Lecture 4 - DNA Sequencing
Study this set o nline at: https://www.cram.co m/flashcards/lecture-4-148 2438 0
Chai n-termi nati ng di deoxynucl eoti des are the key to determi ni ng D N A sequence.
When these are i ncorporated i nto an i n vi tro repl i cati on reacti on, D N A pol ymerase
cannot add any more nucl eoti des and thesynthesi s reacti on ends.
What i s Sanger Sequenci ng? E xpl ai n how i t works
I n cycl e sequenci ng, a PCR reacti on i ncl udes a control l ed amount of f l uorescentl y
l abel ed di deoxynucl eoti des. T aq pol ymerase stops addi ng nucl eoti des when a
di deoxynucl eoti de i s i ncorporated.
T he f l uorescent tag i s used to i denti f y the endi ng base of each f ragment usi ng
anautomated
T hi s methodssequencer.
entai l s sequenci ng many overl appi ng D N A f ragments i n paral l el and
then usi ng a computer to assembl e the smal l f ragments i nto l arger conti gs wi thout
Sangeroni
subcl sequenci
ng i t i nto
ng BACs.
can onl y can onl y be used f or short D N Astrands of 100 to 800
What i s shotgun sequenci ng?
base pai rs.
T hi s doesn't reveal i ndi vi dual nucl eoti des, but i t provi des a way to use, f or
exampl e Sanger Sequenci ng, to research l arger D N A.
Pol ymorphi sm - E ssenti al l y j ust al l el es. T hese are D N A sequences f or geneti c
vari ants at the same l ocus i n a popul ati on
Gi ve qui ck expl anati on of pol ymorphi sm
RF LP - Restri cti on F ragment Length Pol ymorphi sm, a pol ymorphi sm i n a restri cti on
T hen expl ai n RF LP, SN P, VN T R and M i crosatel l i te Pol ymorphi sm enz yme cut-si te (E x: F ather yi el ds a D N A sequence of 1000 bp i n between two
E coRI , M other
yi el ds 450 bp + 550 bp f ragments)
SN P -ne
I magi Siwe
ngl e N ucl
have a eoti de Pol ymorphi
chromosome sm, sequence
wi th f our a si ngl e nucl eoti de
tagged i n a(ST
si tes sequence is ly
S - Basi cal
jchanged
ust f our si tes wi th a tagged sequence we can i denti f y).
VN T Rwe
N ow - Vari
wantabltoemap
N umber
thi s, T andem
physi cal Repeats
l y, on the(mi ni sateltol iftes),
genome sequence
i nd out approximoti f s ythat
matel
H ow does ST S M appi ng Usi ng Restri cti on E nz yme D i gests work?
occur natural l y i n genome and consi
where these sequences are on the chromosome.sts of repeats of 9 to 80 bp i n l ength. Used in
f orensi c medi ci ne/paterni ty.
We j ust start cutti ng wi th restri cti on enz ymes, generati ng pi eces of D N A. T hese
M
pi ieces
crosatel l i te
reveal Pol ymorphi
whether thesesm - Alsisotes
f our tandem
are cl repeats, but smal
osel y pl aced l er Say
or not. (2-5we
bp).
cut the
chromosome
D etermi ni ng 6the
di order
f f erentoftivari
mes, and
ous we have
markers al lsi
owste generati
1, si te 2... (S1,
on of g eS2,
neS3,
t i cS4)
matagged.
ps .
M arkers used i ncl ude RF LPs, SN Ps, VN T Rs, and mi crosatel l i te pol ymorphi sms.
IGeneti
n 4 of cthese
mappicuts we fliind
ng uses S1 and
nkage analS3
ysitogether, and i nby
s of twomarkers 2 ofmati
these cuts we
ng experi f i ndorS2
ments
and
pediS4 together.
gree anal ysi s.N ow we know that S1 and S3 are pl aced cl ose together,
Why do we use/l ook f or vari ous types of geneti c markers?
probabl y cl oser than S2 and S4 are to each other.
P h ys i c a l ma ps l i nk a marker or D N A sequence toa physi cal l ocati on al ong a
We al so know
chromosome orthat
l argeS1conti
andg.S3Mare N OT
arkers cl ose
such as EtoSTeisther
and S2
ST or
Ss S4,
are si nce to
used wedetermi
di dn't ne
fthese
i nd any
di fstances.
ragmentsT contai ni ng these
echni ques pairestri
i ncl ude rs. cti on enz yme di gesti on mappi ng, F I SH ,
radi atina
I l l umi onsequenci
hybri d anal
ng iysi
s as,hiand cytogeneti cDmappi
gh-throughput ng.
N A sequenci ng method based on
sequenci ng by synthesi s.
E xpl ai n I l l umi na sequenci ng D N A i s f i rst f ragmented and adapters are added. T he f ragments bi nd to a f l ow
cel l and are ampl i f i ed i n pl ace to f orm cl usters (bri dge ampl i f i cati on). D uri ng
sequenci ng, f l uorescentl y l abel ed nucl eoti des are added one cycl e at a ti me.
