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