A Method for DNA Sequencing Using RNA Polymerase
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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 3455–3460, March 1998 Biochemistry Transcriptional sequencing: A method for DNA sequencing using RNA polymerase NOBUYA SASAKI*, MASAKI IZAWA*†,MASANORI WATAHIKI†,KAORI OZAWA‡,TAKUMI TANAKA‡,YUKO YONEDA†, SHUJI MATSUURA‡,PIERO CARNINCI*, MASAMI MURAMATSU*, YASUSHI OKAZAKI*, AND YOSHIHIDE HAYASHIZAKI*§ *Genome Science Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan; †Research and Development, Nippon Gene Co., Ltd., 1-29, Tonya-machi, Toyama, 930 Japan; and ‡Osaka Research Laboratories, Wako Pure Chemical Industries, Ltd., 6-1, Takada-cho, Amagasaki, Hyogo 661, Japan Communicated by Webster K. Cavenee, University of California, San Diego, La Jolla, CA, December 24, 1997 (received for review November 20, 1997) ABSTRACT We have developed a sequencing method direct sequencing of PCR products by using Dye-terminator based on the RNA polymerase chain termination reaction with chemistry, unreacted 29-dNTP and primers must be eliminated rhodamine dye attached to 3*-deoxynucleoside triphosphate to avoid interference with the subsequent sequencing reaction. (3*-dNTP). This method enables us to conduct a rapid iso- Although efforts have been made to quickly purify the PCR thermal sequencing reaction in <30 min, to reduce the amount product such as enzymatic degradation using exonuclease I of template required, and to do PCR direct sequencing without and shrimp alkaline phosphatase, most protocols are time- the elimination of primers and 2*-dNTP, which disturbs the consuming, laborious, and expensive (7, 8). Sanger sequencing reaction. An accurate and longer read To overcome the above problems, we pursued a completely length was made possible by newly designed four-color dye- different approach. Based on the property of promoter- 3*-dNTPs and mutated RNA polymerase with an improved dependent RNAP, we predicted that the chain termination incorporation rate of 3*-dNTP. This method should be useful method using this group of enzymes would be useful. First, for large-scale sequencing in genome projects and clinical RNAP does not use primers and cannot incorporate 29-dNTP, diagnosis. but can incorporate nucleoside triphosphate (NTP) and 39- dNTP. This would allow direct sequencing without any puri- High throughput DNA sequencing is essential technology for fication steps. Second, it is much faster to process (240 y y genome projects and clinical diagnosis (1). Recent develop- bases sec) than Taq polymerase (60 bases sec), thus reducing ments for high throughput DNA sequencing include multiple the reaction time (9, 10). Third, the turnover of RNAP allows capillary array sequencer, enzyme and fluorescent primer or us to amplify the signal without temperature cycling. This fluorescent dideoxynucleotide (ddNTP) for sequencing reac- reaction does not require a denaturation step to hybridize the tions (2–5). The ideal sequencing reaction should be accurate sequencing primer to the template. Finally, a large amount of and quick and easy to perform, enabling automation of a large sequencing product can be transcriptionally amplified from a number of reactions. small amount of DNA template. Cunninghum et al. (11) Currently, cycle sequencing chemistry, employing dye prim- reported that T7 RNAP can produce 600 molecules of in vitro ers and dye terminators, is widely used. Dye-primer chemistry transcripts from 1 molecule of DNA template. is useful for long-read sequencing due to the uniform incor- Axelrod and Kramer (12) reported the use of T7 and SP6 poration of four types of ddNTP, resulting in an even peak RNAPs for chain termination reaction with radioisotope- height for each signal. However, this requires four independent internal labeling. However, their data showed a variation in reactions and the sequencing pattern is sometimes flawed by peak heights due to variation in the incorporation of 39-dNTP, false stops at some sites with no incorporation of ddNTPs. On in agreement with our unpublished data. Moreover, the chain the other hand, dye-terminator chemistry was developed as a termination method using the wild-type (wt) RNAP and one-tube reaction without false stops, but shows various in- 39-dNTP produced many false ladders caused by nonspecific corporation rates for the four color terminators resulting in stopping of polymerization. Thus, despite the possibility of failure of long-read sequencing. Recently, ‘‘ThermoSeque- applying RNAP for the sequencing technique, there has been nase’’, a newly developed enzyme mutated to make the no further development due to variation in the incorporation incorporation uniform, allowed the improvement of long read of 39-dNTP and the lack of any fluorescent substrate for the sequencing (5). However, cycle sequencing has the drawback chain termination reaction. of a long reaction time (2- to 3-hr reaction) because of its In this paper, we describe a completely new RNAP-based requirement of temperature cycling. sequencing method named, ‘‘transcriptional sequencing.’’ For At present, two independent methods for preparing DNA accurate, long-read sequencing, we developed the four-color templates are available. One is DNA cloning using a plasmid dye-39-dNTPs (dye terminators), which carry a long carbon vector and the other is PCR. Although PCR has limitations on spacer (n 5 4), connecting nucleotides and fluorescent (rho- the size or sequence to be amplified, it is very convenient to damine) dyes, and mutated T7 RNAPs to improve the uni- prepare template(s) directly from plasmids in Escherichia coli formity of the incorporation rate of 39-dNTP. We also purified and cells from tissues without cloning and library construction the RNase-free yeast pyrophosphatase (PPase) to inhibit (6). From this aspect, PCR allows automation of template pyrophospholysis that leads to degradation of specific 39- preparation because it can amplify very rapidly DNA frag- dNTP-terminated fragments on transcriptional sequencing, ment(s) from a large number of samples. However, in the Abbreviations: 39-dNTP, 39-deoxynucleoside triphosphate; RNAP, 9 9 The publication costs of this article were defrayed in part by page charge RNA Polymerase; dd, 2 ,3 -dideoxy; wt, wild type; pyrophosphatase, PPase; TMR, 6-carboxytetramethylrhodamine; ROX, 6-carboxy-X- payment. This article must therefore be hereby marked ‘‘advertisement’’ in rhodamine; R6G, 5-carboxyrhodamine-6G; R110, 5-carboxyrhodam- accordance with 18 U.S.C. §1734 solely to indicate this fact. ine-110. © 1998 by The National Academy of Sciences 0027-8424y98y953455-6$2.00y0 §To whom reprint requests should be addressed. e-mail: yosihide@ PNAS is available online at http:yywww.pnas.org. rtc.riken.go.jp. 3455 Downloaded by guest on September 24, 2021 3456 Biochemistry: Sasaki et al. Proc. Natl. Acad. Sci. USA 95 (1998) FIG. 2. Effects of linker length and mutated T7 RNAP in the termination pattern with TMR-39-dUTP. Sequencing reactions were performed with human thyroid-stimulating hormone b-subunit unpu- FIG. 1. Structures of four rhodamine-labeled 39-deoxynucleoside rified PCR products for the template. Wild-type or mutated F644Y T7 59-triphosphates. Cn indicates the number of CH2 in the linker arm RNAP for enzyme, C1 or C4 linker arm for dye-39dNTP were tested, between rhodamine dye and base of 39dNTP. respectively. PPase was added to all reactions. All peaks are shown on the same scale. (a) Combination of C1 linker and wt T7 RNAP. (b) resulting in improvement of peak uniformity. This method Combination of C1 linker and mutant T7 RNAP. (c) Combination of made possible a rapid isothermal sequencing reaction in ,30 C4 linker and wt T7 RNAP. (d) Combination of C4 linker and mutant min, based on the high processivity of RNAP and complete T7 RNAP. Arrows indicate false peaks. prevention of false stops by the newly designed dye-39-dNTP. 8 amplification, 100 ng of genomic DNA prepared from human PCR direct sequencing using RNAP can overcome the tedious steps of removing primers and 29-dNTP, thus allowing in great colon carcinoma line HT29 (American Type Culture Collec- tion) or human placenta, 0.1 M T7 promoter appended primer reduction of time and labor to enable preparation of much 9 larger numbers of sequencing samples. (5 -GTAATACGACTCACTATAGGGCACCTACCTGGA- GCTGGAGC-39), and G2 primer (59-CCAAGACTTAGTA- CCTGAAG-39) were used (17). The first cycle was at 96°C for MATERIALS AND METHODS 1 min, 55°C for 30 sec, and 72°C for 1 min. This was followed Synthesis of Rhodamine Dye Attached to 3*dNTP. The by 24 cycles of 96°C for 30 sec, 55°C for 30 sec, and 72°C for N-hydroxysuccinimidyl ester of 6-carboxytetramethylrhodam- 1 min. ine (TMR), 6-carboxy-X-rhodamine (ROX), 5-carboxyrho- Transcriptional Sequencing. Direct sequencing reactions m damine-6G (R6G), and 5-carboxyrhodamine-110 (R110) were were carried out in 10 l containing 10 ng of unpurified PCR z purchased from Molecular Probes. Rhodamine-labeled products, 40 mM Tris HCl (pH8.0), 8 mM MgCl2,5mMDTT, m m 39dNTPs were synthesized according to the method reported 2 mM spermidine-(HCl)3, 500 M GTP and UTP, 250 M m 9 m 9 by Prober et al. (13) with minor modification. ATP and CTP, 0.1 M R6G-3 -dATP, 0.1 M R110–3 - m 9 m 9 Construction and Enzyme Purification of Mutant T7 RNA dGTP, 1 M TMR-3 -dUTP, 5 M ROX-3 -dCTP, 10 units of Polymerases (RNAP). Mutant polymerase genes were con- yeast PPase, and 25 units of wt T7 RNAP or mutant T7 RNAP m structed by PCR-mediated site direct mutagenesis (14). Mu- F644Y. To use 7-deaza-GTP, 500 M 7-deaza-GTP was tant enzyme expression and large-scale purification have been substituted for GTP and 1 mM GMP for the initiator. These described (15). reaction mixtures were incubated at 37°C for 30 min. Next, the Enzyme Purification of Yeast Pyrophosphatase. Baker’s unincorporated dye terminators were separated from the yeast inorganic pyrophosphatase (Sigma) was further purified sequencing products with Sephadex G25 (Pharmacia), dried, by liquid chromatography by using SP-Sepharose and Q- and suspended with 4 ml of formamide dye (95% formamide, Sepharose (Pharmacia).