sequences of nucleotides (sequences of antiparallel base pairs that read the same in opposite directions)
- They cut the DNA at these recognition/palindromic sequences
- Diff enzymes cut at diff specific recognition sequences due to the shape of the recognition site being complementary to an enzymes active site
- DNA incubated with restriction enzyme and so cuts DNA by hydrolysis reaction exposing sticky ends (small tails of unpaired bases)
Describe the process of electrophoresis
• STEP 1:
o Agarose gel poured into a gel tray and left to solidify
o Wells made at one end of tray closest to negative electrode and put into gel box/tank
o Buffer solution added to reservoirs at side of gel box so surface of gel becomes covered in the buffer solution
• STEP 2:
o Use micropipette to add fragmented DNA and dye (same vol) to each well
o Dye helps sample sink into bottom of wells and makes them easier to see
o Ensure micropipette doesn’t go too far into the well and pierce the bottom of it
o Repeat this process using a clean micropipette tip each time
• STEP 3:
o Put lid on gel box and connect leads from box to power supply and turn on power supply to correct voltage
o DNA fragments negatively charged so move to positive electrode being pulled through the gel separating the fragments into size
o Let the gel run for 30 minutes or until dye is about 2cm from end of gel and remove gel tray from box using gloves
o The bands of different DNA fragments should be visible
• Electrophoresis with RNA
o Same as with DNA but mixed with chemical to denature the proteins so they all have the same charge
o Used to identify proteins present in urine or blood samples to diagnose disease
Describe the process of DNA profiling
• Extract the DNA:
o PCR used to extract DNA from a small fragment of tissue to develop a profile
• Digesting the sample:
o Strands of DNA cut into small fragments by restriction endonucleases
o Diff ones cut DNA at specific nucleotide sequence making 2 cuts at the restriction site
• Separating the DNA fragments:
o Electrophoresis separates the cut fragments into a clear recognisable pattern by passing the charged particles through a gel medium under influence of an electric current
o The gel is then immersed in alkali to separate double strands into single strands
o Then transferred onto nylon membrane (southern blotting)
• Hybridization:
o Fluorescent DNA probes added in excess to fragments that bind to known DNA sequence under particular conditions of pH and temp
o DNA probes identify regions more varied than the larger minisatellite regions and excess probes washed off
• Seeing the evidence:
o Radioactive labels/fluorescent label added to DNA probes and X-ray/UV images made
o Fragments give a pattern of bars
Describe how PCR is used
- Allows scientists to produce a lot of DNA from a small sample
- Needs excess of 4 nucleotide bases, primer and enzyme DNA polymerase all mixed in PCR machine
- Temp carefully controlled and changes at rapid intervals triggering different stages
Describe how DNA profiling is used
- Forensic science: performed on sample of saliva, semen, blood, hair roots, skin cells etc
- Prove paternity in immigration cases etc
- Demonstrate evolutionary relationships
- Identifying individuals at risk of particular diseases e.g. genetic disorders
Describe the sanger method
- Leaf cells crushed in buffer solution. DNA is precipitated, washed and suspended in buffer solution
- Primers (short section of DNA around 20 bases long) added to the DNA which has complementary code to the start of the gene and the primer binds there.
- Free nucleotides are added to the DNA and primer. Enzymes are added and many copies of the gene are made, each starting from the primer. The copies separate from the original DNA in the mixture
- Free nucleotides are added. Some of them have been modified in two ways:
a. A dye that will fluoresce when illuminated is joined to the nucleotide – a diff colour for each of the four types of base (they are tagged)
b. The nucleotide is double deoxidised and this nucleotide stops the reaction when by chance it is joined to the growing DNA - A computer records the sequence of colours and therefore bases that pass the window. The shortest lengths will arrive first so these bases were near the start of the DNA. So the sequence in which the bases pass the window matches the sequence of the bases in DNA.
- The mixture of the diff lengths of DNA is pulled along a capillary tube by electrophoresis. The short lengths move fastest. Near the far end a laser light shines on the tube through a window and the ‘tag’color for each bond of DNA is recorded automatically
- The result is many copies of the DNA of lots of diff lengths – each ending in a base which has a colored tag identifying it. If there are enough pieces of DNA every base in the gene will be tagged
What is genetic engineering
• Manipulation of an organism’s DNA
• Genetic engineering involves extracting a gene form one organism and inserting it into another organism
o Genes can also be manufactured (e.g. by PCR)
What are transformed organsims
Organisms that have had their DNA altered and therefore have recombinant DNA
Describe what a transgenic species is
organism that has been genetically engineered to include a gene from a different species
Describe the process of genetic engineering
• PART 1: obtaining DNA containing the desired gene
o The fragment can be isolated from another organism with restriction enzymes
• PART 2: making recombinant DNA (inserting the DNA fragment into vector DNA)
o Vector DNA isolated
o Vector DNA is cut open using same restriction enzyme used to isolate DNA fragment meaning sticky ends of vector DNA are complementary to sticky ends of DNA fragment containing DNA
o Vector DNA and DNA fragment mixed together with DNA ligase joining the sugar phosphate backbones of the two bits of the DNA (process = ligation)
o New combination of bases in the DNA is recombinant DNA
• PART 3: Transforming cells:
o Vector with transformed DNA is used to transfer gene into bacterial host cells
♣ If plasmid vector used then host cells have to persuaded to take in the plasmid vector and it’s DNA
What are the ethical issues of genetically modified soybeans
• POSITIVE:
o Reduce amount of chemical pesticides farmers use on crops that can harm the environment
o GM plants can be designed to be more nutritious
• NEGATIVE:
