6.3 DNA Techniques and Vocabulary Flashcards
Describe the function/use of amplification
Amplification is used to greatly increase the number of copies of a DNA sequence for further laboratory use.
How can amplification be achieved?
It can be achieved either by vivo (research done on living organisms), by inserting the sequence into a cloning vector that replicates it within a host cell or in vitro (research done in a lab dish/test tube) by PCR.
Describe the function/use of annealing
Annealing allows the two DNA primers to join each DNA strands on the 3’ by complementary base paring (joining of sticky ends).
The two pieces are joined by weak hydrogen bonds only, and therefore only temporary
What is the optimal temperature for annealing?
50-60°C
Define genome
A genome is all the genetic material contained in an organism or a cell
Define vector
A vector is a vehicle that transports and introduces foreign DNA/genes into a host organism, where it can be replicated and expressed.
Define Polymerase chain reaction (PCR)
It is a cyclic method used to rapidly amplify small amounts of a particular DNA sequence into an extremely large amount of copies.
What is required for PCR?
-A template of the DNA
-A special DNA polymerase (taq polymerase)
-A supply of the four DNA nucleotides
-Two-sets of single-stranded DNA primers
-A thermal cycler that is able to rotate through the temperatures
What are the 3 steps for PCR?
-Denaturing
-Annealing
-Extension
What is the temperature of the mixture in the denaturing stage?
95°C
What occurs during denaturing in PCR?
Double stranded DNA is heated to around 95°C, breaking the weak hydrogen bonds between complementary bases causing the two template strands to denature (separate).
What is the temperature of the mixture in the annealing stage?
50-60°C
What occurs during annealing in PCR?
The temperature is reduced to 50-60°C, allowing the single-stranded DNA primers to anneal (join via hydrogen bonds) to complementary sequences on opposite ends of each strand (on the 3’ end).
Primers attach to either side of the target DNA to be amplified. They attach to pre-emptied complementary bases
What is the temperature of the mixture during extension in PCR?
72°C
What occurs in extension in PCR?
DNA taq polymerase extends to the new strand, starting from primers. New DNA strands are synthesised using taq polymerase and avaliable complementary nucleotides.
What is the end results of PCR?
After extension, there are two copies of each orginal strand of the double-stranded DNA. (then the cycle repeats)
Define gel electrophoresis
Gel electrophoresis is a technique that can separate large molecules (e.g fragments of DNA) so they can be visualised and identified by comparison with a standard.
What type of gel is used in gel electrophoresis?
Agarose gel
What are the four steps of gel electrophoresis?
-Set up the apparatus
-Pippete samples
-Turn on current
-Visualise and compare
What happens in the ‘set up the apparatus’ step?
-Set an agarose gel and make wells with a comb.
-Place the gel into the apparatus and pour a buffer solution over to regulate pH.
-Cut the DNA fragments with restriction enzymes and dye them with a binding chemical e.g. ethidium bromide, that fluoresces under UV light.
What happens in the ‘pipette solution’ step?
-Pipette the solution and DNA ladder (standard) into the wells of the agar.
-Make sure the negatively charged samples are at the end of the apparatus where the negative electrode is situated
-Discard used micropipette tips after each use.
What happens in the ‘turn on current’ step?
-Turn on the current
-The negatively charged DNA fragments are repelled by the negative electrode and travel (migrate) towards the positive electrode, which they are attracted to.
-The smaller fragments mover faster and further than the large fragments due to its molecular weight.
