Manipulating genomes Flashcards
What does the PCR do?
Can be used to select a fragment of DNA and amplify it to produce millions of copies in just a few hours
What are the 3 key stages in a PCR?
Denaturation, annealing and extension/ elongation
PCR: What happens in denaturation?
The double-stranded DNA is heated to 95°C which breaks the hydrogen bonds that bond the two DNA strands together.
PCR: What happens in annealing?
The temperature is decreased to between 50-60°C so that primers can anneal to the ends of the single strands of DNA.
PCR: What happens in extension/ elongation?
The temperature is increased to 72°C for at least a minute, as this is optimum temperature for Taq polymerase to build complementary strands of DNA to produce the new double-stranded DNA molecules
How many DNA fragment are there after 1 cycle of PCR?
4, because it doubles the amount of DNA (i.e. originally had 2)
Why is Taq polymerase used?
Comes from thermophilic bacterium which means it does not denature at high temperature.
What does each PCR require?
- Target DNA being amplified.
- Primers
- DNA polymerase
- Free nucleotides
- Buffer solution
What is the machine called that a PCR takes place in?
Thermocycler
What is electrophoresis?
Is a procedure that uses an electrical current to seperate out DNA fragments, RNA fragments, or proteins depending on their size.
Electrophoresis: How is the gel tray in the gel box set up?
- Electrophoresis is commonly performed using agarose gel that has been poured into a gel a tray and left to solidify. A row of wells is created at one end.
- Firstly, you need to put the gel tray into a gel box. You need to make sure the end with wells is closest to the negative electrode on the gel box.
- Then add a buffer solution to the reservoirs at the side of the gel box so the surface gel becomes covered in buffer solution.
Electrophoresis: How are the DNA samples loaded into the wells?
1) Take your fragmented DNA samples, and using a micropipette, add the same volume of loading dye to each- loading dye helps the samples sink to the bottom of the wells and makes them easier to see.
2) Next add a set volume of a DNA sample to the first well.
3) Then repeat this process for each of the other DNA samples to other wells in the gel.
Electrophoresis is carried out?
1) put the lid on the gel box and connect the leads from the gel box to the power supply.
2) Turn on power supply- this causes an electrical current to be passed through the gel.
3) DNA fragments are negatively charged, so they’ll move through the gel towards the positive electrode at the far end of the gel (called the anode). Small DNA fragments move faster and travel further through the gel, so DNA fragments will seperate according to their size.
What are palindromic sequences (recognition sequences?
Sequences that consist of antiparallel base pairs (base pairs that read the same in both directions).
What are restriction enzymes ?
Enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places.
What can different restriction enzymes do?
Cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to an enzymes’s active site.
What happens if recognition sequences are present at either side of the DNA fragment you want?
Can use the restriction enzyme to seperate it from the rest of the DNA.
How does restriction enzyme used?
The DNA sample is incubated with the specific restriction enzyme, which cuts the the DNA fragment out via a hydrolysis reaction.
What can restriction enzymes cutting sometimes leave?
Sticky ends- small tails of unpaired bases at each end of the fragment. Sticky ends can be used to bind the DNA fragment to another piece of DNA that ahs sticky ends with complementary sequences
How can PCR be used in forensic science?
1) The DNA is isolated from all the collected samples.
2) PCR is used to amplify multiple areas containing sequence reports- primers are used to bind to either side of these reports so the whole repeat is amplified.
3) The PCR products are then run on an electrophoresis gel and the DNA profiles are comapred to see if any match .
4) If the samples match it links a person to the crime scene.
How can DNSA profiling be used in medical diagnosis?
In a medical diagnosis, a DNA profile can refer to a unqiue pattern of several alleles.
It can be used to analyse the risk of genetic disorders. It’s useful when the specific mutation isn’t known or where several mutations could have caused one disorder, because it identifies broader, altered genetic pattern.
What is a transformed organisms?
Organisms that have had their DNA altered by genetic enginnering.
What is a transgenic organism?
An organism that has been genetically engineered to include a gene from a different species.
Genetic enginnering: How is the desired DNA fragment inserted into a vector?
1) The DNA fragment is inserted into vector DNA. They can be plasmids (small circular molecules of DNA in bacteria) or bacteriophages (viruses that infect bacteria).
2) The vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment containing the desired gene. So the ends are complementary.
3) The vector and the DNA are mixed together with DNA ligase. DNA ligase joins up the sugar-phosphate backbones of the two bits. This process is called ligation.
4) The new combination of bases in the DNA (vector DNA + DNA fragment ) is called recombination DNA.
