Highly Sensitive Pyrosequencing Based on the Capture of Free

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Highly Sensitive Pyrosequencing Based on the Capture of Free Adenosine 50 Phosphosulfate with Adenosine Triphosphate Sulfurylase Haiping Wu,† Wenjuan Wu,† Zhiyao Chen,‡ Weipeng Wang,† Guohua Zhou,*,†,‡ Tomoharu Kajiyama,§ and Hideki Kambara§ †

Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, China Medical School, Nanjing University, Nanjing 210093, China § Central Research Laboratory, Hitachi, Ltd., 1-280 Higashi-Koigakubo, Kokubunji-shi, Tokyo 185-8601, Japan ‡

ABSTRACT: In pyrosequencing chemistry, four cascade enzymatic reactions with the catalysis of polymerase, adenosine triphosphate (ATP) sulfurylase, luciferase, and apyrase are employed. The sensitivity of pyrosequencing mainly depends on the concentration of luciferase which catalyzes a photoemission reaction. However, the side-reaction of adenosine 50 phosphosulfate (APS, an analogue of ATP) with luciferase resulted in an unavoidable background signal; hence, the sensitivity cannot be much higher due to the simultaneous increase of the background signal when a larger amount of luciferase is used. In this study, we demonstrated a sensitive pyrosequencing using a large amount of ATP sulfurylase to lower the concentration of free APS in the pyrosequencing mixture. As the complex of ATP sulfurylase and APS does not react with luciferase, a large amount of luciferase can be used to achieve a sensitive pyrosequencing reaction. This sensitivity-improving pyrosequencing chemistry allows the use of an inexpensive light sensor photodiode array for constructing a portable pyrosequencer, a potential tool in a point-of-care test (POCT).

P

yrosequencing, which is based on the real-time bioluminometric quantification of pyrophosphate (PPi) released from deoxyribonucleoside triphosphate (dNTP) incorporation,1 has been widely applied to single-nucleotide polymorphism (SNP) typing,2 microbe genotyping,3 gene expression analysis,4 and gene methylation analysis.5 The next-generation sequencing platform was first constructed using pyrosequencing chemistry as 454 GS FLX.6 Thus, pyrosequencing technology has offered us a powerful tool in the field of postgenomic research. In the conventional pyrosequencing chemistry, four enzymatic reactions are employed, polymerization with Klenow, conversion of PPi to adenosine triphosphate (ATP) with ATP sulfurylase in the presence of adenosine 50 phosphosulfate (APS), photoemission through luciferase reaction, and degradation of ATP and excess dNTPs with apyrase. This chemistry arises from the enzymatic luminometric PPi detection assay (ELIDA)79 but has a lower background than ELIDA because ATP generated from PPi contamination in the reagents is degraded by the coexisting apyrase. Thus, the background signal of pyrosequencing is only from the side-reaction of APS (an analogue of ATP) with luciferase.10 The sensitivity of pyrosequencing mainly depends on the background level and the luciferase concentration. Although the signal intensity in pyrosequencing increases in proportion to luciferase concentration, the background due to free APS in the sequencing mixture increases with luciferase as well.10 r 2011 American Chemical Society

Table 1. Sequences of Primers Used for Gene-Specific PCR sequence (50 to 30 )

gene

primer

M

InfA forward

gaccratcctgtcacctctgac

NP

InfA reverse SW InfA forward

biotin-agggcattytggacaaakcgtcta gcacggtcagcacttatyctrag

SW InfA reverse

biotin-gtgrgctgggttttcatttggtc

HA

SW H1 forward

gtgctataaacaccagcctycca

SW H1 reverse

biotin-cgggatattccttaatcctgtrgc

Therefore, the sensitivity of conventional pyrosequencing chemistry is greatly limited. To use an inexpensive and compact photodiode (PD)-array based instrument for pyrosequencing,11 the sensitivity of pyrosequencing chemistry should be improved to compensate the sensitivity insufficiency of PD sensor. We have previously reported a method enabling pyrosequencing with a high sensitivity using pyruvate orthophosphate dikinase (PPDK) instead of ATP sulfurylase to convert PPi into ATP in the presence of adenosine 5'-monophosphate (AMP) which does not react with luciferase.12 Although several fmols of DNA Received: January 11, 2011 Accepted: March 25, 2011 Published: March 25, 2011 3600

