DNA RESEARCH 2, 225-227 (1995)
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`A Non-radioactive DNA Sequencing Method Using Biotinylated
`Dideoxynucleoside Triphosphates and ATth DNA Polymerase
`
`Katsunori IKEDA, Hiroaki INOUE, Masanori OKA, Bunsei KAWAKAMI,* and Yoshihisa KAWAMURA
`Tsuruga Institute of Biotechnology, Toyobo Co., Ltd. Tsuruga, Fukui 914, Japan
`
`(Received 3 October 1995; revised 25 October 1995)
`Abstract
`We synthesized a set of four biotinylated dideoxynucleoside triphosphates (biotin-9-ddNTPs) and
`optimized the reaction conditions for non-radioactive cycle sequencing using modified Tth DNA poly-
`merase (ATth) and a chemiluminescent detection system. The resulting sequencing ladders showed lower
`background compared to those with the conventional non-radioactive sequencing method which uses 5'-
`biotinylated primers, especially when PCR products were analysed. With our method, DNA sequences
`can be determined at any primer positions without preparing 5'-biotinylated primers for dideoxy chain-
`termination.
`Key words: non-radioactive DNA sequencing; cycle DNA sequencing; thermostable DNA polymerase;
`biotinylated dideoxynucleoside triphosphate
`
`1.
`
`Introduction
`
`Dideoxy DNA sequencing has become a widely
`used method for DNA sequencing, and non-radioactive
`dideoxy sequencing methods using biotinylated primers
`and the chemiluminescent detection system has been
`developed.1'2 However, one limitation of the previous
`non-radioactive method is that one has to prepare 5'-
`biotinylated primers for each sequencing reaction. It
`would seem to be more convenient if a DNA sequenc-
`ing method using biotin-dideoxynucleoside triphosphates
`can be developed.
`We have previously cloned a thermostable DNA poly-
`merase gene from Thermus thermophilus HB83'4 and iso-
`lated a deletion mutant polymerase (ATth) lacking the
`5'-3'exonuclease activity (to be published). This enzyme
`works well with the dideoxy DNA sequencing method,
`and has enough thermostability to perform cycle DNA
`sequencing. In this report, we synthesized a set of four
`biotin-9-ddNTPs and established the sequencing condi-
`tions which use four biotin-9-ddNTPs, ATth DNA poly-
`merase and the chemiluminescent detection system.
`
`2. Materials and Methods
`
`2.1. Enzymes and chemicals
`ATth DNA polymerase, M13mpl8 single-stranded
`DNA (M13mpl8 ssDNA), 2'-deoxy-, and 2',3'-dideoxy-
`nucleoside 5'-triphosphates (dNTPs and ddNTPs) used
`
`Communicated by Mituru Takanami
`* To whom correspondence should be addressed. Tel. +81-770-
`22-7643, Fax. +81-770-22-7671
`
`(Osaka, Japan).
`were the products of Toyobo Co.
`Oligonucleotide primers for DNA sequencing and PCR
`were synthesized on a DNA synthesizer (Model 392,
`Applied Biosystems, USA). 7-Deaza-dGTP, dITP, and
`dUTP were obtained from Boehringer Mannheim (Ger-
`many). Other chemicals were of reagent grade obtained
`from commercial sources.
`
`2.2. PCR amplification of DNA gyrase gene fgyrA)
`A single colony of Escherichia coli HB101 harboring
`pGYRA was suspended in 10 ill of H2O, heat denatured
`at 95°C for 5 min and centrifuged at 12,000 rpm for
`3 min. The supernatant was added to a reaction mix-
`ture (100 fjl) containing 20 pmol of each primer (5'-
`CGCCAGGGTTTTCCCAGTCACGAC-3', 5'-CGGA-
`TAACAATTTCACACAGGAAAC-3'), 200 /xM each
`dNTPs, 2.5 units ATth DNA polymerase, 50 mM KC1,
`10 mM Tris-HCl (pH 8.3) and 1.5 mM MgCl2. After
`an initial denaturaion at 94°C for 5 min, the reaction
`mixture was incubated for 30 cycles of 30 sec at 94°C, 1
`min at 60°C and 2 min at 72°C using the Thermal Cy-
`cler Model 480 (Perkin-Elmer, USA). Amplified products
`were purified and concentrated to 20 //I by a Centricon-
`100 (Amicon, USA) at 500 rpm for 30 min. Five mi-
`croliters of concentrated DNA solution (approximately
`250 fmol) was used for the sequencing reaction. Plasmid
`pGYRA containing the DNA gyrase gene of Neisseria
`gonorrhoeae was donated by Dr. Deguchi.