E ach i ncorporated base emi ts a speci f i c col or, whi ch i s detected by a camera.
T he f l uorescence si gnal reveal s whi ch base was added at each posi ti on, bui l di ng
the
Ad asequence
pt e rs one nucl eoti de at a ti me across mi l l i ons of cl usters i n paral l el .
Adapters are short, syntheti c D N A sequences l i gated to both ends of D N A
fI ragments.
n short: massi velaly lparal
T hey l el , short-read
ow f ragments to bi ndsequenci
to the fng wicel
l ow th lhi, be
gh accuracy.
ampl i f i ed, and be
E xpl ai n: Adapter, Bri dge Ampl i f i cati on, and Reversi bl e T ermi nator N ucl eoti des sequenced (they al so contai n pri mer and i ndex sequences).
(Used duri ng I l l umi na)
B ri d g e a mpl i f i c a t i o n
Bri dge ampl i f i cati on i s how I l l umi na makes many copi es of each f ragment. A
f ragment bi nds to the f l ow cel l vi a an adapter, bends over to f orm a “bri dge” wi th
a nearby
I on prisequenci
T orrent mer, and ing s copi
i s a ed.
D N ARepeated
sequenci cycl es create
ng method thata cl uster ofpHi denti
detects cal
changes
iDnstead
N A molofecul es i n one spot.
f l uorescence.
R
DeNve
A frs i b l e tare
ragments e rmi naitfoi ed
ampl r nu
onc beads
l e ot i d e spl aced i nto ti ny wel l s on a
and
E xpl ai n I on T orrent sequenci ng
T hese are f l uorescentl y l abel ed nucl eoti
semi conductor chi p. D uri ng sequenci ng, nucl deseoti
thatdes
bl ock
are fflurther
owed extensi
over theonchi
af ter
p one
oneabase
at ti me.i When
s added.
a nuclAf ter
eotii magi
de i sng, the bl ocki ng
i ncorporated group
i nto and dye
the growi ng Dare
N Aremoved,
strand, a
al l owi ng the
hydrogen next
i on (H ⁺ ) base
i s rel to be added.
eased, causi ng a smal l drop i n pH . T hi s pH change i s
detected el ectroni cal l y and tel l s whi ch base was added.
Key i dea: adapters anchor D N A, bri dge ampl i f i cati on makes cl usters, and
reversi bl e termi nators ensure one-base-at-a-ti me readi ng.
Study this set o nline at: https://www.cram.co m/flashcards/lecture-4-148 2438 0
Chai n-termi nati ng di deoxynucl eoti des are the key to determi ni ng D N A sequence.
When these are i ncorporated i nto an i n vi tro repl i cati on reacti on, D N A pol ymerase
cannot add any more nucl eoti des and thesynthesi s reacti on ends.
What i s Sanger Sequenci ng? E xpl ai n how i t works
I n cycl e sequenci ng, a PCR reacti on i ncl udes a control l ed amount of f l uorescentl y
l abel ed di deoxynucl eoti des. T aq pol ymerase stops addi ng nucl eoti des when a
di deoxynucl eoti de i s i ncorporated.
T he f l uorescent tag i s used to i denti f y the endi ng base of each f ragment usi ng
anautomated
T hi s methodssequencer.
entai l s sequenci ng many overl appi ng D N A f ragments i n paral l el and
then usi ng a computer to assembl e the smal l f ragments i nto l arger conti gs wi thout
Sangeroni
subcl sequenci
ng i t i nto
ng BACs.
can onl y can onl y be used f or short D N Astrands of 100 to 800
What i s shotgun sequenci ng?
base pai rs.
T hi s doesn't reveal i ndi vi dual nucl eoti des, but i t provi des a way to use, f or
exampl e Sanger Sequenci ng, to research l arger D N A.
Pol ymorphi sm - E ssenti al l y j ust al l el es. T hese are D N A sequences f or geneti c
vari ants at the same l ocus i n a popul ati on
Gi ve qui ck expl anati on of pol ymorphi sm
RF LP - Restri cti on F ragment Length Pol ymorphi sm, a pol ymorphi sm i n a restri cti on
T hen expl ai n RF LP, SN P, VN T R and M i crosatel l i te Pol ymorphi sm enz yme cut-si te (E x: F ather yi el ds a D N A sequence of 1000 bp i n between two
E coRI , M other
yi el ds 450 bp + 550 bp f ragments)
SN P -ne
I magi Siwe
ngl e N ucl
have a eoti de Pol ymorphi
chromosome sm, sequence
wi th f our a si ngl e nucl eoti de
tagged i n a(ST
si tes sequence is ly
S - Basi cal
jchanged
ust f our si tes wi th a tagged sequence we can i denti f y).
