Novel Cyanine Dye-Labeled Dideoxynucleoside Triphosphates for

Jun 26, 2002 - Single color cyanine dye-labeled (Cy 5.0 and Cy 5.5) dideoxynucleoside-5'-triphosphates, or 'terminators', containing different spacer ...
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JULY/AUGUST 2002 Volume 13, Number 4 © Copyright 2002 by the American Chemical Society

ARTICLES Novel Cyanine Dye-Labeled Dideoxynucleoside Triphosphates for DNA Sequencing R. Scott Duthie, Inta M. Kalve,† Sui Bi Samols,‡ Scott Hamilton, Inna Livshin,‡ Mahesh Khot, Satyam Nampalli, Shiv Kumar,* and Carl W. Fuller* Amersham Biosciences, 800 Centennial Avenue, Piscataway, New Jersey 08855. Received June 27, 2001; Revised Manuscript Received March 24, 2002

Single color cyanine dye-labeled (Cy 5.0 and Cy 5.5) dideoxynucleoside-5′-triphosphates, or ‘terminators’, containing different spacer lengths were synthesized and evaluated for efficacy in DNA sequencing methods using a modified thermally stable DNA polymerase. The single color cyanine dye terminators were formulated into two separate sets of sequencing mixes, one for Cy 5.0 and the other for Cy 5.5, and evaluated on different automated sequencing platforms. Each set of mixes included two pyrimidine terminators with 17-atom linkers and two purine terminators with 10-atom linkers between the dye and the nucleotide. The two sets of cyanine dye-labeled terminators chosen for this cycle sequencing study produced improved band patterns with band uniformity similar to that obtained with dyeprimer sequencing methods.

INTRODUCTION

Fluorescent sequencing was first reported using dyelabeled primers (1), but methods using dye-labeled dideoxynucleoside triphosphates (ddNTPs; terminators) (2, 5) have also gained acceptance in recent years. The advantage of using dye-labeled ddNTPs instead of labeled primers is that only properly terminated fragments are labeled (2). Strong stops associated with polymerase pausing or falling off the template without proper termination of the nascent DNA strand is not seen. Use of dye-labeled ddNTPs in automated sequencing has helped * To whom correspondence should be addressed. Fax: (732) 457-8353, E-mail: [email protected] or [email protected]. † Present address: Pel-Freez Clinical Systems, LLC, 9099 N. Deerbrook Trail, Brown Deer, WI 53223. ‡ Present address: USB Corporation, 26111 Miles Rd., Cleveland, OH 44128.

reduce the need to resequence problematic areas where polymerase stops are prevalent. Additionally, synthesis of labeled, specific primers is not required for the sequencing reactions. One major weakness of using dyelabeled terminators is the unevenness of the resulting sequencing peak heights or areas with cycle sequencing methods (4-6). Most of the fluorescent dyes used for dye-terminator DNA sequencing have been applied to the four-color instruments of Applied Biosystems and E. I. Du Pont (2, 5). These instruments have 488 nm lasers, so the dyes were all chosen for excitation at this wavelength. Most of these dyes have been either fluorescein or rhodamine derivatives. Since the instruments must distinguish four colors of fluorescence at once, there have even been attempts to use fluorescence energy transfer to obtain efficient four-color sets of dyes that can all be excited at 488 nm (7-9). These approaches have required the use

