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`About the Journal Home > Science Magazine > 17 July 1998 >
`
`Ronaghi et al. , pp. 363 - 365
`Science 17 July 1998:
`Vol. 281. no. 5375, pp. 363 - 365
`DOI: 10.1126/science.281.5375.363
`TECH.SIGHT
`DNA SEQUENCING:
`A Sequencing Method Based on Real-Time Pyrophosphate
`Mostafa Ronaghi, Mathias Uhlén, and Pål Nyrén*
`DNA sequencing is one of the most important technologies in bioscience today.
`Whole-genome approaches (1) and human expressed sequence tag (EST)
`sequencing (2) have started to exert profound in�uence on biology and medicine.
`The need for robust, high-throughput methods to replace the elegant Sanger
`method, described more than 20 years ago (3), has led to the development of
`several new principles, such as array methods based on sequencing by hybridization
`(4). New applications, such as population-based biodiversity projects and
`brute-force genotyping using single-nucleotide polymorphism, make such e orts
`ff
`even more urgent, in particular, for simple and robust methods for sequencing short
`"tags" (1 to 20 bases) such as ESTs or biallelic markers and methods suitable for
`routine diagnostic applications.
`Sequencing-by-synthesis is based on the detection of nucleotide incorporation,
`using a primer-directed polymerase extension. The sequence can be deduced
`iteratively (5). During the last decade, many researchers, including the groups of
`Rosenthal (6), Gibbs (7), and Jones (8), described various protocols based on
`�uorescently labeled nucleotides. The level of incorporation of these �uorescent
`nucleotides is low, however, as shown by Metzker et al. (7), and therefore, the
`protocols only permit detection of a few bases.
`Recently, Ronaghi et al. (9) showed that natural nucleotides can be used to obtain
`e cient incorporation during a sequencing-by-synthesis protocol. The detection
`ffi
`was based on the pyrophosphate (PPi) released during the DNA polymerase reaction,
`the quantitative conversion of pyrophosphate to ATP by sulfurylase, and the
`subsequent production of visible light by �re�y luciferase. However, this PPi-based
`sequencing method is not without drawbacks: The template must be washed
`
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`Columbia Ex. 2022
`Illumina, Inc. v. The Trustees
`of Columbia University
`in the City of New York
`IPR2020-01177
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`thoroughly between nucleotide additions to remove unincorporated nucleotides.
`Also, templates not bound to a solid support are di cult to sequence, and the
`ffi
`addition of new enzymes to each cycle of deoxynucleotide (dATP, dTTP, dGTP, and
`dCTP) is required.
`
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`Fig. 1. In the new DNA sequencing method, four nucleotides are added stepwise to
`the template hybridized to a primer. The PPi released in the DNA polymerase-
`catalyzed reaction is detected by the ATP sulfurylase and luciferase in a coupled
`reaction. The added nucleotides are continuously degraded by a nucleotide-
`degrading enzyme. After the �rst added nucleotide has been degraded, the next
`nucleotide can be added. As this procedure is repeated, longer stretches of the
`template sequence are deduced. dXTP, one of the four nucleotides.
`
`Here, we address these problems by a modi�cation in which the sequencing cycles
`can be performed without intermediate washing steps. This is achieved by the
`addition of a nucleotide-degrading enzyme to obtain a four-enzyme mixture. The
`principle of pyrosequencing is outlined in Fig. 1. The DNA fragment of interest
`(sequencing primer hybridized to a single-stranded DNA template) is incubated with
`DNA polymerase, ATP sulfurylase, �re�y luciferase, and a nucleotide-degrading
`enzyme (such as apyrase). Repeated cycles of deoxynucleotide addition are
`performed. A deoxynucleotide will only be incorporated into the growing DNA
`strand if it is complementary to the base in the template strand. The synthesis of
`DNA is accompanied by release of PPi equal in molarity to that of the incorporated
`deoxynucleotide. Thereby, real-time signals are obtained by the enzymatic
`inorganic pyrophosphate detection assay (10). In this assay the released PPi is
`converted to ATP by ATP sulfurylase and the concentration of ATP is then sensed by
`the luciferase. The amount of light produced in the luciferase-catalyzed reaction can
`readily be estimated by a suitable light-sensitive device such as a luminometer or a
`CCD (charge-coupled device) camera. Unincorporated deoxynucleotides and the
`produced ATP are degraded between each cycle by the nucleotide-degrading
`enzyme. The nucleotide-degrading enzyme must possess the following properties:
`First, the enzyme must hydrolyze all deoxynucleotide triphosphate at approximately
`the same rate. This includes the a-thio-dATP, which is used instead of dATP to
`improve the background in sequencing reactions (9). Second, it should also
`hydrolyze ATP to prevent the accumulation of ATP between cycles. Third, the time
`for nucleotide degradation by the nucleotide-degrading enzyme must be slower
`than nucleotide incorporation by the polymerase. Obviously, these two enzymes
`compete for the same substrate, and it is important that the yield of primer-directed
`incorporation is as close to 100% as possible before the nucleotide-degrading
`enzyme can degrade the nucleotide to a concentration below the KM for the
`polymerase. Finally, the rate of ATP synthesis by the sulfurylase should preferably be
`
`

