US 20040142330A1
(19) United States
(12) Patent Application Publication (10) Pub. No.: US 2004/0142330 A1
(43) Pub. Date:
Nyren et al.
(54) METHOD OF SEQUENCING DNA
(76) Inventors: Pal Nyren, Stockholm (SE); Mostafa
Ronaghi, Palo Alto, CA (US); Annika
Jul. 22, 2004
Publication Classi?cation
(51)
Int. Cl? ..................................................... ..C12Q 1/68
(52)
Us. 01. ................................................................ .. 435/6
Tallsjo, Uppsala (SE)
(57)
Correspondence Address:
DORSEY & WHITNEY LLP
INTELLECTUAL PROPERTY DEPARTMENT
250 PARK AVENUE
NEW YORK, NY 10177 (US)
(21) Appl. No.:
10/363,231
(22) PCT Filed:
Sep. 7, 2001
(86) PCT No.:
PCT/GB01/04015
ABSTRACT
The present invention provides a method of identifying a
base at a target position in a sample nucleic acid sequence,
said method comprising: subjecting a primer hybridised to
said sample nucleic acid immediately adjacent to the target
position, to a polymerase primer extension reaction in the
presence of a nucleotide, Whereby the nucleotide Will only
become incorporated if it is complementary to the base in the
target position, and determining Whether or not said nucle
otide is incorporated by detecting Whether Ppi is released,
the identity of the target base being determined from the
identity of any nucleotide incorporated, Wherein, Where said
nucleotide comprises an adenine base, an (X-ihiO triphos
(30)
Foreign Application Priority Data
Sep. 7, 2000
(GB) ....................................... .. 0021977.4
phate analogue of said nucleotide is used, ant the Rp isomer
of said analogue and/or the degradation products of said
analogue are eliminated from the polymerase reaction step.
Patent Application Publication
com
o ow
Jul. 22, 2004 Sheet 1 0f 22
oom
US 2004/0142330 A1
Patent Application Publication Jul. 22, 2004 Sheet 2 0f 22
US 2004/0142330 A1
2
FIG.
Sdof+A5OTPitor.ns
C)
O
O
C)
(0
lo
O
O
O
O
0')
Light Intensify
200
100
Patent Application Publication Jul. 22, 2004 Sheet 3 0f 22
oom omw oov 0mm
O
O
0")
o
to
N
o
o
N
Light Intensify
om? o2
US 2004/0142330 A1
om
Patent Application Publication Jul. 22, 2004 Sheet 4 0f 22
US 2004/0142330 A1
w.OI
omw oov 0mm com
O
LO
0
0
m
N
Light Intensify
om? oor
om
Patent Application Publication Jul. 22, 2004 Sheet 5 0f 22
US 2004/0142330 A1
dTP+A
doTA5Pi+ftA0ToPns
600
500
O
O
00
u
m
00
n
Llght lntensnfy
100
FIG.5
Patent Application Publication Jul. 22, 2004 Sheet 6 0f 22
US 2004/0142330 A1
wi Et
02 cm? 03 cm?
O
O
(I)
2
Light Intensity
0
(O
0
v
om
Patent Application Publication Jul. 22, 2004 Sheet 7 0f 22
US 2004/0142330 A1
86990418E3PN+kinase2mu+AP+256sup Well:25
ollllllllllllI'IIIITTFIIFIIIIIIIII
-GCGCGCGCATCGCATCGCé é
25'
i ll
OlllllllllIIIllllllllllllllllllllllll
-GCGCGCATCGCGCGCATCATC
FIG. 7B
86990418E3PN+kinase2mu+AP+256sup Well143
25
15
10
Patent Application Publication Jul. 22, 2004 Sheet 8 0f 22
US 2004/0142330 A1
(kitA) OpJ
A
TACGTA
TACGTACGTACGTACGTA
TACGTA
(kitA) 5m
B
A
T
TACGTACGTACGTACGTACGTACGT
(kitA) 10w
(3
ACGTACGTACGTACGT
(kitA) 20w
D
FIG. 8
T
Patent Application Publication Jul. 22, 2004 Sheet 9 0f 22
T
TA
TA
US 2004/0142330 A1
TACGTACGTACGTACGTA
T
(Super Sp) 5p!
