1. US008632975B2
(12) Ulllted States Patent (10) Patent N0.: US 8,632,975 B2
Vander Horn et a]. (45) Date of Patent: Jan. 21, 2014
(54) NUCLEOTIDE TRANSIENT BINDING FOR 7,264,934 B2 9/2007 Fuller
SEQUENCING METHODS 7,270,951 B1 9/2007 Stemple et a1.
7,279,563 B2 10/2007 KWiatkoWski
. . 7,329,492 B2 2/2008 Hardin et al.
(75) Inventors: Peter B. Vander Horn, Enc1mtas, CA 7,361,466 B2 4/2008 Korlach et a1‘
(U$);CheI1g-Ya0 Chen, Carlsbad, CA 7,393,640 B2 7/2008 Kumar etal.
(US); Guobin Luo, Oceanside, CA 7,405,281 B2 7/2008 Xu et al.
(US); Michael Previte, Carlsbad, CA 3,: golrlaclé et a1~ _ tal. - - , , a asu ramanlan e .
(US), Jarnshld Te.rn.1rov,GermantoWn, 7,476,504 B2 V2009 Turner
TN NlklfOl‘OV, Carlsbad, 7,482,120 B2 V2009 BuZby
(US); Zhaohul Zhou, San Ramon, CA 7,485,424 B2 2/2009 Korlach et al.
(US); Hongye Sun, Belmont, CA (US); 7,541,444 B2 6/2009 Milton et al.
Yufang Wang. San Carlos. CA (Us); Egg/3Z3, g5: gggg 2101111811184 ~~~~~~~~~~~~~~~ 355/169},- - - - - , , ar e a . ................. ..
slefame Yuklk” Nlshuflura’ Mountam 2005/0100932 A1 5/2005 Lapidus et al.
VIeW, CA 0J$);H0I1gyl Wang, 2006/0003383 A1 1/2006 Graham
Pearland, TX (US); Marian Peris, 2007/0072196 A1 3/2007 Xu et al.
Belmont, CA (US); Barnett B. 2007/0148645 A1 * 6/2007 Hoser ............................. .. 435/6
. 2007/0196846 A1 8/2007 HanZel et al.
?lqsilnbiléllll’ lsan Ewe’ CA5(éiB’US 2008/0009007 A1 1/2008 Lyle et al.1° “6 e an’ aywar ’ ( ) 2008/0050780 A1 2/2008 Lee et al.
_ _ _ _ 2008/0091005 A1 4/2008 Wang et al.
(73) Ass1gnee: Life Technologies Corporation, 2008/0103053 A1 5/2008 Siddiqi et a1,
Carlsbad, CA (US) 2008/0108082 A1 5/2008 Rank et al.
2008/0132692 A1 6/2008 Wu et al.
* ' . ~ ~ ~ - 2008/0138804 A1 6/2008 BuZby
( ) Not1ce. Subject'to any d1scla1mer, the term ofthis Zoos/0227970 Al 9/2008 Siddiqiet a1‘
patent 1s extended or adjusted under 35 _
U.S.C. 154(1)) by 617 days. (Con?rmed)
(21) App1_ NO; 12/790,760 FOREIGN PATENT DOCUMENTS
' . WO WO-91/05060 4/1991
(22) Wed‘ May 28’ 2010 WO WO-91/06678 5/1991
(65) Prior Publication Data (Continued)
US 2010/0330570 A1 Dec. 30, 2010 OTHER PUBLICATIONS
Bebenek et al., Dissecting the Fidelity ofBacteriophage RB69 DNA
Related U-S-Applwatwn Data Polymerase: Site-Speci?c Modulation of Fidelity by Polymerase
(60) Provisional application No. 61/184,774, ?led on Jun. €ccesstorly $15211; lGenencs Sign“? ofATmeniat’ 2003:) _ _~ ~ ~ ~ erg e a ., O yrnerases equlre a emp a e an a r1mer,1n
5’ 20809’ prilsllslogézlgapphcatlqn Nlo' 61/2142’762’ f?Ied Biochemistry, 5th edition, NewYork: W H Freeman; 2002.*On eP- 5 s PrOVlSlOna app manor} _ 0' Arzumanov, Andrey A. et al., “y-Phosphate-substituted
61083320’ ?led on NOV‘ 20’ 2009’ provlslonal 2‘-Deoxynucleoside 5‘-Triphosphates as Substrates for DNA
appl1cat1on NO- 61095533, ?led 011 Jan 15, 2010- Polymerases”, J. Biol. Chem., vol. 271(40), 1996, pp. 24389-24394.
(51) Int. Cl. (Continued)
C12Q 1/68 (2006.01)
(52) US. Cl. Primary Examiner * Christopher M Babic
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Assislanz Examiner i Aaron Priest
(58) Field of Classi?cation Search
None . ~ _ (57) ABSTRACT
See appl1cat1on ?le for complete search h1story.
Provided herein are compositions and systems for use in
(56) References Cited polymerase-dependent, nucleotide transient-binding meth
U.S. PATENT DOCUMENTS
5,001,050 A 3/1991 Blanco et al.
5,198,543 A 3/1993 Blanco et al.
5,576,204 A 11/1996 Blanco et al.
5,707,804 A 1/1998 Mathies et al.
5,798,210 A 8/1998 Canardet al.
6,309,836 B1 10/2001 Kwiatkowski
6,482,590 B1* 11/2002 Ullman et al. ............. .. 435/612
6,982,146 B1 1/2006 Schneider et al.
7,041,812 B2 5/2006 Kumar et al.
7,052,839 B2 5/2006 Nelson et al.
7,125,671 B2 10/2006 Sood et al.
7,169,560 B2 * 1/2007 Lapidus et al. .............. .. 435/61
7,223,541 B2 5/2007 Fuller et al.
ods. The methods are useful for deducing the sequence of a
template nucleic acid molecule and single nucleotide poly
morphism (SNP) analyses. The methods rely on the fact that
the polymerase transient-binding time for a complementary
nucleotide is longer compared to that of a non-complemen
tary nucleotide. The labeled nucleotides transiently-binds the
polymerase in a template-dependent manner, but does not
incorporate. The methods are conducted under any reaction
condition that permits transient binding of a complementary
or non-complementary nucleotide to a polymerase, and
inhibits nucleotide incorporation.
85 Claims, 41 Drawing Sheets

