Manipulating Genomes

Sanger’s Approach:

• gene isolated form bacterium using restricitve enzymes

• inserted into plasmid + then into e.coli

• e.coli divides, copying plasmid

• copies isolated form plasmids + put into 4 test tubes

• each tube also contains: dna poymerase, nucleotides, modified nucleotides (radioactively labelled)

• during normal process, when modified nucleotide attaches, sequencing stops + fragment breaks off

High Throughput Sequencing:

• DNA cut into fragments + degraded into single immobilised strands

• add primer, enzymes and substrates

• add small amounts of activated nucleotides

• when activated nucleotide is incorporated into

  complementary strand, pyrophosphate released

• pyrophosphate converted to ATP via enzyme

• ATP causes luciferin → oxyluciferin, gives out visible light

Applications of gene sequenicng:

• comparison between species

• study evolutionary relationships

• varaitions between indiiduals

PCR = artificial DNa replication - differs to normal as only short sequences can be rplicated as opposed to whole chromosomes - apps = detecting mutations