Chapter 8: DNA Sequencing: Cloned Genes and Genomes

Two early techniques for DNA sequencing Maxam Gilbert (now only for footprint experiments) Sanger (common)

Sanger

Uses 2’ 3’ ddNTP, single stranded DNA is the template for reaction that is terminated by ddNTP, invitro

  • Can sequence the insert of plasmid without purifying from the plasmid
  • Add primer complementary to sequence before the insert, long enough to be specific to target
  • Carry out invitro DNA synthesis with ddNTP and labelled nucleotide
  • In each tube carry out reaction starting with same template, 10% of each tube contains ddNTP
  • Each tube dATP dGTP dTTP dCTP, ddNTP, template, primer with one letter being labelled
  • After synthesis denature the strands and perform acrylamide gel electrophoresis and expose to x-ray to look at products (polyacrylamide resolves smaller fragments better)
  • Read it bottom up 5 to 3 on strand being made, and then complementary for original sequence
  • To validate use a primer going the other way

Automated

Each nt tagged with different fluorescent tag which is detected by laser beam,

  • Errors: Runs of same nt, runs of GC, hairpin loops, can improve by sequencing overlapping fragments

Next Generation Sequencing

High throughput, provides short stretches of data from many DNA fragments simultaneously and can be applied to cDNA and the sequence is determined directly from fragment without cloning

  • High volume relies on detection of pyrophosphate which is released when each nt added, it is detected via coupled enzymatic reaction producing a flash of light
  • Machines differ in how the sequence is generated and the length: chemistry of sequencing done as detected
  • Several fragments attached to separate beads via adaptors with water containing reagents for PCR
  • PCR creates many copies of fragment all attached to the bead, place each bead in well containing primers for DNA synthesis and enzymes/substrate required for detection of nt
  • Individuals nt flowed over the wells with beads in cycled and when a nt is added pyrophosphate is released and luciferase produces light, different intensity of light is different nt, wash off and add next nt


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