Sanger Sequencing and NGS Flashcards
What are the limitations of current sequencing technology?
1) Limit to accuracy.
2) Practical limit to the length of DNA that can be sequenced.
3) Need for a molecular “handle” to “grab” a target DNA molecule.
4) Cost.
What are the core aims of sequencing?
1) DNA base order.
2) DNA base variants.
3) DNA base chemical modification.
4) RNA transcripts and splicing.
How does Sanger Sequencing work?
1) in vitro DNA synthesis reaction using DNA polymerase.
2) Primed by short oligonucleotides complementary to the DNA sequence.
3) Labelled modified bases are inserted at random that stop DNA synthesis (ddNTPs).
4) Bases are read out according to the sizes of the resulting DNA molecules.
What is a pro and a con of Sanger Sequencing?
Extreme accuracy (0.001-1% error rate). Very slow.
How do ddNTPs differ from dNTPs?
They lack the crucial 3’ OH- group required for DNA synthesis.
How does Illumina NGS differ to Sanger Sequencing?
Far more sequences per run.
Shorter read lengths.
Much cheaper.
Lower accuracy - around a 2% error rate.
Describe Cycle 1 of NGS.
1) DNA synthesis incorporates dNTPs with a dye molecule.
2) The dye group lacks a 3’ OH- so synthesis terminates/halts.
3) Flow cell surface is washed leaving dye incorporated at the end of the halted DNA chain.
4) A laser scans the whole flow cell, logging each spot’s fluorescence colour.
5) Chemicals flow through the cell removing dye groups to leave a 3’ OH-.
Describe Cycle 2 of NGS.
1) The reversible dye-terminators and DNA polymerase are added back and the next base is incorporated.
2) The dye group is NOT a 3’ OH- so synthesis terminates/halts.
What are NGS adaptors?
Short synthetic DNAs that contain non-complementary sequence at one end causing them to form a Y shape.
What is a pro and a con of NGS?
Cheap.
Short reads - assembly into whole genomes is difficult.