2. US 8,632,975 B2
Page 2
(56) References Cited
U.S. PATENT DOCUMENTS
2008/0269476 A1
2008/0286837 A1
2008/0287305 A1
2008/0293071 A1
2009/0061437 A1
10/2008 Siddiqi
11/2008 Siddiqi
11/2008 Fuller et al.
11/2008 Gelfand et a1.
3/2009 Efcavitch et a1.
2009/0081686 A1 3/2009 Wu et al.
2009/0087850 A1* 4/2009 Eid et al. ......................... .. 435/6
2009/0176233 A1 7/2009 Clark et a1.
2010/0075328 A1* 3/2010 Bjornson et a1. ................ .. 435/6
2010/0075332 A1* 3/2010 Patel et al. ...................... .. 435/6
2010/0311144 A1
2011/0014612 A1
12/2010 Peris et al.
1/2011 Hendricks et al.
FOREIGN PATENT DOCUMENTS
WO WO-00/70073 11/2000
WO WO-2005/080605 9/2005
WO WO-2007/048033 4/2007
WO WO-2009/091847 7/2009
WO WO-2010/141390 12/2010
OTHER PUBLICATIONS
Bai, Xiaopeng et al., “Design and synthesis of a photocleavable
biotinylated nucleotide for DNA analysis by mass spectrometry”,
Nucleic Acids Research, vol. 32, No. 2, 2004, 535-541.
Bakhtina, Marina et al., “Contribution of the Reverse Rate of the
Conformational Step to Polymerase [3 Fidelity”, Biochem., vol. 48,
2009, 3197-3208.
Beaucage, S. et al., “Advances in the Synthesis of Oligonucleotides
by the Phosphoramidite Approach”, Tetrahedron Report No. 309,
vol. 48 (12), 1992, pp. 2223-2311.
Bentley, D et al., “Accurate whole human genome sequencing using
reversible terminator chemistry”, Nature, vol. 456(6), 2008, pp.
53-59.
Berman, Andrea J. et al., “Structures of phi29 DNA polymerase
complexed with substrate: the mechanism oftranslocation in B-fam
ily polymerases”, The EMBO Journal, vol. 26, 2007, 3494-3505.
Braslavsky, Ido et al., “Sequence information can be obtained from
single DNA molecules”, Proc. Natl. Acad. Sci. , vol. 100( 7), 2003, pp.
3960-3964.
Castro, Christian et al., “Nucleic acid polymerases use a general acid
for nucleotidyl transfer”, Nature Structural & Molecular Biology,
vol. 16 No.2, 2009, 212-218.
Dos Remedios, Cristobal G. et al., “Fluorescence Resonance Energy
Transfer Spectroscopy is a Reliable “Ruler” for Measuring Structural
Changes in Proteins”, Journal ofStructural Biology, vol. 115, 1995,
pp. 175-185.
Johnson, K. “Rapid kinetic analysis of mechanochemical
adenosinetriphosphatases”, Methods Enzymol, vol. 134, 1986, pp.
677-705.
Ju, J et al., “Four-color DNA sequencing by synthesis using cleavable
?uorescent nucleotide reversible terminators”, PNAS, vol. 103 (52),
2006, pp. 19635-19640.
Kumar, Amarendra et al., “Inhibition ofT7 RNA Polymerase: Tran
scription Initiation and Transition from Initiation to Elongation Are
Inhibited by T7 Lysozyme via a Ternary Complex with RNA
Polymerase and Promoter DNA”, Biochemistry, vol. 36, No. 45,
1997, pp. 13954-13962.
Kumar, Shiv et al., “Terminal Phosphate Labeled Nucleotides: Syn
thesis, Applications, and Linker Effect on Incorporation by DNA
Polymerasea”, Nucleosides, Nucleotides andNucleic Acids, vol. 24,
Nos. 5-7, 2005, 401-408.
Laitala, Ville et al., “Homogeneous Assay Based on Anti-Stokes’
Shift Time-Resolved Fluorescence Resonance Energy-Transfer
Measurement”, Analytical Chem., vol. 77, 2005, 1483-1487.
Marshall, P. N., “Rules for the visible absorption spectra ofhaloge
nated Fluorescein dyes”, Histochemical Journal, vol. 7, 1975, pp.
299-303.
Meijer, Wilfried et al., “Phi29 Family of Phages”, Microbiology and
Molecular Biology Reviews, vol. 65, No. 2, 2001, 261-287.
Patel, et al., “Insights into DNA Polymerization Mechanisms from
Structure and Function Analysis of HIV-1 Reverse Transcriptase”,
Biochemistry, vol. 34, No. 16, Apr. 1995, 5351-5363.
PCT/US2010/036755, International Search Report and Written
Opinion mailed Jun. 23, 2011, 12 pgs.
Pelletier, Huguette et al., “Structures of Ternary Complexes of Rat
DNA Polymerase beta, a DNA Template-Primer, and ddCTP”, Sci