F
TACGTACGTACGT
T
(Super Sp) 10p]
G
(Super Sp) 20p!
H
8 cont'd
Patent Application Publication Jul. 22, 2004 Sheet 10 0f 22
US 2004/0142330 A1
50
45
40
35
30
25
20
15
10
5
O
ACGTACGTACGTACGTACGTACGTAOGTACGTACGTACGTACGTACGTACGTACGTACGT
40
35
30
25
20 _
15
10
5
0
ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGT
25
l
20
K
10-;
5
\
“WWW
\\.
0lTlllllllllllllllllllll?lllllnjlllllllllllllllllllllllwll
ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGT
ACGTACGTACGTACGTACGTACGTAQ'STACGTACGTACGTACGTACGTACGTACGTACGT
8 cont'd
Patent Application Publication Jul. 22, 2004 Sheet 11 0f 22
US 2004/0142330 A1
6996
9026
L988
L678
1mM0tpAdPSLhlBa.rUopmeT0b7Cc2nhPaitq1gk9
68LL
SEVL
LQOL
LZLQ
SL219
6L09
9999
LL89
L967
809’?
67217
9688
N798
L9l€
‘8982
6LVZ
SZLZ
LLLL
LLVL
890i
60L
99$
L
(D
(D
800
700
600
o
o
no
400
300
200
100
Patent Application Publication Jul. 22, 2004 Sheet 14 0f 22
US 2004/0142330 A1
aGE£8
Patent Application Publication Jul. 22, 2004 Sheet 16 0f 22
US 2004/0142330 A1
LZLZL
EOVZL
SQOZL
LQLH
6177M
L‘EILH
€L8OL
96VOL
LLLOL
6988
M796
8ZZ6
9068
L898
6928
[86L
889i
Sl-SL
L669
6L99
L989
8709
GZLG
1.0179
6mM0ptAdPhBla.Ur0pmTbC72aohcP1itk9
6809
LLLV
89W
98W
LL88
66119
L8L€
9982
91793
[.332
606L
L6Sl
SLZL
996
L99
6L9
L
800
700
600
500
400
300
200
100
CD
G
9
l
F
cont'd.
Patent Application Publication Jul. 22, 2004 Sheet 17 0f 22
US 2004/0142330 A1
mm
206st‘
mMp0tAdPBahl.Ur0pTbRC72toahPE1ck9
CGTA g c
€917£L
660€L
QVLZL
L6€ZL
120a
€89LL
GZSLL
SLSOL
LZ9OL
.LQZOL
H66
6996
9026
L988
1.6178
ewe
6822
98172
L802
LZLQ
use
6L09
9999
LL89
L967
20911
617217
9682
M798
L8H)
sesz
GLVZ
SZLZ
LLLL
1m
€9OL
60L
66
800
700
600
500
998
L
400
300
200
100
Patent Application Publication Jul. 22, 2004 Sheet 18 0f 22
US 2004/0142330 A1
Icont'd
mMp0tAdPaBlh.Urp0TbRC7t2ahoPE1ck9
CGTA CT
o
o
o
O
O
o
N
(D
LO
Q’
0‘)
(\l
"
Patent Application Publication Jul. 22, 2004 Sheet 19 0f 22
US 2004/0142330 A1
LOSSL
SEVSL
6608L
SVLZL
LGEZL
L 8 0 3 ll?
63814
SLSOL
LZQOL
LQZOL
H66
6996
9086
L988
A6178
(9
8W8
22mE25t3Q32“m20%8x50%R28
O2:8
luv 8 0 l.
L c... 9
H89
L961’
80917
W98
28%?
<
8882
61.173
0
SZLZ
9
LLVL
LLLL
998
com
com
com
oom
oov
com
com
oov