`
`Illumina Ex. 1123
`IPR Petition - USP 10,435,742
`
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`226
`
`Non-radioactive DNA Sequencing Method
`
`[Vol. 2,
`
`2.3. Sequencing reaction using biotin-9-ddNTPs
`Unless otherwise noted, 250 fmol of M13mpl8 ss-
`DNA or PCR product was added
`to
`the reac-
`tion mixture (17 (A) containing 5 pmol of primer
`(5'-CGCCAGGGTTTTCCCAGTCACGAC-3'), 50 mM
`Tris-HCl (pH 8.8), 1.5-4 mM MgCl2, 10 /xM each set of
`four dNTPs (7-deaza-dGTP, dATP, dTTP and dCTP)
`and 4 units ATth DNA polymerase. Aliquots (4 /il) were
`added to 4 tubes each containing 2 /xl of either 60 /xM
`biotin-9-ddGTP, 90 /xM biotin-9-ddATP, 900 /xM biotin-
`9-ddUTP or 600 /xM biotin-9-ddCTP. The mixture was
`incubated for 30 cycles of 30 sec at 95°C, 30 sec at 60°C
`and 2 min at 72°C using the Thermal Cycler Model 480
`(Perkin-Elmer, USA). The reaction was terminated by 4
`(il of stop solution (deionized formamide containing 0.1%
`bromophenol blue, 0.1% xylene cyanol and 20 mM dis-
`odium EDTA, pH 7.0).
`
`2.4- Sequencing reaction using 5'-biotinylated primers
`The reaction was carried out under conditions sim-
`ilar to those used for biotin-9-ddNTPs. 250 fmol of
`M13mpl8 ssDNA or PCR product was added to the
`reaction mixture (17 /xl) containing 5 pmol primer (5'-
`biotin-CGCCAGGGTTTTCCCAGTCACG AC-3'), 50
`mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 10 /xM of each
`dNTP (7-deaza-dGTP, dATP, dTTP and dCTP) and 4
`units ATth DNA polymerase. Aliquots (4 /xl) were added
`to 4 tubes each containing 2 /xl of 60 /xM ddGTP, 90 /iM
`ddATP, 900 fiM ddTTP or 600 /zM ddCTP. Then the
`mixture was incubated as in the above section.
`
`2.5. Other methods
`The sequencing reaction products (2 /xl each) were elec-
`trophoresed at 30 W for 2 hr on 8% polyacrylamide gel,
`and blotted onto a dryed, positive-charged nylon mem-
`brane (Imaging High, Toyobo) according to the manufac-
`turer's instructions. The chemiluminescent detection re-
`action was performed using a horizontal rolling apparatus
`(Rolling Mixer, Toyobo) at room temperature according
`to the manufacturer's instructions.
`
`3. Results and Discussion
`
`3.1. Synthesis of biotin-9-ddNTPs
`Biotin-9-ddNTP was synthesized by a previously re-
`ported method using fluorescence-tagged ddNTP5 with
`some modification. 5-Halogeno-2',3'-dideoxypurine or
`-pyrimidine was coupled to TV-trifluoroacetyl propargy-
`lamine under catalyst in dimethylformamide. The result-
`ing nucleoside was converted to the 5'-triphosphate, and
`coupled with biotinyl-iV-hydroxy succinimide and depro-
`tected to generate the corresponding biotin-9-ddNTP.
`The structure of one of the biotin-9-ddNTPs (biotin-9-
`ddGTP) is shown in Fig. 1. ATth DNA polymerase
`
`O
`
`4(H2C)
`
`C—HN
`
`OO
`O
`II
`II
`II
`HO— P—O— P—O— P—O
`I
`I
`I
`OH
`OH
`OH
`
`NHj
`
`Figure 1. Chemical structure of biotin-9-ddGTP.
`
`could incorporate biotin-9-ddNTPs as well as ddNTPs,
`but there were some differences in the incorporation rate
`among the four biotin-9-ddNTPs, as in the following sec-
`tion. Recently, we learned that biotin-11-ddNTPs were
`listed in the latest catalogue of NEN Research (USA).