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Figure 1. Effect of the concentration of ATP sulfurylase on both signals and background of pyrosequencing at 2 μM APS (A) and on background of pyrosequencing at various concentrations of APS (0.5, 1, 2, 5, and 7.5 μM), respectively (B). The sequences of the template (50 fmol) and the sequencing primer are 50 -CAG ATG AGA GAA CAA GAA GTG GGG-30 and 50 -CCC CAC TTC TTG TTC TCT CAT-30 , respectively.

Figure 2. Pyrosequencing sensitivity at various amounts of luciferase with the ATP sulfurylase concentration of 0.1 μM (A) and 5.3 μM (B), respectively. GLU means 108 relative light units.

template have been successfully sequenced in the PD-sensor pyrosequencer, it is difficult to obtain the commercialized PPDK on market for the moment. In addition, the optimum temperature for PPDK catalyzed reaction is 5560 °C, and the activity of PPDK drops to 25% of the maximum value at room temperature;13 therefore, PPDK-AMP based chemistry is not in good accordance with the other three enzymatic reactions in pyrosequencing. Furthermore, we found that the yield of PPDK expressed from Microbispora rosea subsp.aerata in E.coli is much lower than that of ATP sulfurylase,14 suggesting that PPDKAMP based pyrosequencing may require expensive reagents. Accordingly, the widespread application of the PD-array pyrosequencer in molecular diagnosis is greatly limited by the reagents. It is preferable to take advantage of the low cost of conventional pyrosequencing chemistry while keeping a higher sensitivity. Because the sensitive pyrosequencing using ATP sulfurylase and APS needs a low background, a straightforward way is to use a small amount of APS; however, this is not practical due to the decrease in the rate of PPi conversion reaction. Here, we improved the conventional pyrosequencing chemistry using a large amount of ATP sulfurylase to capture the free APS, allowing

the use of a high concentration of luciferase in the sequencing mixture for increasing pyrosequencing sensitivity.

’ EXPERIMENTAL SECTION Reagents. ATP sulfurylase and Klenow fragment were obtained by gene engineering in our lab. DNA polymerase was purchased from TaKaRa (Dalian, China). Polyvinylpyrrolidone (PVP) and QuantiLum recombinant luciferase were purchased from Promega (Madison, WI). Bovine serum albumin (BSA), APS, and apyrase-VII were obtained from Sigma (St. Louis, MO). Dynabeads M-280 streptavidin was from Dynal Biotech ASA (Oslo, Norway). Sodium 20 -deoxyadenosine-50 -O-(1triphosphate) (dATPRS), 20 -deoxyguanosine-50 -triphosphate (dGTP), 20 -deoxythymidine-50 -triphosphate (dTTP), and 20 deoxycytidine-50 -triphosphate (dCTP) were purchased from MyChem (San Diego, CA). Other chemicals were of a commercially extra-pure grade. All solutions were prepared with deionized and sterilized water. Primers and Target Sequences. All of the oligomers were synthesized and purified by Invitrogen (Shanghai, China). Two 3601

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Figure 3. Pyrograms of 0.1 pmol of a synthesized template on a commercialized pyrosequencing platform (PyroMark ID) with a standard kit from Qiagen (A) and our homemade kit based on 2 μM APS and 2 μM ATP sulfurylase (B). The sequences of the template and the pyrosequencing primer are 50 -GGT TCC AAG TCA ATG AGA GAA CAA GAA GTG GGG-30 and 50 -CCC CAC TTC TTG TTC TCT CAT-30 , respectively.

