T4 Module 3 - Applications of DNA replication

Question 1

describe PCR technique

Answer 1

used to replicate large amounts of DNA

INGREDIENTS
- sample DNA
- short, single stranded DNA primers (15-30 nucleotides)
- 4 free deoxyribonucleotides (dNTP) added
- high temp resistant DNAP (e.g. taq polymerase)

PROCESS
1) DENATURATION
- heated to separate strands
2) ANNEALING
- thermocycler cools solutions so primers can bind to each strand
3) EXTENSION
- DNAP extends + polymerizes daughter strands using dNTP

*after each cycle, copies are doubled - exponential growth

Question 2

explain gel electrophoresis

Answer 2

separates DNA fragments or macromolecules from PCR. Defines their size.

PROCESS
- contents placed in charged electric field
- DNA + RNA are negative, move towards positive end
- speed of molecule movement depends on its size
- known DNA ladder used to determine sizes
- UV activated fluorescent dye used to show DNA

Question 3

Sanger/dideoxy sequencing

Answer 3

components (same as DNA replication)
- single stranded template DNA
- DNA primers
- dNTPs
- ddNTPs (all 4 dyed a different colour)
- DNAP

PROCESS
- DNA replication, but dyed ddNTP stops growth (chain termination)
- gel electrophoresis
- different coloured strands distinguished by fluorescence detector

Question 4

shotgun sequencing

Answer 4

uses small sequences DNAs to sequence larger segments of DNA via overlap.

1) sequence DNA
2) assemble sequences
3) Annotate the sequences

CONTIGS: overlapping regions of DNA assembles into a consensus regions

Question 5

sequence annotation

Answer 5

reading the DNA sequence
- 6 possible reading frames
- sequences identified via sequence motifs
e.g. coding regions identified by long stretches caped by start and stop codons