In vitro cloning Flashcards
1
Q
Polymerase Chain Reaction
A
- Method of copying fragments of DNA
- Automated process - rapid and efficient
2
Q
Why is it valuable for CSI
A
- Very small DNA samples of DNA can be copied using PCR
3
Q
What does PCR need?
A
- DNA fragments to be copied
- DNA polymerase
- Free nucleotides
- DNA primers
- Thermocycler
4
Q
DNA polymerase
A
- Join together the nucleotides
- Forms phosphodiester bonds = phosphate sugar backbone on DNA
5
Q
Free nucleotides
A
A, T, C and G
6
Q
DNA primers
A
- Short sequences of DNA with a set of bases that are complementary to those at the end of the DNA fragments
7
Q
Thermocycler
A
- Computer-controlled machine that varies temperatures precisely over a set period of time
8
Q
Stage 1
A
- Separation of the DNA strand
- DNA fragments, primers, nucleotides and DNA polymerase - put in a vessel in the thermocycler
- 95 degrees
- Causes the hydrogen bonds to break causing the 2 strands of DNA to separate
9
Q
Stage 2
A
- Annealing of the primers
- 55 degrees
- Causes the primers to anneal to complementary bases of the DNA fragment at the 3’ end
= Starting sequence for DNA polymerase to begin copying DNA - Primers prevent strands re-joining
10
Q
Stage 3
A
- Synthesis of DNA
- 72 degrees
- Optimum temperature for DNA polymerase to add complementary bases to each of the single stranded sections of DNA
- Begins at primer and adds nucleotides until it reaches the 5’ end
11
Q
Result
A
- Number of fragments doubles as both strands are copied simultaneously