ence, vol. 264, Jun. 24, 1994, 1891-1903.
Piston, David W. et al., “Fluorescent protein FRET: the bad and the
ugly”, Trends Biochem. Sci.,, vol. 32, No. 9, 2007, 407-414.
Rienitz, Axel et al., “On the ?delity of DNA polymerase alpha: the
in?uence ofalpha-thio dNTPs, Mn2+ and mismatch repair”, Nucleic
Acids Research, vol. 13, No. 15, 1985, 5685-5695.
Roettger, Michelle P et al., “Mismatched and Matched dNTP Incor
poration by DNA Polymerase [3 ProceedviaAnalogous Kinetic Path
ways”, Biochemistry, vol. 47, No. 37, 2008, 9718-9727.
Rothwell, Paul J. et al., “Structure and Mechanism of DNA
Polymerases”, Advances in Protein Chemistry, vol. 71, 2005, 401
440.
Ruparel, H. et al., “Design and synthesis of a 3‘-a-allyl photocleav
able ?uorescent nucleotide as a reversible terminator for DNA
sequencing by synthesis”, vol. 102 (17), Apr. 1, 2005, 5932-5937.
Selvin, Paul R., “Fluorescence Resonance Energy Transfer”, Meth
ods in Enzymology, vol. 246, 1995, 300-334.
Shimkus, M et al., “A chemically cleavable biotinylated nucleotide:
Usefulness in the recovery of protein-DNA complexes from avidin
a?inity columns”, PNAS, vol. 82, 1985, pp. 2593-2597.
Sood, Anup et al., “Terminal Phosphate-Labeled Nucleotides with
Improved Substrate Properties for Homogeneous Nucleic Acid
Assays”, J Am. Chem. Soc., vol. 127, No. 8, 2005, 2394-2395.
Stryer, “Fluorescence Energy Transfer as a Spectroscopic Ruler”,
Ann. Rev. Biochem.,, vol. 47,, 1978, 819-846.
Tsai, Yu-Chih et al., “A New Paradigm for DNA Polymerase Speci
?city”, Biochemistry, vol. 45, No. 32, 2006, 9675-9687.
Turcatti, Gerardo et al., “A new class of cleavable ?uorescent
nucleotides: synthesis and optimization as reversible terminators for
DNA sequencing by synthesis”, NucleicAcids Research, vol. 36, No.
4, e25, 2008, 1-13.
Wu, J et al., “3‘-O-modi?ed nucleotides as reversible terminators for
pyrosequencing”, PNAS, vol. 104 (42), 2007, pp. 16462-16467.
Wu, J et al., “Termination of DNA synthesis by N6-alkylated, not
3‘-O-alkylated, photocleavable 2‘-deoxyadenosine triphosphates”,
Nuc Acids Research, vol. 35(19), 2007, pp. 6339-6349.
Wu, Pengguang et al., “Resonance Energy Transfer: Methods and
Applications”,Anab/ticalBiochemistry, vol. 218, No. 1, 1994, 1-13.
Barone, A. D. et al., “Novel Nucleoside Triphosphate Analogs for the
Enzymatic Labeling ofNucleic Acids”, Nucleosides, Nucleotides &
Nucleic Acids, 20(4-7), 2001, 1141-1145.
Beese, Lorena et al., “Structural basis for the 3‘-5‘ exonuclease activ
ity of Escherichia coli DNA polymerase I: a two metal ion mecha
nism”, The EMBO Journal, vol. 10 No. 1, 1991, 25-33.
Burgers, Peter et al., “Eukaryotic DNA Polyrnerases: Proposal for a
Revised Nomenclature”, The Journal ofBiological Chemistry, vol.
276, No. 47, 2001, 43487-43490.
Castro, Christian et al., “Two proton transfers in the transition state
for nucleotidyl transfer catalyzed by RNA- and DNA-dependent
RNA and DNA polymerases”, PNAS, vol. 104, No. 11, 2007, 4267
4272.
Dunaway-Mariano, Debra et al., “Investigations of Substrate Speci
?city and Reaction Mechanism of Several Kinases Using
Chromium(III) Adenosine 5‘-Triphosphate and Chromium(III)
Adenosine 5‘-Diphosphate”, Biochemistry, 19, 1980, 1506-1515.
Dunaway-Mariano, Debra et al., “Preparation and Properties of
Chromium(III) Adenosine 5‘-Triphosphate, Chromium(III)
Adenosine 5‘-Diphosphate, and Related Chromium(III) Com
plexes”, Biochemistry, 19, 1980, 1496-1505.
Forster, T., “Intermolecular Energy Migration and ?uorescence”,
Ann. Physik (Leipzig), vol. 2, 1948, 55-75.
Gangurde, Rajiv et al., “Participation ofActive-Site Carboxylates of
Escherichia coli DNA Polymerase I (Klenow Fragment) in the For
mation of a Prepolyrnerase Ternary Complex”, Biochemistry, 41,
2002, 14552-14559.