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of complex, multiccolor optics for fluorescence detection. The use of fluorescein and rhodamine dyes has also been limited by their extinction coefficients (approximately 80000 M-1 cm-1). Furthermore, it is desirable to use longer wavelength light for excitation because background fluorescence is considerably lower with 635 or 675 nm excitation than it is with 488 nm excitation (12-14). We have instead focused on the use of simpler, singlecolor instruments, such as ALFexpress and SEQ4X4 (Amersham Biosciences, Piscataway, NJ) and Clipper and Long-Read Tower (Visible Genetics, Toronto, Canada). These instruments use inexpensive 635 and 675 nm lasers, which are ideally suitable for excitation of the cyanine dyessCy 5.0 and Cy 5.5 (12). While these instruments require 2-4 lanes for determination of a single DNA sequence, they are much simpler and less expensive than the four-color instruments that need only a single lane per sequence. Additionally, the cyanine dyes have significantly higher extinction coefficients (250000 M-1 cm-1) than the fluorescein and rhodamine dyes, so they act as more efficient collectors of the excitation light. To produce fully functional sets of cyanine-labeled dideoxynucleotides, a number of criteria had to be met. Preliminary experiments indicated that direct coupling of the cyanine dye NHS esters with simple propargylamino-modified pyrimidine nucleotides resulted in dyelabeled nucleotides that were relatively poor substrates for DNA polymerases. In addition, dyes with different charges resulted in labeled fragments that had widely differing mobilities upon electrophoresis through gels. After considerable experimentation with many combinations of dyes, linkers and nucleotides (10, 11), we have come up with two optimal sets of novel labeled dideoxynucleotides for DNA sequencing on single-color instruments (vide supra). Herein, we describe these two sets of dye-labeled terminators and show their performance with Thermo Sequenase DNA polymerase (15, 16) as monitored using the single-color sequencing instruments. These sequences have remarkably even band intensities for dye-terminator sequencing methods (18). In fact, they rival better dye-primer sequences for high data quality. MATERIALS AND METHODS

The following reagents were obtained from Amersham Biosciences: Thermo Sequenase DNA polymerase, M13mp18(+) strand DNA, four-color dye terminators, HLA DRBplus Typing Kit, GFX PCR DNA and Gel Band Purification Kit. Genomic DNA from immortalized B-lymphocyte cell lines AMAI and WT51 was obtained from ProtoProbe, Inc., Milwaukee, WI. Synthesis of Dye-Labeled ddNTPs. The propargylamino dideoxy nucleotides 1-6 (Scheme 1), required for synthesis of Cy 5.5 and Cy 5.0 conjugates 7-14 (Scheme 2), were prepared following the procedure described by Hobbs and Cocuzza (3). Cy 5.5- and Cy 5.0-NHS esters used in these conjugation reactions were purchased from Amersham Biosciences (Piscataway, NJ). Preceding the name of each ddNTP are numbers which denote how many atoms are between the dye moiety and the attachment site on the base, either the C5 position of pyrimidines or the C5 position of pyrrolopyrimidines. Yields of the dye-nucleotide conjugates were determined by UV-vis spectrophotometer readings in TE buffer, pH 7.5, using a λmax of 648 nm for Cy 5.0 conjugates ( ) 250000 M-1 cm-1) or 675 nm for Cy 5.5 conjugates ( ) 250000

