McLaughlin Final Flashcards
What is needed for Sanger sequencing?
DNA template
primer that is complementary to the 3’ end of template
flurorescent ddNTPs and normal dNTPs
capillary gel electrophoresis
can do ~1000 bases
Name 2 2nd Gen DNA sequencing technologies
Ilumina/Solexa
Ion torrent
Name 2 3rd Gen DNA sequencing technologies
Pacific Biosciences (SMRT) Oxford Nanopore
What is different in 2nd Gen DNA sequencing from 1st Gen?
no bacterial cloning required i.e. you can use DNA from any source and amplify it in vitro
millions or billions of parallel reactions
don’t need electrophoresis
faster and cheaper
BUT shorter read lengths
How does the Ion Torrent amplify and sequence DNA?
emulsion PCR for amplification
pH sensor for sequencing
How does the Solexa/Illumina amplify and sequence DNA?
solid-phase amplification
reversible terminators for sequencing
How do Pacific Biosences and Oxford Nanopore amplify DNA?
they don’t because 3rd Gen doesn’t need to be amplified
How does Pacific Biosciences sequence DNA?
fluorescence in DNAP active site
How does Oxford Nanopore sequence DNA?
nanopore current sequencing
Explain how the Ion torrent works
emulsion PCR:
DNA is randomly sheared and fragments of known sequence are added to each end (different sequence on each side)
beads have complement of 3’ end of the ssDNA on them
make an emulsion with little water droplets in oil
each droplet has one bead and one fragment (hopefully)
do a version of PCR on them i.e. anneal, extend, denature repeat
sequencing:
put one bead per well on a plate
add primer complementary to the 5’ end and polymerase
wash dNTPs over the plate one at a time
when a nucleotide is incorporated H+ is released
change in pH is detected by an ion sensor
can do about 400 bases
Explain how Illumina/Solexa works
solid-phase amplification:
DNA is randomly sheared and known sequence is added to each end
amplification surface is covered with ss primers
DNA fragments are added and will anneal to the primers (there is a primer for each end)
fragments are PCR amplified in clusters on the surface
end up with clusters of strands with the same sequence, both strands (i.e. complement strand is there too)
reversible termination sequencing: add one of the primers all 4 dNTPs are added at once each with a different dye and blocked to stop further synthesis colour shows which dNTP was added terminating group is cleaved add dNTPs again etc then can do again with the other primer
can do ~300 bases
What is different in 3rd Gen sequencing from 2nd?
no DNA amplification is required which reduces bias (i.e. GC strands won’t denature as easily, won’t get as much amplification of them etc)
very long reads are possible (up to 60kb)
high error rates
Explain Pacific Biosciences DNA sequencing
DNA is randomly sheared and is ligated to hairpin primers at both ends
this makes a ss circular DNA templates with known sequence on either side
immobilize a modified DNAP that works very slowly with the template and a primer in the bottom of a well
(many wells per chip)
add all fluorescent dNTPs at the same time
there is a little light shining on the bottom of the well
when the correctly bound dNTP binds it lingers in the active site and the fluorescence is detected
when the base is added the fluorescent part is cleaved off and diffuses away
can go around the same circular piece multiple times
will keep reading as long as there is template
eventually the polymerase will fall off
typically read is ~5000-10000 bases
they say you can do up to 60 000 bases
Note: doesn’t need to be a circle, but this is usually how you do it when its genomic
Explain Oxford Nanopore DNA sequencing
DNA fragments are ligated to a hairpin at one end a sequence that will be recognized by a motor protein (probably a helicase) at the other end
motor protein pushes the sequence through a small pore through which there is a current flowing
the bases being pushed through disrupt the current being passed through the pore
different bases cause different disruptions
this is how it reads the sequence
basically read it twice if you do it this way because you will go through the sequence, then the linker and then through the complement
can also get epigenetic info this way because you can tell a methylated C from a normal one
Note: that its actually combinations of bases that are influencing the current because more than one can fit in at once
up to 60 000 bases
How long is the max read for each type of sequencing?
Sanger- 1000
Ion torrent- 400
Illumina- 300
PacBio and OxNano- 60000