RNA specimens of avian influenza A virus subtype H1N1 (A/ Jiangsu/212/2009, A/Jiangsu/214/2009) were provided by the Centers for Disease Control and Prevention in Jiangsu Province of China. Three pairs of gene-specific primers were designed for the amplification of a 106-bp DNA fragment in the Membrane protein gene (the M gene), a 195-bp DNA fragment in the Nucleocapsid Protein gene (the NP gene), and a 116-bp DNA fragment in the Haemagglutinin gene (the HA gene), respectively. The primer sequences are listed in Table 1. RNA Reverse Transcription. First, total RNA in RNase-free water was heated to 65 °C for 5 min and snap chilled on ice for at least 1 min. Then, a reaction mixture containing 0.5 mM of each dNTP, 1 RT buffer, 1 μM random primer, 0.5 U/μL RNase inhibitor, and 0.2 U/μL Omniscript reverse transcriptase was added to each tube containing RNA, mixed gently, collected by a brief centrifugation, and incubated at 37 °C for 60 min, followed by 93 °C for 5 min to terminate the reaction. PCR. Fifty microliters of each PCR reaction mixture contained 1.5 mM MgCl2, 0.2 mM of each dNTP, 1.25 U of Taq polymerase, 20 pmol of the biotinylated PCR primer, and 20 pmol of the other PCR primer. Amplification was performed on an EDC-810 thermocycler PCR system (Eastwin Life Science Inc., China) according to the following protocol: denaturing at 94 °C for 2 min, followed by 35 thermal reaction cycling (94 °C for 30 s; 56 °C for 60 s; 72 °C for 60 s). After the thermal cycle reaction, the product was incubated at 72 °C for 7 min to ensure the complete extension of the amplified DNA. ssDNA Preparation. Streptavidin-coated magnetic Dynabeads M280 were used to prepare ssDNA template for pyrosequencing. Biotinylated PCR products were captured onto the magnetic beads. After sedimentation, the remaining components of the PCR reaction can be removed by washing to obtain pure double-stranded DNA followed by alkali denaturation to yield ssDNA. The immobilized biotinylated strand was used as the sequencing template. Pyrosequencing. Pyrosequencing was performed in 100 μL of pyrosequencing mixture containing 0.1 M Trisacetate (pH 7.7), 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM magnesium acetate, 0.1% BSA, 1 mM dithiothreitol, 2 μM APS, 0.4 mg/mL PVP, 0.4 mM D-luciferin, 0.15 μM ATP sulfurylase, 5.7  108 RLU QuantiLum recombinant luciferase, 18 U/mL Exo- Klenow fragment, and 1.6 U/mL apyrase. Sequencing reaction starts by a stepwise elongation of primer strands through sequential additions of four kinds of deoxynucleotide triphosphates.

Figure 4. Pyrograms of different amounts of synthesized ssDNA with a conventional pyrosequencing system (A) and an improved pyrosequencing system (B). The amount of template was indicated in each pyrogram. Pyrosequencing was performed in a 100 μL mixture using a PD array based portable bioluminescence analyzer.

Apparatus for Pyrosequencing. We used a portable bioluminescence analyzer (HITACHI, Ltd. Central Research Laboratory, Japan) for pyrosequencing.15 This apparatus has a portable size of 140 mm (W)  158 mm (H)  250 mm (D), which used an array of 8 photodiodes (S1133; Hamamatsu Photonics K.K) to detect photo signals, and employed 4 separate capillaries for dispensing small amounts of dNTPs into the reaction chamber. To vibrate the reaction chamber, a cell phone motor fixed on the base support was employed, and a plate holding the reaction chambers was vibrated with the motor. In addition, a commercialized pyrosequencing platform of PyroMark ID System (Biotage Co., Sweden) together with the pyrosequencing kit (Qiagen, Germany) was used for the method evaluation.

’ RESULTS AND DISCUSSION In conventional pyrosequencing, the concentration of APS and ATP sulfurylase was around 5 μM and 0.1 μM (in the case of the specific activity of 42 U/mg ATP sulfurylase), respectively.12,16 3602

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Figure 5. Typical pyrograms and the corresponding theoretical sequence histograms of the M gene, the NP gene, and the HA gene of influenza A 2009 virus (H1N1). The detected sequence is on the top of each peak. Each dNTP was dispensed with the order of A-T-C-G.