`
`3.2. Optimization of sequencing reaction conditions
`When the sequencing reaction was performed by using
`biotin-9-ddNTPs under conditions similar to those used
`for the 5'-biotinylated primers, the band intensities in
`lanes T and C were weaker compared with those of lanes
`A and G. As this is likely caused by differences in the
`activity of ATth DNA polymerase incorporating biotin-
`9-ddNTPs, we investigated the effect of the reaction con-
`ditions on activity. As shown in Fig. 2, the optimum
`MgCl2 concentrations were different for the four biotin-
`9-ddNTPs, and the weak band intensities in lanes C and
`T could be improved by increasing the MgCl2 concentra-
`tion.
`Another problem found in the biotin-9-ddNTP se-
`quencing was that the band intensities became relatively
`weak in the stretch of T or G, but this was essentially
`solved by replacing 7-deaza-dGTP and dTTP with dITP
`and dUTP, respectively. Although the mechanism in-
`volved in the difference of band intensities is not known,
`it is apparent that the activity of ATth DNA polymerase
`incorporating biotin-9-ddNTPs is influenced by the sur-
`rounding DNA sequences. In Fig. 3A, the sequence pat-
`terns obtained when the MgCl2 concentration for the C
`and T reactions was increased 3 and 4 mM, respectively,
`and that 7-deaza-dGTP and dTTP were replaced with
`dITP and dUTP are shown (right four lanes), in com-
`parison with those obtained by the biotin-primer method
`(left four lanes).
`To test whether the biotin-9-ddNTP method can be
`applicable to PCR products, a part of the gyrA gene was
`amplified and used as a template. The resulting sequence
`patterns are shown in Fig. 3B (right four lanes) in com-
`parison with the patterns produced by the biotin-primer
`method (left four lanes). Essentially identical patterns
`were detected by both methods, but it was noted that
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`No. 5]
`
`K. Ikeda et al.
`
`227
`
`4mM
`3mM
`2mM
`1mM
`GATC GATC GATC GATC
`
`(A)
`
`(B)
`
`GATC GATC GATC GATC
`
`1OObp-
`
`m.
`
`ae
`
`m m#
`
`»
`
`100bp-
`
`m
`
`-
`
`Figure 2. Effect «£ MgCi^ tancen'l-raft.iiOIlS tm -sequencing 'ladders
`by the biotin-terminator method. Sequencing was carried out
`as in Materials and Methods except for the MgCb concentra-
`tions.
`
`the patterns produced by the biotin-terminator method
`gave lower background levels.
`In summary, the biotin-terminator sequencing method
`that we established by synthesis of four biotin-9-
`ddNTPs offers two advantages over the conventional non-
`radioactive sequencing method which uses 5'-biotinylated
`primers: (I) DNA sequences can be determined from any
`primer site without preparing 5'-biotinylated primers,
`and (II) the resulting sequence ladders have lower back-
`ground levels, so that PCR products can be used as tem-
`plates.
`
`References
`
`1. Beck, S., O'Keeffe, T., Coull, J. M. et al. 1989, Chemi-
`luminescent detection of DNA: application for DNA se-
`
`Figure 3. Comparison of sequencing ladders between the bi-
`otin-primer method and biotin-terminator methods. M13mpl8
`ssDNA(A) and PCR products of the gyrA gene(B) were used
`as templates.
`
`quencing and hybridization, Nucleic Acid Res., 17, 5115-
`5123.
`2. Tizard, R., Cate, R. L., Ramachandran, K. L. et al. 1990,
`Imaging of DNA sequence with chemiluminescence, Proc.
`Natl. Acad. Sci. USA, 87, 4514-4518.
`3. Oshima, T. and Imahori, K. 1974, Description of Ther-
`mus thermophilus comb, nov., a nonsporulating ther-
`mophilic bacterium from a Japanese thermal spa, Int.
`J. Syst. BacterioL, 24, 102-112.
`4. Asakura, K., Komatsubara, H., Soga, S. et al. 1993,
`Cloning, nucleotide sequence, and expression in Es-
`cherichia co/i of DNA polymerase gene (polA) from Ther-
`mus thermophilus HB8 Ferment. Bioeng., 76, 265-269.
`5. Prober, J. M., TVainor, G. L., Dam, R. J. et al. 1987, A
`system for rapid DNA sequencing with fluorescent chain-
`terminating dideoxynucleotides, Science, 238, 336-341.
`
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