6.3.1 DNA sequencing

Question 1

define DNA sequencing

Answer 1

technique which allows genes to be isolated & read

Question 2

what did sanger’s DNA sequencing approach allow

Answer 2

scientists to sequence whole genomes

Question 3

outline fred sanger’s DNA sequencing approach (1975)

Answer 3

1. single strand of DNA used as template for 4 experiments in separate dishes
2. dishes contain solution with 4 bases (A, C, T, G) & DNA polymerase
3. modified bases added - labelled with radioactive isotopes
4. DNA fragments of varying lengths created
5. DNA fragments passed through gel by electrophoresis (smaller fragments travel further = sorted by length)
6. nucleotide bae at end of each fragments read according to radioactive label

Question 4

describe fred sanger’s DNA sequencing approach

Answer 4

• efficient & safe
• time-consuming & costly

Question 5

what was the first DNA based organism to have it’s genome sequenced

Answer 5

phi-X174 (phage virus)

Question 6

application of fred sanger’s DNA sequencing approach

Answer 6

• 1981 = sanger published sequencing of human mitochondrial genome (37 genes & 16,569 base pairs)
• 1984 = scientists sequenced 170 kilo base pair-long genome of epstein-barr virus
• 1995 = genome of bacterium haemophilus influenzae was sequenced

Question 7

how is DNA cloned

Answer 7

1. gene being sequenced is isolated from bacterium using restriction enzymes
2. DNA inserted into bacterial plasmid (vector) & into escherichia coli bacterium host = divides many times once cultured (enables plasmid with DNA inserted too be copied many times)
3. each new bacterium contains copy of candidate gene
4. lengths of DNA isolated using plasmid preparation then sequenced

Question 8

when was the first DNA sequencing machine developed & where

Answer 8

california institute of technology 1986

Question 9

describe the first DNA sequencing machine

Answer 9

• based on fred sanger’s method
• fluorescent dyes used to label bases (instead of radioactivity)
• dyes glowed when scanned with laser beam & light signature identified by computer

Question 10

describe high throughput sequencing t

Answer 10

• cost-effective, rapid & simple to operate
• generates huge amounts of data = bioinformatics store & analyse data

Question 11

example of high throughput sequencing

Answer 11

pyrosequencing

Question 12

describe pyrosequencing

Answer 12

• involves synthesising single DNA strand (complementary to strand being sequenced) 1 base at a time, whilst detecting (by light emission which base was added at each step
• unincorporated activated nucleotides degraded by apyrase & reaction begins again with another nucleotide
• 1 million reads happen simultaneously
• software packaging assemble sequences into longer sequences (bioinformatics)