4.3 Flashcards
1
Q
components for in vitro replication/PCR
A
- 4 dNTPs
- template DNA
- pair of primers
- DNA polymerase
- essential ions and salts
- thermocycler
2
Q
PCR (polymerase chain reaction) technique
(in vitro replication)
A
- sample DNA in tube of buffered solution with essential ions and salts and DNA primers
- primers bind to template DNA, starting points for copying
- free deoxiribonuclieotides added (dNTPs)
- thermocycler heats and cools to facilitate replication, DNA polymerase also added (eg, Taq polymerase)
3
Q
cycle of amplification
A
Denaturation: heat DNA to separate strands
Annealing: cool so primers anneal to DNA
Extension: DNA polymerase synthesizes new DNA (medium heat)
4
Q
visualizing DNA on a gel
A
gel electrophoresis
- molecules loaded into wells of gel
- electric field +ive to -ive (top - bottom +)
- DNA is attracted to +ive because it’s -ive, small DNA travels faster
5
Q
genome sequencing
A
shotgun sequencing
- sequence small segments
- assemble sequences (computational software used)
- annotate sequences
6
Q
Sanger sequencing
A
- includes all components for PCR + modified dNTPs (ddNTPs)
- removed OH group from 2 bonded dNTPs, prevents further elongation
- leads to interrupted daughter strands broken at every site the ddNTP bonded to
- ddNTPs are fluorescently labelled
7
Q
Assembly
A
- identify overlapping sequences and assemble them using complex algorithms
- overlapping segments are called contigs
8
Q
Annotation
A
- use 6 different reading frames to find the correct one
- identifying binding sites for transcription, hairpin structure codes, open reading frames, non-coding RNA, single-copy genes