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DNA Sequencing - Coggle Diagram
DNA Sequencing
Process of Pyrosequencing
Based on
"sequencing by synthesis"
principle instead of chain termination with dideoxy nucleotides
Immobilise a single template DNA molecule on a bead/substrate & synthesise complementary strand
Detect which nucleotide is added at each step. Sequencing (polymerisation) doesn't stop
Requires template DNA, primer, DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate (APS), and luciferin
As with dideoxy sequencing,
base incorporation recorded when light emitted at particular wavelengths
Process of Sanger Sequencing
Sanger Technique
Uses dideoxynucleotides
Molecules that resemble normal nucleotides but lack normal -OH group
Steps
1.
DNA template denatured
to single strands
2.
Single DNA primer
(3' end near sequence of interest)
annealed to template DNA
& extended with DNA polymerase
Four reactions set up,
each containing: DNA template, Primer annealed to template DNA, DNA polymerase, dNTPs (dATP, dTTP, dCTP, and dGTP)
A different labelled
dideoxynucleotide
(ddATP, ddTTP, ddCTP, or ddGTP)
added
to each of four reaction tubes
ddNTPs possess a 3' -H
instead of 3' -OH, compete in reaction with normal dNTPs, & produce no phosphodiester bond
Whenever labelled ddNTPs are incorporated in chain, DNA synthesis terminates
Dideoxy DNA sequencing (
dye terminator sequencing
)
Each of four reaction mixtures produces a population of DNA molecules with DNA chains terminating at all possible positions
Extension products in each of four reaction mixtures also end with a different labelled ddNTP (depending on base)
Each reaction mixture
electrophoresed
in a separate lane (4 lanes) at high voltage on a polyacrylamide gel
Polyacrylamide gels can be thinner --> higher voltage --> faster
Pattern of bands in each of 4 lanes is visualised on X-ray film or automated sequencer
Compare & contrast sanger & pyrosequencing
Sanger sequencing
Relies on electrophoretic separation of end-stage PCR products
Physical limits on number of capillaries
Relatively expensive
Pyrosequencing
Very expensive
Not easy to run many reactions at once
Describe multiple approaches to next-gen sequencing
NGS
Steps
Fragment target DNA & ligate universal adaptors
Amplify single molecules (beads vs free)
Sequence clonal amplicons
Each system differs in the way DNA is sequenced
Computer assemble the data
Reference-based assemblies
De novo assemblies
Advantages & limitations of sanger sequencing
Provide examples of how NGS can be used to identify disease genes
Compare & contrast low & high throughput sequencing