Although the yeast ATP sulfurylase has a homohexameric structure,17 the concentration of ATP sulfurylase was calculated using the molecular weight of a subunit. As the Kd (dissociation constant) for the complex of APS and ATP sulfurylase is 0.09 μM,18 the concentration of free APS was calculated as 4.902 μM (approximately 4.9 μM) when 5 μM APS and 0.1 μM ATP sulfurylase were added in the conventional pyrosequencing mixture; this free APS could be the substrate of luciferase. As apyrase does not degrade APS, the background signal due to the free APS will result in a pyrogram baseline, which increases in direct proportion to the amount of luciferase used; 4.9 μM APS produces the signal equivalent to about 1.2 nM ATP.10 Usually, the concentration of a DNA template used for pyrosequencing is around 2 nM, so the background due to APS is at the same level of the signals; consequently, a higher sensitivity by increasing the luciferase concentration is impossible in conventional pyrosequencing chemistry.

The effect of ATP sulfurylase concentration on the background due to APS and the signals from DNA extension reaction was investigated. As shown in Figure 1A, the background signal intensities due to APS decreased dramatically when the concentration of ATP sulfurylase increased from 0.1 to 2 μM, and no obvious decrease of the background is observed when the concentration of ATP sulfurylase is more than 2 μM, which is equal to that of added APS. On the other hand, the signal intensity due to dNTP incorporation also increased a little with the concentration of ATP sulfurylase (around 80% from 0.1 to 1 μM) and stayed constant after 1 μM (see Figure 1A for details). These results indicate that a highly sensitive pyrosequencing is possible by optimizing the ATP sulfurylase concentration to allow a low background and the largest signal; therefore, the best concentration of ATP sulfurylase was selected as 2 μM in the case of 2 μM APS. To further investigate the optimum ratio in concentration between APS and ATP sulfurylase, pyrosequencing with different 3603

dx.doi.org/10.1021/ac2000785 |Anal. Chem. 2011, 83, 3600–3605

Analytical Chemistry amounts of ATP sulfurylase was individually performed at the APS concentration of 0.5, 1, 2, 5, and 7.5 μM, respectively. As shown in Figure 1B, the background signals at each APS concentration decreased with the increase of ATP sulfurylase concentration. Using the background signal of 300 as a criteria, the optimum concentrations of ATP sulfurylase are around 0.5, 1, 2, 5, and 7.5 μM at 0.5, 1, 2, 5, and 7.5 μM APS, respectively; we thus concluded that a sensitive pyrosequencing reaction could be achieved by keeping ATP sulfurylase concentration as high as that of APS in a sequencing mixture. From the viewpoint of reagent cost, a small amount of ATP sulfurylase is preferable; hence, APS concentration in pyrosequencing should be optimized. Pyrosequencing on various amounts of APS showed that sequencing signals become constant when more than 1 μM APS was employed (data not showed), indicating that 2 μM APS is enough for a pyrosequencing reaction. At this condition (2 μM APS and 2 μM ATP sulfurylase), the concentration of free APS in the pyrosequencing mixture was theoretically calculated to be 0.38 μM by taking the Kd of 0.09 μM into consideration. The background signal from this amount of APS corresponds to the signal from 0.09 nM ATP, much lower than the template amount (2 nM) used in conventional pyrosequencing. As the concentration of free APS was significantly reduced by adding a large amount of ATP sulfurylase, the sensitivity of conventional pyrosequencing can be increased using a high concentration of luciferase. For comparison, pyrosequencing on 50 fmol of synthesized ssDNA at various amounts of luciferase (0.57  108 RLU, 1.1  108 RLU, 2.8  108 RLU, 5.7  108 RLU, and 11.3  108 RLU) was carried out at 0.1 μM and 5.3 μM ATP sulfurylase, respectively. The results in Figure 2A showed that the backgrounds increased significantly with the increase of luciferase concentration when 0.1 μM ATP sulfurylase was employed, resulting in a low signal-to-noise ratio and a low detection sensitivity. On the other hand, pyrosequencing with 5.3 μM ATP sulfurylase (Figure 2B) gave a very low background and a higher signal-to-noise ratio, while the signal increased in proportion to the luciferase concentration. Therefore, it is feasible to employ a higher ATP sulfurylase concentration to achieve a sensitive pyrosequencing. Comparison of pyrosequencing reactions on a commercialized pyrosequencing platform (PyroMark ID System) between a standard kit from Qiagen and our homemade kit (2 μM APS and 2 μM ATP sulfurylase) was shown in Figure 3, indicating a significant increase of signal intensities by the homemade kit. For a sensitive pyrosequencing, a sensitive light sensor is necessary to the instrumentation. The light sensors currently available on the market are photomultiplier tube (PMT), charge coupled device (CCD), and PD (photodiode). Among these three sensors, PD sensor is the most inexpensive (several dollars) and the smallest (6W  2H  8D mm) one and can be readily compacted and arrayed, but PD sensor is less sensitive. We have constructed a prototype of an 8-channel pyrosequencer using a PD array sensor with the dimensions of 140W  158H  250D (mm).11,15 Different amounts of ssDNA template were detected by conventional and improved pyrosequencing systems, respectively. As shown in Figure 4, about 250 fmol of DNA template was required for an accurate sequencing with conventional pyrosequencing chemistry, while 25 fmol of DNA template gave the similar sequencing intensity with the improved pyrosequencing chemistry. Therefore, the pyrosequencing sensitivity increased 10 times in the improved pyrosequencing chemistry, resulting in a 10-fold reduction in the amount of DNA template for pyrosequencing. We have applied this chemistry in the