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M-1 cm-1). The formula for this determination was the following: Concentration ) (absorbance at either λ648 for Cy 5.0 or λ675 for Cy 5.5 ÷ extinction coefficient of 250000)(dilution factor). Micro Mass LCT was used to analyze the dye-nucleotide terminators. Synthesis of 11-ddUTP (3) 11-ddCTP (4). A 50 mg (63 µmol) amount of 5-propargylamino-ddUTP (4-ddUTP, 1) (10) or 5-propargylamino-ddCTP (4-ddCTP, 2) in 3 mL of 0.1 M Na2CO3-NaHCO3 (pH 8.5) was stirred individually at room temperature with 2.5 equiv of N-TFAaminocaproic acid-NHS ester (51 mg; 160 µmol) in 1 mL of anhydrous DMF. After 1 h of stirring, the solutions were evaporated under reduced pressure. The resulting residues were treated with 5 mL of 30% aqueous NH4OH for 2 h at RT to remove the TFA group and then dried as before. The products were purified by HPLC using a Delta Pak (Waters) C18 column (5 cm × 30 cm, 15 µm, 100 Å) eluted with a gradient of 0.1 M TEAB (buffer A, pH 7.0) and 3:1 0.1 M TEAB:CH3CN (buffer B, pH 7.0) in 30 min with a flow rate of 130 mL/min. Identical fractions were pooled and evaporated under reduced pressure to obtain 11-ddUTP, 3 (40 mg, 70%), and 11-ddCTP, 4 (30 mg, 52%). Conjugation of 11-ddUTP (3) with Cy 5.5-NHS ester: Formation of Cy 5.5-17-ddUTP (7, Scheme 2). To a magnetically stirred solution of 11-ddUTP (12 µmol) in 0.1 M Na2CO3-NaHCO3 (1 mL, pH 8.5) at room temperature was added 1 mL of an anhydrous DMF solution of Cy 5.5-NHS ester (20 mg, 18 µmol, 1.6 equiv). After 1 h, the reaction mixture was evaporated under reduced pressure and the product purified by silica gel column (32 cm × 2 cm) chromatography. Excess dye and unwanted byproducts were eluted with 1:1 MeOH/CHCl3 (3 × 30 mL) followed by neat MeOH (2 × 30 mL). The desired Cy 5.5-17-ddUTP (7) was then eluted with 6:3:1 iso-PrOH:NH4OH:H2O (4 × 40 mL). Identical fractions were pooled and evaporated, and the resulting residue was further purified by HPLC using a Delta Pak (Waters) C18 column (19 mm × 30 cm, 15 µm, 100 Å) using a gradient of 0.1 M TEAB (buffer A, pH 7.0) and 1:1 0.1 M TEAB:CH3CN (buffer B, pH 7.0) in 30 min at a flow rate of 20 mL/min. The HPLC retention time for this compound was 19 min. Identical fractions were pooled and evaporated under reduced pressure to obtain 4.36 µmol (39%) of Cy 5.5-17-ddUTP (7). TOF MS ES-, m/z in water, MS 1516.25, observed M-1 1515.24. Synthesis of Cyanine Dye-Nucleotide Conjugates (Scheme 2). Following the same methods used for Cy 5.5-17-ddUTP, syntheses of Cy 5.5-17-ddCTP (8), Cy 5.5-10-ddATP (9), and Cy 5.5-10-ddGTP (10) were accomplished with yields ranging from 30 to 45%. HPLC retention times for these compounds were 19, 22, and 23 min, respectively. 8: TOF MS ES-, m/z in water, MS 1515.26, observed M-1 1514.25; 9: TOF MS ES-, m/z in water, MS 1425.19, observed M-1 1424.19; 10: TOF MS ES-, m/z in water, MS 1441.19, observed M-1 1440.18. Nucleotide derivatives 11-ddUTP, 11-ddCTP, 4-ddATP, and 4-ddGTP were also conjugated with Cy 5-NHS ester to synthesize their conjugates, Cy 5.0-17-ddUTP (11), Cy 5.0-17-ddCTP (12), Cy 5.0-10-ddATP (13), and Cy 5.0-10-ddGTP (14). Yields from these reactions were in the range of 30-50%. HPLC retention times for these compounds were 20, 7, 9, and 10 min, respectively. 11: TOF MS ES-, m/z in water, MS 1256.30, observed M-1 1255.29; 12: TOF MS ES-, m/z in water, MS 1255.32, observed M-1 1254.31; 13: TOF MS ES-, m/z in water,

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Scheme 1. Synthesis of 5-Propargylamidoaminocaproic-ddUTP (11-ddUTP) and 5-PropargylamidoaminocaproicddCTP (11-ddCTP)

