4.3

Question 1

components for in vitro replication/PCR

Answer 1

• 4 dNTPs
• template DNA
• pair of primers
• DNA polymerase
• essential ions and salts
• thermocycler

Question 2

PCR (polymerase chain reaction) technique
(in vitro replication)

Answer 2

• 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)

Question 3

cycle of amplification

Answer 3

Denaturation: heat DNA to separate strands
Annealing: cool so primers anneal to DNA
Extension: DNA polymerase synthesizes new DNA (medium heat)

Question 4

visualizing DNA on a gel

Answer 4

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

Question 5

genome sequencing

Answer 5

shotgun sequencing
- sequence small segments
- assemble sequences (computational software used)
- annotate sequences

Question 6

Sanger sequencing

Answer 6

• 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

Question 7

Assembly

Answer 7

• identify overlapping sequences and assemble them using complex algorithms
• overlapping segments are called contigs

Question 8

Annotation

Answer 8

• 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