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`faster than the rate of ATP hydrolysis to obtain ATP concentrations and light
`production in proportion to the amount of PPi released.
`
`To optimize the assay, several parameters were tested by using synthetic
`oligonucleotides as template (11). The assay solution consisted of the template-
`primer plus varying amounts of the four enzymes (12). The protocol consisted of
`simply adding a new nucleotide every other minute in an iterative manner and
`detecting the visible light produced. In Fig. 2 (Left), an example of the results is
`shown. Clear specific signals can be observed with low background. Note the higher
`signals obtained at cycles 21, 23, and 24 when two nucleotides were incorporated
`because of the presence of two identical adjacent bases in the template. Similar
`high-quality sequencing results were obtained for other oligonucleotide templates
`(13).
`
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`Fig. 2. Pyrosequencing was performed on a 35-base-long oligonucleotide template
`(Left) and a 130-base-long PCR product (Right). About 2 pmol of the template-
`primer was used in the assay. The reaction was started by the addition of the
`indicated deoxynucleotide, and the PPi released was detected by the described
`method. The DNA sequence after the primer is indicated.
`
`For direct sequencing of polymerase chain reaction (PCR) products, single-stranded
`template was obtained by biotin capture on magnetic beads (14). In Fig. 2 (Right),
`the amount of light produced during approximately 40 subsequent cycles is shown.
`Sequence data covering 34 bases is obtained (15), and the signal-to-noise ratio
`remains relatively high even at 40 cycles. The low signals in the early cycles (2, 3, 6,
`and 7) show that the misincorporation of bases, which would produce PPi and
`consequently light, is not a major problem, despite the presence of "wrong"
`noncomplementary nucleotides. The background does increase during the later
`cycles, which is not surprising because relatively crude enzyme preparations were
`used in this work. Low amounts of contaminating enzymes such as exonucleases or
`kinases will give rise to nonsynchronized extensions on some templates, causing
`increased background and lower specific signal. Obviously, more work is needed to
`purify these enzymes from present contaminants. In addition, apyrase activity is
`decreased in later cycles, which is because of accumulation of intermediate products
`(such as deoxynucleoside diphosphate, or dNDP) and eventually undegraded dNTP.
`Removal of these nucleotides by enzymes such as nonspecific nucleoside
`diphosphatases (16) will increase the e(cid:2)ciency of apyrase in degradation of
`nucleoside triphophates and thereby allow longer reads. However, it is reassuring
`that the nonoptimal enzyme mixture used here allows accurate determination of
`more than 20 bases for many di(cid:1)erent PCR products tested (13).
`
`An inherent problem with the described method is the di(cid:2)culty in determining the
`number of incorporated nucleotides in homopolymeric regions due to the nonlinear
`light response following incorporation of more than three or four identical
`nucleotides. This can be demonstrated by the relatively low signal at cycle 16 (Fig. 2,
`Right) when four T bases are incorporated. However, this nonlinear response can
`most likely be compensated for by software algorithms. In addition, for most
`
`