MS 1165.25, observed M-1 1164.24; 14: TOF MS ES-, m/z in water, MS 1181.24, observed M-1 1180.24. Polymerase Chain Reactions. Typical reaction volumes were 25-50 µL containing 15 mM Tris-HCl, pH, 8.3, 200 µM each dATP, dCTP, dGTP, and dTTP (Amersham Biosciences), 1.5-2.5 units of Taq DNA polymerase (Amersham Biosciences), 1-200 ng of template, and 1-10 pmol of primers. Typical thermal cycling conditions consisted of a 1-2 min initial heat denaturation step at 95 °C and then 25-30 cycles, each consisting of the following steps: 95 °C for 30 s, 45- 60 °C for 30 s, 72 °C for 120 s. Reactions were held at 4 °C once thermal cycling had been completed. PCR products were purified using GFX PCR DNA and Gel Band Purification Kit. Sequencing Reactions. The basic procedure involved mixing 10-20 units of Thermo Sequenase DNA polymerase, 1-10 pmol of primer, 50-500 ng of template, deoxynucleoside triphosphates (dNTPs; Amersham Biosciences), and a specific dye-labeled ddNTP in a total volume of 6-8 µL. Four separate reactions were required to sequence a template, each reaction containing a different dye-labeled ddNTP. Reactions were subjected to a thermal cycling protocol followed by postreaction workup of the cyanine dye terminator-labeled Sanger fragments by either ethanol precipitation or column

chromatography. Purified products were then analyzed on an automated DNA sequencing instrument. A typical Cy 5.0 dye-labeled terminator sequencing reaction contained the following mixture: 14.6 mM Tris-HCl, pH 9.5; 3.4 mM MgCl2; 150 µM each of dATP, dCTP, dGTP, and dTTP; and either 0.2 µM Cy 5.0-10-ddATP, 0.16 µM Cy 5.0-17-ddCTP, 0.4 µM Cy 5.0-10 ddGTP, or 0.325 µM Cy 5.0-17-ddUTP. A typical DNA sequencing reaction using Cy 5.5 dyelabeled terminators contained the following mixture: 14.6 mM Tris-HCl, pH 9.5; 3.4 mM MgCl2; 75 µM each of dATP, dCTP, dGTP, and dTTP; and either 1.5 µM Cy 5.5-10-ddATP, 1.5 µM Cy 5.5-17-ddCTP, 1.5 µM Cy 5.5-10-ddGTP, or 1.5 µM Cy 5.5-17-ddUTP. Typical thermal cycling conditions consisted of a 1-2 min initial heat denaturation step at 95 °C, and then 25-30 cycles each consisting of 95 °C for 30 s, 45-60 °C for 30 s, and 72 °C for 120 s. Reactions were held at 4 °C until purification and analysis. Reaction products were purified by precipitation with the addition of 2 µL of 7.5 M ammonium acetate and 30 µL of chilled absolute ethanol. After being mixed and incubated on ice for 20 min, DNA was pelleted by centrifugation at room temperature for 15 min using a standard lab bench microcentrifuge set on high speed (10000-16000g). Supernatants were carefully removed

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Scheme 2. Synthesis of Cy-Dye Conjugates

by aspiration, and each DNA pellet was rinsed with 200 µL of ice-cold 70% ethanol. The tubes were recentrifuged for 5 min, and supernatants were again removed. DNA pellets were stripped of liquid under vacuum at room temperature for about 2 min without taking them to complete dryness. Each tube had 6-8 µL of formamide loading dye (95% formamide, 50 mM EDTA, pH 8.0, 0.2 mg/mL new fuchsin) added and was then mixed, using a vortex mixer fitted with a tube holder, for 10-15 min at room temperature. Resuspended DNA was denatured at 70-72 °C for 2.5-3 min, and the tubes were immediately quenched on ice. Aliquots, 1.5-8 µL, were then loaded in wells of sequencing gels for analysis. Alternatively, sequencing reaction products were purified using AutoSeq G-50 columns (Amersham Biosciences). In this alternative procedure, each completed reaction mixture was diluted with distilled water to a volume of 15-25 µL and the entire sample loaded on a prespun column. The separate column effluents were stripped of liquid using a speed-vac, treated, and loaded on sequencing gels for analysis (vide supra).

