Chapter Twenty-One: Manipulating genomes Flashcards
1
Q
What is Polymerase Chain Reaction (PCR)?
A
- artificial DNA replication. it’s a technique that allows DNA fragments of interest to be copied many times (amplification)
- the amplified material can be used for many other genetic techniques
2
Q
What are the reagents for PCR?
A
- DNA
- primers
- DNA nucleotides
- DNA polymerase (Taq)
3
Q
What happens in the first stage of PCR?
A
- the DNA sample is mixed with primers, DNA nucleotides, and Taq (DNA polymerase)
- the vial is placed in a PCR machine
4
Q
What happens in the second stage of PCR?
A
- the mixture is heated to 95°c, breaking the hydrogen bonds that hold the complementary strands together
- the double stranded DNA sample denatures to make simple stranded
DNA - DNA polymerase dose to denature since it’s a ‘thermophilic’ version (Taq)
5
Q
What happens in the third stage of PCR?
A
- mixture is cooled to around 55°c, allowing the primers to anneal
- the primers (short sequences of nucleotide bases) must join to the start of the separated DNA strands so the full copying process can start. Taq is able to bind to these double stranded areas
6
Q
What happens in the fourth stage of PCR?
A
- Mixture is heated up t0 72°C as this is the optimum temperature for the DNA polymerase enzyme
- the DNA polymerase extends the small sections of double stranded DNA by adding free nucleotides to the unwound DNA
7
Q
What happens in the fifth stage of PCR?
A
- two new copies of the fragment of DNA are formed and one cycle of PCR is completed
- the cycle starts again
8
Q
What are three advantages of PCR?
A
- automated process making it more efficient
- rapid process, 100 billion copies can be made in a few hours
- it doesn’t require living cells making it quicker and less complex techniques are needed
9
Q
What are restriction enzymes?
A
- another way to get a DNA fragment from an organisms DNA
10
Q
What are palindromic sequences?
A
- sequences that consist of antiparallel base pairs (base pairs that read the same in opposite directions)
11
Q
What do restriction enzymes do?
A
- they recognise specific palindromic sequences (recognition sequences) and cut (digest) the DNA at these places
12
Q
Why do different restriction enzymes cut at different specific recognition sequences?
A
- because the shape of the recognition sequence is complementary to an enzymes active site
13
Q
How to use restriction enzymes to separate the DNA fragment you want that’s in between recognition sequences?
A
- DNA sample is incubated with the specific restriction enzyme, this cuts the DNA fragment via a hydrolysis reaction
- the cut can leave sticky ends (small tails of unpaired bases at each end of the fragment) that can be used to anneal the DNA fragment to another piece of DNA that has sticky ends with complementary sequences
14
Q
What does gel electrophoresis do?
A
- it uses an electrical current to separate out: different fragments of DNA, RNA fragments, and proteins according to their sizes
15
Q
What happens in the first step of gel electrophoresis?
A
- DNA fragments are treated with restriction enzymes to cut up the fragments and the DNA samples are placed unto the wells cut in one end of the gel (closest to the negative electrode)