3. US 8,632,975 B2
Page 3
(56) References Cited
OTHER PUBLICATIONS
Guo, Jia et al., “Four-color DNA sequencing With 3‘-O-modi?ed
nucleotide reversible terminators and chemically cleavable ?uores
cent dideoxynucleotides”, PNAS, vol. 105, No. 27, Jul. 8, 2008,
9145 -9150.
Joyce, Catherine et al., “Fingers-Closing and Other Rapid
Conformational Changes in DNA Polymerase I (Klenow Fragment)
and Their Role in Nucleotide Selectivity”, Biochemistry, 47, 2008,
6103 -61 16.
Kaushik, Neerja et al., “Biochemical Analysis of Catalytically Cru
cial Aspartate Mutants of Human Immunode?ciency Virus Type 1
Reverse Transcriptase”, Biochemistry, 35, 1996, 11536-11546.
Lee, Harold et al., “The Reopening Rate ofthe Fingers Domain Is a
Determinant of Base Selectivity for RB69 DNA Polymerase”, Bio
chemistry, 2009, 2087-2098.
SaWaya, Michael et al., “Crystal Structures of Human DNA
Polymerase [5 Complexed With Gapped and Nicked DNA: Evidence
for an Induced Fit Mechanism”, Biochemistry, 36, 1997, 11205
1 12 1 5.
Scaringe, Stephen et al., “Novel RNA Synthesis Method Using 5‘-O
Silyl-2‘-O-orthoester Protecting Groups”, .1. Am. Chem. Soc., 120,
1998,11820-11821.
Seo, Tae Seok et al., “Four-color DNA sequencing by synthesis on a
chip using photocleavable ?uorescent nucleotides”, PNAS, vol. 102,
No. 17, 2005, 5926-5931.
SteitZ, Thomas et al., “A general tWo-metal-ion mechanism for cata
lytic RN ”, Proc. Natl. Acad. Sci. USA, vol. 90, 1993, 6498-6502.
SteitZ, Thomas, “A mechanism for all polymerases”, Nature, vol.
391, 1998, 231-232.
Wang, Mina et al., “Effect ofA and B Metal Ion Site Occupancy on
Conformational Changes in an RB69 DNA Polymerase Ternary
Complex”, Biochemistry, 48, 2009, 2075-2086.
Zhang, H. et al., “Fluorescence of 2-aminopurine reveals rapid
conformational changes in the RB69 DNA polymerase-primer/tem
plate complexes upon binding and incorporation of matched
deoxynucleoside triphosphates”, Nucleic Acids Research, vol. 35,
No. 18, 2007, 6052-6062.
Zhong, Xuejun et al., “DNA Polymerase [5. 5. Dissecting the Func
tional Roles of the Two Metal Ions With Cr(III)dTTP”, J'. Am. Chem.
Soc., 120, 1998, 235-236.
* cited by examiner

