11.3 Flashcards
how do phosphodiester bonds form?
DNA poly catalyzes
P-P is released from DNTP
condensation reaction
what is ddNTP?
doesn’t have an OH on the 3’ so cannot add anything to it
what happens in Sanger dideoxy sequencing
ddNTPs are added into reaction with fluorescent colours added to each type
what is the problem with Sanger’s technique?
it is slowwwww
and used radioactive tags (fluorescence is so much better)
requires purification of each individual DNA sequence
what was the human genome project?
sequencing the first human genome
was very expensive and time-consuming
what are next generation sequencing methods?
ion torrent
illumina
makes it faster!!!
what are the steps of illumina DNA sequencing?
prepare genomic DNA sample
attach DNA to surface
bridge amplifcation
fragments become double-stranded
denature the double-stranded molecules
complete amplification
determine first base
image first base
determine second base
image secondchemistry cycle
sequence reads over multiple chemistry cycles
align data
what happens when preparing genomic DNA sample in illumina?
randomly fragment the DNA and ligate the adaptors to both ends
what happens when DNA is attached to surface during illumina?
ss fragments are randomly bound to inside of flow cell channels
what happens during bridge amplification in illumina?
unlabeled nucleotides and enzyme to initiate solid-phase bride amplification
what happens when fragments become ds in illumina?
enzyme incorporates nucleotides to build ds bridges on solid-phase substrate
what happens when ds molecules are denatured in illumina?
denaturation leaves the ss templates anchored to substrate (no more bridges)
what happens when amplification is complete in illumina?
several millions of dense clusters of dsDNA are generated in each channel of the flow cell
what happens during the first chemistry cycle in illumina?
initates sequencing cycle, all four labelled reversible terminators, primers, and DNA poly are added
how happens when imaging the first base in illumina?
after laser excitation, image is taken of emitted fluorescence
what happens when you read the sequence in illumina?
cycles of sequence reap to determine the sequences of bases in fragment each base at a time
how big does DNA need to be for illumina?
short segments
what are adaptor sequences in illumina?
they are added by litigation to ends of segments and add sites for the primers and enable attachment to oligonucleotides on surface of flow cell
how are DNA sequences arrayed on illumina?
randomly arrayed across flow cell surface
what is the point of bridge amplification?
amplifies single DNA molecules into clusters of identical DNA molecules
what type of sequencing is illumina?
massively parallel
what is nanopore sequencing?
3rd gen sequencing using an enzyme (no synthesis)
what happens in nanopore sequencing?
enzyme unwinds DNA and pulls a ss through a pore in membrane using an electric current
bases produce characteristic disturbance in electrical current when passing through pore which can be read
what are the pros of nanpore?
long reads
no amplification step
small portable machine
can detect methylated bases
what are the cons of nanopore?
it is slightly less accurate than other methods
which sequencing methods are massively parallel?
illumina and nanopore
which sequencing methods use synthesis?
snager and illumina
which which sequencing methods are use a single molecule?
nanopore
which sequencing methods use chan terminators?
sanger (nonreversible)
illumina (reversible)