TECHNICAL NOTE

8-channel pyrosequencer for genotyping the 2009 influenza A virus (2009 H1N1) by sequencing the segments of the M gene, the NP gene, and the HA gene which is species specific. The results were shown in Figure 5, and it is observed that the pyrograms contain some unexpected signals (short peaks). The pyrogram of a negative control (data not showed) suggests that these short peaks are from the contributions of both dNTP’s impurities and the background due to incompletely extended templates. Comparison between the theoretical histograms and the obtained pyrograms indicates that these short peaks have less impact on the base-calling accuracy. Therefore, the sensitive pyrosequencing system coupled with a PD-array based portable DNA analyzer is feasible.

’ CONCLUSION In short, this study demonstrated that the concentration of free APS in the pyrosequencing mixture can be significantly lowered using a large amount of ATP sulfurylase. The complex formed by APS and ATP sulfurylase does not react with luciferase; a large amount of luciferase can thus be used to achieve a sensitive pyrosequencing reaction. One possible concern arising from the use of a large amount of ATP sulfurylase is the higher cost than that of conventional pyrosequencing chemistry; we, hence, tried to express ATP sulfurylase from Saccharomyces cerevisias in E. coli19 and found that it is very easy to obtain a high expression yield of this protein without any tedious optimization and purification,20 suggesting that a sensitive pyrosequencing kit can be very inexpensive. An alternative way to achieve sensitive pyrosequencing at a low cost is to develop a new reaction to convert PPi to ATP. Recently, a ATP regeneration system in luciferase reaction was proposed using ADPglc pyrophosphorylase (AGPPase) to catalyze the synthesis of ATP from PPi in the presence of ADPglc, yielding much lower luminescence background than the APSATP sulfurylase reaction.21 An inexpensive and sensitive pyrosequencing should be possible if the ADPglcAGPPase system is commercially available, has low cost, and is well coupled with other reactions in pyrosequencing. The combination of the sensitivity-improving pyrosequencing chemistry with a PD-array based portable DNA analyzer should be a potential tool in a point-of-care test (POCT) for various clinical applications as well as on-site screening of pathogenic agents. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: þ86 25 84514223. Tel: þ86 25 84514223.

’ ACKNOWLEDGMENT The first two authors contributed equally to this work. This research is funded by the National Natural Science Foundation of China (project numbers: 20975113, 21005088), the Science and Technology Development Project of Jiangsu Province (project number: BE2010604), and Central Research Laboratory of Hitachi, Ltd., Japan. ’ REFERENCES (1) Ronaghi, M.; Uhlen, M.; Nyren, P. Science 1998, 281, 363. (2) Ahmadian, A.; Gharizadeh, B.; Gustafsson, A. C.; Sterky, F.; Nyren, P.; Uhlen, M.; Lundeberg, J. Anal. Biochem. 2000, 280, 103. 3604

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