`

`tag-sequencing applications, such as brute-force EST-sequencing, biallelic marker
`analysis, and confirmatory sequencing, this problem is not a major concern, because
`the number of bases, if present, will be known.
`
`With this method, parallel processing of large numbers of samples can easily be
`envisioned with the use of high-density microtiter plates and microinjector
`technology. An automated instrument has recently been developed based on the
`precise delivery of submicroliter volumes of the four nucleotides by "ink-jet"
`technology into a microtiter plate coupled with simultaneous detection of all
`samples by a single CCD unit (17). Together with a robot (17) performing single-
`strand template preparation (from double-stranded PCR products), ready for
`pyrosequencing, it would be possible to analyze thousands of samples daily with
`little manual intervention.
`
`References and Notes
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`7.
`
`8.
`
`9.
`
`R. D. Fleischmann et al., Science 269, 496 (1995); C. M. Fraser et al., ibid. 270,
`397 (1995); C. J. Bult et al., ibid. 273, 1058 (1996); J. Tomb et al., Nature 388,
`539 (1997); H. W. Mewes et al., ibid. 387, 7 (1997).
`
`V. E. Velculescu, L. Zhang, B. Vogelstein, K. W. Kinzler, Science 270, 484 (1995).
`
`F. Sanger, S. Nicklen, A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463
`(1977).
`
`R. Drmanac, I. Labat, I. Brukner, R. Crkvenjakov, Genomics 4, 114 (1989); H.
`Köster et al., Nature Biotechnol. 14, 1123 (1996).
`
`E. D. Hyman, Anal. Biochem. 174, 423 (1988).
`
`A. Rosenthal, International Patent Application Publication 761107 (1989).
`
`M. L. Metzker et al., Nucleic Acids Res. 22, 4259 (1994).
`
`D. H. Jones, Biotechniques 22, 938 (1997).
`
`M. Ronaghi, S. Karamohamed, B. Pettersson, M. Uhlén, P. Nyrén, Anal. Biochem.
`242, 84 (1996).
`
`10.
`
`P. Nyrén and A. Lundin, ibid. 151, 504 (1985).
`
`11.
`
`12.
`
`The oligonucleotides E3PN
`(5¢-GCTGGAATTCGTCAGACTGGCCGTCGTTTTACAAC-3¢), NUSPT
`(5¢-GTAAAACGACGGCCAGT-3¢), and JA80
`(5¢-GATGGAAACCAAAAATGATAGG-3¢) were synthesized by phosphoramidite
`chemistry (Interactiva).
`
`The oligonucleotide E3PN and the PCR product generated from cloned HIV-V3
`were used as templates for DNA sequencing. The oligonucleotides and single-
`stranded PCR product were hybridized to the primers NUSPT and JA80,
`respectively. The hybridized DNA fragments were incubated with exo- Klenow or
`exo- T7 DNA polymerase (Sequenase 2.0), respectively (Amersham). The
`sequencing procedure was carried out by stepwise elongation of the primer-
`strand upon sequential addition of the di(cid:1)erent deoxynucleoside triphosphates
`and simultaneous degradation of nucleotides by apyrase (nucleoside
`5¢-triphosphatase and nucleoside 5¢-diphosphatase; EC 3.6.1.5) (Sigma). The
`sequencing reaction was performed at room temperature and was started by
`adding a specific amount of one of the deoxynucleotides. The PPi released due
`to nucleotide incorporation was detected as described (9).
`
`13.
`
`M. Ronaghi and P. Nyrén, data not shown.
`
`14.
`
`The biotinylated PCR products were immobilized onto streptavidin-coated super
`paramagnetic beads [Dynabeads M280-Streptavidin (Dynal)]. Elution of single-
`stranded DNA and hybridization of sequencing primers was carried out as
`
`

`

`15.
`
`16.
`17.
`
`described (9).
`The sequencing data obtained from the pyrosequencing method was con�rmed
`by semiautomated solid-phase Sanger sequencing (18).
`H. D. Doremus and D. G. Belvins, Plant Physiol. 87, 36 (1988).
`The pyrosequencing instrument was based on a cassette containing the four
`separate nucleotides on an x-ray robotic arm (B. Ekström, M. Ronaghi, T.
`Nordström, P. Nyrén, M. Uhlén, unpublished data). The sample preparation robot
`was based on streptavidin-coated magnetic particles for PCR-capture and
`handling (A. Holmberg and M. Uhlén, unpublished data).
`T. Hultman, S. Ståhl, E. Hornes, M. Uhlén, Nucleic Acids Res. 17, 4937 (1989).
`We thank K. Nourizad for very helpful technical assistance and A. Scott for
`critical review. Supported by grants from PyroSequencing AB and the Swedish
`Research Council for Engineering Science (TFR).
`Mailbox:www.sciencemag.org/dmail.cgi?53491
`
`18.
`19.
`
`The authors are in the Department of Biochemistry and Biotechnology, The Royal
`Institute of Technology, SE-10044 Stockholm, Sweden. E-mail:
`paaln@biochem.kth.se
`*To whom correspondence should be addressed.
`
`