Primers. Various primers were employed during the course of this study for both PCR and cycle sequencing reactions. Their sequences and how they are defined in the body of the text are as follows: M13-40 primer, 5′-d[CGC CAG GGT TTT CCC AGT CAC GAC]-3′. Reverse sequence primer (RSP), 5′-d[CAG GAA ACA GCT ATG AC]-3′. M13 Universal primer, 5′-d[GTA AAA CGA CGG CCA GT]-3′. Universal cycle primer, 5′-d[GTT TTC CCA GTC ACG ACG TTG TA]-3′. Sequencing Instruments. The following DNA sequencing instruments were used in this study: ALFexpress DNA Analysis System (Cy 5.0 dye; Amersham Biosciences, Piscataway, NJ), SEQ4X4 Personal Sequencing System (Cy 5.5 dye; Amersham Biosciences, Piscataway, NJ), and both Clipper and Long-Read Tower System (Visible Genetics, Inc., Toronto, Ontario, Canada; both Cy 5.0 and Cy 5.5 dyes) (17).

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Figure 1. Visual representation of peak height variation using different dye terminators and DNA polymerases.

Figure 2. A 600-base PCR product generated by amplification of a pUC18 clone containing a 385-base insert of AT rich Tetrahymena thermophilia genomic DNA. The product was gel-purified and 300 ng of the resulting material sequenced using Thermo Sequenase DNA polymerase and Cy 5.0 dye terminators with detection on the ALFexpress DNA Analysis System. RESULTS AND DISCUSSION

Thermo Sequenase DNA polymerase (Amersham Biosciences) contains a single amino acid modification which

increases the rate of ddNTP incorporation by more than 1000-fold (15). When used in cycle sequencing reactions with unmodified ddNTPs, DNA polymerases containing this amino acid modification produce band patterns with

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Figure 3. Sequence of pUC18 containing about an 800-base insert of p53 DNA using Thermo Sequenase DNA polymerase and Cy 5.0 dye terminators with detection on ALFexpress DNA Analysis System.

Figure 4. Heterozygote determination using Cy 5.0 dye terminators and Thermo Sequenase DNA polymerase with detection on ALFexpress DNA Analysis System.

very uniform peak intensities. In contrast, when performing similar reactions with dye-labeled ddNTPs, the same DNA polymerases generate peak heights that can fluctuate over approximately a 20-fold range in intensity (18).

A comparative peak height variation study (5) was performed between the Cy 5.5 terminators and other fluorophore reporting systems used in DNA sequencing. The peak height variation was standardized using bases between 50 and 350 of M13mp18(+) DNA sequenced with

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Figure 5. PCR products of M13mp18(+) strand sequenced with the indicated primers. Note that the first base of each product is readable using SEQ4X4 Personal Sequencing System.

Figure 6. Last base determination of a PCR product of human β-globin DNA using Cy 5.5 dye terminators with SEQ4X4 Personal Sequencing System.

the M13-40 primer. Sequence data within this region was examined prior to peak variation analysis to ensure that the sequence was correct. Briefly, peak height data were fit to a second-degree polynomial equation comparing height to scan plot position. This corrected for the normal systematic variation in intensities found in DNA sequences. The data were then normalized by dividing the peak height at a given scan position by the polynomial estimate at that same position. The values reported are the variance of the normalized peak heights. Thus, smaller values indicate better, more uniform band intensities. Figure 1 represents some examples showing peak height variation obtained with different terminators and different DNA polymerases. Sequencing reactions using dye-primers and T7 DNA polymerase had the best peak height variation with a value of 0.07. Peak height variation for the Cy 5.5 terminators was found to be considerably better than the value (approximately 0.3) found for the four-color dye terminators currently used in the majority of high throughput sequencing facilities (8, 9). We investigated the ability of four new dye terminators to resolve stretches of either GC- or AT-rich base pairs. The Cy 5.0 dye-terminator protocol was tested with two plasmid constructs, one highly AT-rich that was used as