VN T Rwe
N ow - Vari
wantabltoemap
N umber
thi s, T andem
physi cal Repeats
l y, on the(mi ni sateltol iftes),
genome sequence
i nd out approximoti f s ythat
matel
H ow does ST S M appi ng Usi ng Restri cti on E nz yme D i gests work?
occur natural l y i n genome and consi
where these sequences are on the chromosome.sts of repeats of 9 to 80 bp i n l ength. Used in
f orensi c medi ci ne/paterni ty.
We j ust start cutti ng wi th restri cti on enz ymes, generati ng pi eces of D N A. T hese
M
pi ieces
crosatel l i te
reveal Pol ymorphi
whether thesesm - Alsisotes
f our tandem
are cl repeats, but smal
osel y pl aced l er Say
or not. (2-5we
bp).
cut the
chromosome
D etermi ni ng 6the
di order
f f erentoftivari
mes, and
ous we have
markers al lsi
owste generati
1, si te 2... (S1,
on of g eS2,
neS3,
t i cS4)
matagged.
ps .
M arkers used i ncl ude RF LPs, SN Ps, VN T Rs, and mi crosatel l i te pol ymorphi sms.
IGeneti
n 4 of cthese
mappicuts we fliind
ng uses S1 and
nkage analS3
ysitogether, and i nby
s of twomarkers 2 ofmati
these cuts we
ng experi f i ndorS2
ments
and
pediS4 together.
gree anal ysi s.N ow we know that S1 and S3 are pl aced cl ose together,
Why do we use/l ook f or vari ous types of geneti c markers?
probabl y cl oser than S2 and S4 are to each other.
P h ys i c a l ma ps l i nk a marker or D N A sequence toa physi cal l ocati on al ong a
We al so know
chromosome orthat
l argeS1conti
andg.S3Mare N OT
arkers cl ose
such as EtoSTeisther
and S2
ST or
Ss S4,
are si nce to
used wedetermi
di dn't ne
fthese
i nd any
di fstances.
ragmentsT contai ni ng these
echni ques pairestri
i ncl ude rs. cti on enz yme di gesti on mappi ng, F I SH ,
radi atina
I l l umi onsequenci
hybri d anal
ng iysi
s as,hiand cytogeneti cDmappi
gh-throughput ng.
N A sequenci ng method based on
sequenci ng by synthesi s.
E xpl ai n I l l umi na sequenci ng D N A i s f i rst f ragmented and adapters are added. T he f ragments bi nd to a f l ow
cel l and are ampl i f i ed i n pl ace to f orm cl usters (bri dge ampl i f i cati on). D uri ng
sequenci ng, f l uorescentl y l abel ed nucl eoti des are added one cycl e at a ti me.
E ach i ncorporated base emi ts a speci f i c col or, whi ch i s detected by a camera.
T he f l uorescence si gnal reveal s whi ch base was added at each posi ti on, bui l di ng
the
Ad asequence
pt e rs one nucl eoti de at a ti me across mi l l i ons of cl usters i n paral l el .
Adapters are short, syntheti c D N A sequences l i gated to both ends of D N A
fI ragments.
n short: massi velaly lparal
T hey l el , short-read
ow f ragments to bi ndsequenci
to the fng wicel
l ow th lhi, be
gh accuracy.
ampl i f i ed, and be
E xpl ai n: Adapter, Bri dge Ampl i f i cati on, and Reversi bl e T ermi nator N ucl eoti des sequenced (they al so contai n pri mer and i ndex sequences).
(Used duri ng I l l umi na)
B ri d g e a mpl i f i c a t i o n
Bri dge ampl i f i cati on i s how I l l umi na makes many copi es of each f ragment. A
f ragment bi nds to the f l ow cel l vi a an adapter, bends over to f orm a “bri dge” wi th
a nearby
I on prisequenci
T orrent mer, and ing s copi
i s a ed.
D N ARepeated
sequenci cycl es create
ng method thata cl uster ofpHi denti
detects cal
changes
iDnstead
N A molofecul es i n one spot.
f l uorescence.
R
DeNve
A frs i b l e tare
ragments e rmi naitfoi ed
ampl r nu
onc beads
l e ot i d e spl aced i nto ti ny wel l s on a
and
E xpl ai n I on T orrent sequenci ng
T hese are f l uorescentl y l abel ed nucl eoti
semi conductor chi p. D uri ng sequenci ng, nucl deseoti
thatdes
bl ock
are fflurther
owed extensi
over theonchi
af ter
p one
oneabase
at ti me.i When
s added.
a nuclAf ter
eotii magi
de i sng, the bl ocki ng
i ncorporated group
i nto and dye
the growi ng Dare
N Aremoved,
strand, a
al l owi ng the
hydrogen next
i on (H ⁺ ) base
i s rel to be added.
eased, causi ng a smal l drop i n pH . T hi s pH change i s
detected el ectroni cal l y and tel l s whi ch base was added.
Key i dea: adapters anchor D N A, bri dge ampl i f i cati on makes cl usters, and
reversi bl e termi nators ensure one-base-at-a-ti me readi ng.
