11.3

Question 1

how do phosphodiester bonds form?

Answer 1

DNA poly catalyzes
P-P is released from DNTP
condensation reaction

Question 2

what is ddNTP?

Answer 2

doesn’t have an OH on the 3’ so cannot add anything to it

Question 3

what happens in Sanger dideoxy sequencing

Answer 3

ddNTPs are added into reaction with fluorescent colours added to each type

Question 4

what is the problem with Sanger’s technique?

Answer 4

it is slowwwww
and used radioactive tags (fluorescence is so much better)
requires purification of each individual DNA sequence

Question 5

what was the human genome project?

Answer 5

sequencing the first human genome
was very expensive and time-consuming

Question 6

what are next generation sequencing methods?

Answer 6

ion torrent
illumina
makes it faster!!!

Question 7

what are the steps of illumina DNA sequencing?

Answer 7

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

Question 8

what happens when preparing genomic DNA sample in illumina?

Answer 8

randomly fragment the DNA and ligate the adaptors to both ends

Question 9

what happens when DNA is attached to surface during illumina?

Answer 9

ss fragments are randomly bound to inside of flow cell channels

Question 10

what happens during bridge amplification in illumina?

Answer 10

unlabeled nucleotides and enzyme to initiate solid-phase bride amplification

Question 11

what happens when fragments become ds in illumina?

Answer 11

enzyme incorporates nucleotides to build ds bridges on solid-phase substrate

Question 12

what happens when ds molecules are denatured in illumina?

Answer 12

denaturation leaves the ss templates anchored to substrate (no more bridges)

Question 13

what happens when amplification is complete in illumina?

Answer 13

several millions of dense clusters of dsDNA are generated in each channel of the flow cell

Question 14

what happens during the first chemistry cycle in illumina?

Answer 14

initates sequencing cycle, all four labelled reversible terminators, primers, and DNA poly are added

Question 15

how happens when imaging the first base in illumina?

Answer 15

after laser excitation, image is taken of emitted fluorescence

Question 16

what happens when you read the sequence in illumina?

Answer 16

cycles of sequence reap to determine the sequences of bases in fragment each base at a time

Question 17

how big does DNA need to be for illumina?

Answer 17

short segments

Question 18

what are adaptor sequences in illumina?

Answer 18

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

Question 19

how are DNA sequences arrayed on illumina?

Answer 19

randomly arrayed across flow cell surface

Question 20

what is the point of bridge amplification?

Answer 20

amplifies single DNA molecules into clusters of identical DNA molecules

Question 21

what type of sequencing is illumina?

Answer 21

massively parallel

Question 22

what is nanopore sequencing?

Answer 22

3rd gen sequencing using an enzyme (no synthesis)

Question 23

what happens in nanopore sequencing?

Answer 23

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

Question 24

what are the pros of nanpore?

Answer 24

long reads
no amplification step
small portable machine
can detect methylated bases

Question 25

what are the cons of nanopore?

Answer 25

it is slightly less accurate than other methods

Question 26

which sequencing methods are massively parallel?

Answer 26

illumina and nanopore

Question 27

which sequencing methods use synthesis?

Answer 27

snager and illumina

Question 28

which which sequencing methods are use a single molecule?

Answer 28

nanopore

Question 29

which sequencing methods use chan terminators?

Answer 29

sanger (nonreversible)
illumina (reversible)