a template to generate a 600-base pair PCR product for sequencing, and the other GC-rich where the plasmid was itself sequenced. Figure 2 presents sequence data from a 600-base pair PCR product containing Tetrahymena thermophilia DNA cloned into pUC18. A PCR product was first generated with this template using both the M13-40 primer and RSP. The purified material (∼300 ng) was sequenced using the M13 universal primer. Between bases (Figure 2) 110 and 298 the number of AT base pairs compared with the total number of base pairs is 91%, with the entire fragment comprising 76% AT. Of the 406 bases of sequence data (Figure 2), only one ambiguity at position 83 was observed to be caused by the base-calling software, and that may be resolved with manual intervention as a ‘G’. Also notice that the base-calling software has added an extra base at position 79 (n). Figure 3 presents sequence data from an 807-base insert in pUC18 of a p53 cDNA clone covering part of exon 4, all of exons 5 through 9, and part of exon 10. Between base 163 and 285 in Figure 3 is an area that is 71% GC-rich, with an overall GC content of the presented sequence at 58%. The first ambiguity was identified at base 718 and could not be resolved by manual intervention because of gel resolution.

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Another important characteristic of these dye-terminator systems is unambiguous and reliable detection of heterozygotes. PCR products of separate amplifications of AMAI and WT51 genomic DNA were performed according to the manufacturer’s instructions found in the HLA DRBplus Typing Kit. These products were purified with GFX PCR DNA and Gel Band Purification Kit and the concentration determined by spectrophotometer measurement. A mix of two of the PCR products was then sequenced using Cy 5.0 terminators. Figure 4 demonstrates heterozygote determination when 120 ng of WT51 and 80 ng of AMAI PCR products were mixed and sequenced. The ‘Y’ at position 179 in Figure 4 represents either a ‘C’ or ‘T’. With this mixture of genomic DNA templates both a ‘C’ and a ‘T’ would be expected at this position as observed. We also investigated the ability of our sequencing system to elucidate either the first base following the primer or the last base of an amplicon. For the first base determination 50 ng of M13mp18(+) strand DNA was amplified and then sequenced using three different M13 primers: -40, Universal Cycle, or Universal. Figure 5 demonstrates that the first base following the primer is visible in each reaction, even when the primer was immediately followed by a multiplet of the same base. Last base determination was accomplished using a PCR product of the human beta globin locus purified by Exo/ SAP treatment. All sequence from the 128 base pair fragment is visible in Figure 6, demonstrating our system has the capability of elucidating as much sequence data as possible from a PCR template. CONCLUSIONS

In this paper we have reported the synthesis of cyanine dye-labeled dideoxynucleoside triphosphates and their utility in single-color DNA sequencing methods. We have also demonstrated the ability of these two sets of cyanine dye-labeled terminators, when combined with a suitable DNA polymerase, to generate excellent quality sequencing data using simple and inexpensive instrumentation. ACKNOWLEDGMENT

The authors thank Dr. Kathy Karrer, Marquette University, Milwaukee, WI, for the kind gift of Tetrahymena thermophilia DNA, Dr. Maria Ortiz-Rivera and Steve Marshall for their technical assistance, and Dr. Anup Sood for mass spectral analysis. LITERATURE CITED (1) Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C., Connell, C. R., Heiner, C., Kent, S. B. H., and Hood, L. E. (1986) Fluorescence detection in automated DNA sequence analysis. Nature 321, 674-679. (2) Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A., and Baumeister, K. (1987) A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238, 336-341. (3) Hobbs, F. W., Jr., and Cocuzza, A. J. (1991) Alkynylaminonucleotides. United States Patent No. 5047519. (4) Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A. (1988) DNA sequencing with Thermus aquaticus DNA

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