4. US. Patent Jan. 21, 2014 Sheet 1 0141 US 8,632,975 B2
268
267
266 FIG.1
265
NoDNA
11 1O 9

7. U S. Patent Jan. 21, 2014 Sheet 4 0f 41 US 8,632,975 B2
@535
S
Nago
awwngmnwa.N096
@5353m09m

9. US. Patent Jan. 21, 2014 Sheet 6 0f 41 US 8,632,975 B2
4.3K; -
4.32 -
4.30 -
4,25‘ -
4.2a »
Fluumscenca{volta}
3‘ i i
was {1.85 {1,1 0 0.15 8,28
Time {secands}
FEG. 5A

10. US. Patent Jan. 21, 2014 Sheet 7 0f 41 US 8,632,975 B2
.
nu,5
15a4i3
w
I21a...
%.3
11£1:34%EIU
na~HwMwu
?0"in“50,5,554.4332
Time (seconds)

11. US. Patent Jan. 21, 2014 Sheet 8 0f 41 US 8,632,975 B2
5.5~
w
a”32
5
I
3.3
F
8,5
1
GA
5.1
1.60.20.3
Time (seconds)

12. US. Patent
Fiuarasceme{wits} 35 ~
341“
25 ~
Jan. 21, 2014 Sheet 9 0f 41 US 8,632,975 B2
z I I ' i ' I ' z
"W 15 20
Time (seconds)

14. US. Patent Jan. 21, 2014 Sheet 11 0141 US 8,632,975 B2
53 ~
Carrect 664?
0
5
3.8 *
Time (sacunds)

15. US. Patent Jan. 21, 2014 Sheet 12 0141 US 8,632,975 B2
5.13 -
Fluurescance(volts)
I ‘ E ' i ' l
a 2 4 a a 10
Time (seconds)
FEG. 5G

16. US. Patent Jan. 21, 2014 Sheet 13 0f 41 US 8,632,975 B2
01:1: 01 I
A O s
aFlumesseme(wits)
C?ma;in
3
i X 1 * | ' i ‘ E

17. US. Patent Jan. 21, 2014 Sheet 14 0f 41 US 8,632,975 B2
4.8 ~
533mucmumwhommw
.KM.
Tima (secunds)

18. US. Patent
Fluorescance{waits}
Jan. 21, 2014
5.2 ~
m ~ g
5.3 -
5.6— 1
Sheet 15 0f 41 US 8,632,975 B2
Time (secunds)
FEG. $8

19. US. Patent
?uorescence(wits)
4,3 -
4.6 ~
4.4 w
4.2 -
3.6 -
Jan. 21, 2014 Sheet 16 0f 41 US 8,632,975 B2
- l ‘ I ' I ‘ 1
1a 26 so 4a
Time {amends}
HG. $3
5.0

20. US. Patent Jan. 21, 2014 Sheet 17 0141 US 8,632,975 B2
5.5
“mamamu=muwm32m
Time (semnds)