Genetic tools Flashcards
1
Q
DNA cloning:
A
- making copies of specific fragments of DNA
2
Q
how is DNA cloning achieved:
A
- DNA of interest inserted into plasmid (recombinant DNA molecule)
- plasmids: cloning vectors
3
Q
plasmid:
A
- small circular piece of DNA that can replicate
4
Q
PCR:
A
- polymerase chain reaction
- amplifies DNA
- performed in thermocycler
- using primers, can target specific regions of genome and specifc taxa
5
Q
list steps of PCR:
A
- denaturation
- annealing
- extension
= 1 cycle - repeated around 30 times
6
Q
denaturation:
A
- separates double helix into 2 single strands
- high temp 95˚ for 30 secs
- breaks hydrogen bonds btw complimentary base pairs
7
Q
annealing:
A
- primers bind to DNA molecule
- temp depends (50-60˚ for 30 secs)
- site for Taq (DNA) polymerase to bind
- primers target species (or group) and or gene of interest
8
Q
extension:
A
- DNA replication
- DNA polymerase binds to 3’ end of primer
- Taq (DNA) polymerase adds (extends) nucleotides via complimentary base pairing
- 72˚ for 1 min
9
Q
list PCR components:
A
- buffer
- magnesium chloride
- Taq polymerase
- forward primer
- reverse primer
- dNTPs (deoxyribonucleotide triphosphate)
- template DNA
10
Q
Taq polymerase moves in which direction:
A
always Taq moves 3’ to 5’
11
Q
amplicon:
A
PCR product
- 82 bp
- will accumulate exponentially
12
Q
chain termination:
A
- radiolabelled dNTPs
- incorporates ddNTPs which terminate DNA synthesis
- 1 lane/ tube per base
- determines sequence using gel electrophoresis
- 100 bp at a time
13
Q
colour ddNTPs:
A
- dye- labelled ddNTPs
- 1000bp at a time
- capillary electrophoresis
14
Q
Sanger sequencing:
A
- capillary electrophoresis
- aka chain termination sequencing
15
Q
how does capillary electrophoresis work:
A
- extension produces series of ddNTP terminated products each 1 base different in length
- each ddNTP labelled different colour fluorescent dye
- sequence read by noting peak colour in electropherogram