o May encourage monoculture decreasing biodiversity and leaving the whole crop vulnerable to disease as all plants are genetically identical
o Could interbreed with wild plants creating superweeds weeds resistant to herbicides
What are the ethical issues from producing drugs from GM animals
• POSITIVE:
o Drugs can be made in large quantities compared to other methods making them more available to more people
• NEGATIVE:
o Concern that manipulating an animals gene could cause harmful side-effects for the animal
Using an animal in this way is enforcing idea that animals are assets that can
What are the ethical issues for using pathogens in research
• POSITIVE
o Could mean untreatable diseases can now be treated, reducing suffering
• NEGATIVE
o Worries that the scientists researching the pathogens could become infected with the live pathogen and potentially cause a mass outbreak of disease
o Concern that genetically modified version of a pathogen could revert to its original form and cause an outbreak of disease
o Worries that in the wrong hands knowledge of how to genetically engineer dangerous pathogens could be used maliciously to create agents for Bio-warfare
• HOWEVER:
o Researchers using live pathogens have strict protocols they must follow
What are the ethical issues of ownership of GM organisms
• POSITIVE:
o Owner of patent will get money from selling the product encouraging scientists to compete to be the first to come up with new beneficial genetic engineering ideas so we get genetic engineered products faster
• NEGATIVE:
o Farmers in poor countries may not be able to afford patented genetically modified seeds which could be seen as unfair and that the big GE companies should relax the rules to help farmers in poor countries
How does gene therapy work
• Could be used to cure genetic disorders
• Is not being used widely yet but a form of somatic gene therapy is available
• Involves altering alleles inside cells to cure genetic disorders but depends on whether the disorder is caused by a mutated dominant allele or two mutated recessive alleles
o If dominant you can silence it e.g. sticking a bit of DNA in middle of allele so it doesn’t work anymore
o If recessive you can add a working dominant allele to make up for them supplementing the faulty ones
• Allele is inserted using vectors e.g. altered viruses, plasmids or liposomes
What is somatic therapy
• involves altering alleles in body cells
o Doesn’t affect individuals sex cells so any offspring could inherit disease
What is germ line therapy
• involves altering the alleles in sex cells
o Every cell of any offspring produced from these cells will be affected by gene therapy and won’t inherit the disease
o Currently illegal
What are the positive ethical issues of gene therapy
- Could prolong lives of people with life-threatening genetic disorders
- Could give people with genetic disorders a better quality of life if it helps ease symptoms
- GLT: Allows the carriers of genetic disorders to conceive a baby without the disorder
- GLT: Decrease no. people that would suffer from genetic disorders and cancer benefitting the society as a whole
What are the negative ethical issues of gene therapy
- Body could identify vectors as foreign bodies and start immune response against them
- Allele could be inserted into wrong place in DNA causing more problems e.g. cancer
- Inserted allele could get overexpressed producing too much of the missing protein and causing other problems
- Effects of the treatment may be short-lived in somatic therapy
- Patient might have to undergo multiple treatments with somatic therapy
- Difficult to get allele into specific body cells
Describe DNA sequencing (chain termination method)
• STEP 1:
o Mixture of following is added to 4 separate tubes:
♣ Single stranded DNA template
♣ DNA polymerase
♣ DNA primer
♣ Free nucleotides
♣ Fluorescently labelled modified nucleotide
• STEP 2:
o Tube undergoes PCR amplifying the DNA producing many strands of different lengths
• STEP 3:
o DNA fragments separated by electrophoresis and visualized under UV light and the complementary base sequence can be read
Describe the process of genome sequencing
• Used if you want to sequence the entire genome of an organism
• STEP 1:
o Genome cut into smaller fragments using restriction enzymes
• STEP 2:
o Fragments are inserted into bacterial artificial chromosomes (man-made plasmids). Each fragment is inserted into a different BAC
• STEP 3:
o BACs are then inserted into bacteria – each bacteria contains a BAC with a different DNA fragment
• STEP 4:
o Bacteria divide creating colonies of cloned cells that all contain specific DNA fragment
o Together different colonies make a complete genomic DNA library
• STEP 5:
o DNA is extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNA
• STEP 6:
o Each piece of DNA is sequenced using CTM
• STEP 7:
o Pieces are put back in order to produce the full sequence from that BAC (using powerful computer systems)
• STEP 8:
o DNA fragments from all the BACs are put in order by computers to complete the genome
Describe the advances in sequencing
• CTM commonly used but is now automated and faster
o Nowadays tube contains all modified nucleotides each with different colored fluorescent label and a machine reads the sequence for you
o Instead of running a gel manually and determining sequence from that the sequence is read automatically by a computer
• High-throughput sequencing
o Techniques that can sequence a lot faster than original methods at a fraction of the cost
• Newer faster techniques e.g. pyrosequencing mean scientists can now sequence whole genomes much more quickly
What are the uses of sequencing and comparing genomes
• Studying phenotype-genotype relationships:
o Useful to predict an organisms phenotype by analyzing its genotype
o E.g. Marfan syndrome
• Epidemiological studies
o Some gene mutations have been linked to a greater risk of disease and computerized comparisons between genomes of people that have a disease and those that don’t can be used to detect particular mutations that could be linked to the increased risk of disease
• Understanding evolutionary relationships
o Whole genomes of diff species can be sequenced and then analyzed using computer software to tell us how closely related different species are
o Comparing genomes of members of same species can also tell us about §evolutionary relationships
