Sequencing genes and genomes

• DNA sequencing means finding out the order of bases in all of an organism's DNA or in a specific gene
• The first methods used to determine the order of bases in a section of DNA were chain termination methods
• Chain termination DNA sequencing

  1. Add a single-stranded DNA template, DNA polymerase, DNA primer, free nucleotides, and a fluorescently-labelled modified nucleotide to four separate tubes

  2. Undergo PCR to produce DNA strands of different lengths terminated by the modified nucleotides

  3. Separate the DNA fragments by electrophoresis and visualise under UV light

  4. Read the complementary base sequence from the gel
• The chain-termination method can only be used for DNA fragments up to about 750 base pairs long
• Genome sequencing using the chain-termination method


  1. Cut the genome into smaller fragments using restriction enzymes

  2. Insert the fragments into bacterial artificial chromosomes (BACs)

  3. Insert the BACs into bacteria and allow them to divide, creating colonies of cloned cells

  4. Extract DNA from each colony, cut into overlapping pieces, and sequence each piece

  5. Put the sequenced pieces back in order using powerful computers to complete the entire genome sequence
• Advances in technology have led to rapid advancements in gene sequencing, including automation and higher throughput
• Automated DNA sequencing

  • The tube contains all the modified nucleotides with different coloured fluorescent labels, and a machine reads the sequence automatically
• High-throughput DNA sequencing


  • Can sequence 1000 times more bases in a given time, at a fraction of the cost of original methods
• Pyrosequencing


  1. Cut DNA into fragments, split into single strands, and attach a strand from each fragment to a bead

  2. Amplify the DNA fragments on each bead using PCR

  3. Put each bead into a separate well

  4. Add free nucleotides that attach to the DNA strands via complementary base pairing

  5. Enzymes cause light to be emitted when a nucleotide is added, with intensity varying based on how many are added

  6. Computers analyse the light intensities to interpret the DNA sequence
• Newer, faster techniques like pyrosequencing allow scientists to sequence whole genomes much more quickly