ELSEVIER

JOUR]IAL OF

TICROBIOLOGICAL

TETHODSElsevier Journal 19 (1994) 145-154

Automated sequencing of PcR-amplified 165 rDNA on'Hydrolink' gels

Akira Hiraishi''*, Yo.rg Kook Shinb, Yoko Uedau, Junta SugiyamaboLaboratorl' of Environmental Biotechnology, Konishi Co. Ltd., Yokokata 5-6-3, Sumida-ku Tokyo 1j0,

Japan,blnstitute of Molecular ancl Cellular Biosciences, University of Tokl'6, Tokyo 113, Japan

(Received 21 April 1993; revision received 16 July 1993; accepted 2l July 1993)

Abstract

A commercially available high-performance gel solution, 'Hydrolink Long Ranger', was usedfor direct automated sequencing of PCR-amplified double-stranded l65 rDNA. The 1.5 kbrDNA fragments were amplified directly from bacterial cell lysates and sequenced by using?"rfr DNA polymerase with the linear PCR sequencing protocol. Sequencing reactions wereanalyzed on an automated laser fluorescent DNA sequencer with Hydrolink gels compared tostandard polyacrylamide gels. Automated Hydrolink gel electrophoresis allowed highsequencing speeds and resulted in a marked increase in readable sequences. Upon thiselectrophoresis, an average of 540 bases of nucleotides was resolved by automated reading in asingle sequencing run, and the resolved sequence was extended up to 600 to 700 bases bymanual correction of the errors and ambiguities. This sequencing strategy is useful for theanalysis of long sequences of more than 500 bases and makes it possible to obtain entire 165

rDNA sequence data for several strains within 2 days, using an automated DNA sequencer

with the one dye detection system.

Key words:Automated sequencing; PCR; Phylogenetic study; l65 rDNA

l. Introduction

Comparative sequence analysis of 165 rRNA is currently one of the most power-ful methods for elucidating phylogenetic and evolutionary relationships among pro-karyotes [1,2]. This approach has come into wide use since the development of thereverse transcriptase sequencing method [3] and is being more accelerated by the

tor."rp"raing author. Fax: 03-3625-5660.

016'1-7012194/507.00 O 1994 Elsevier Science B.V. All rights reserved

.s.sD/ o t 67 -1 0 | 2(93) E 0o 5 6 -X

146 A. Hiraishi et al.lElsefier Journal, 19 (1994) 145-154

application of PCR technology to 165 rDNA amplificationl4-l2l and subsequentdirect sequencing [10-12]. While general strategies to sequence l65 rRNA or PCR-amplified 165 rDNA involve using radioactive materials as sequencing labels, auto-mated DNA sequencing systems using fluorescent dyes U3,14] have found an in-creasing number of applications in molecular biology. Although the automatedsequencing method requires sophisticated and expensive equipment, this approachto routine phylogenetic studies is convenient and attractive because of its advantages

of releasing experimenters from radioisotope use, shortening time for sequence ana-

lysis, minimizing human errors, and making it possible to perform on-line dataprocessing throughout the sequencing-phylogenetic study.

A recent report introduced a protocol for PCR amplification of 165 rDNA with-out the need to purify DNA used as the template and subsequent direct sequencingol the double-stranded PCR product by using an automated fluorescent DNA se-

quencer [11]. The combined method of linear PCR sequencing [15-17] and auto-mated sequence detection was shown to allow the resolution of about 400 nucleo-tides of the rDNA in a single sequencing run. Recently, a manufacturer has pro-duced a high performance gel solution, 'Hydrolink', as an alternative to standardacrylamide mixtures for DNA sequencing, and this gel system has been applied toautomated DNA sequencing with one dye label [18,19]. In the present paper, we

report the use of Hydrolink gels for automated sequencing of PCR-amplified 165

rDNA to improve sequence resolution. Some modihcations in the procedure ofdirect automated 165 IDNA sequencing are described.

2. Materials and methods

2.1. Bacterial stvains and cultivationThe organisms used in this study were the type strains of Comamanas testosteroni,

Escherickia coli, and Pseudomonas aeruginosa which were obtained from the CultureCollection Center of the Institute of Molecular and Cellular Biosciences, Universityof Tokyo (Tokyo, Japan). An unknown phototrophic bacterium which is of owncollection (A.H.) and designated tentatively Rhodopseudomonas sp. MB314 was

also used for partial sequencing. The test organisms were grown in 5 ml of an

appropriate medium, harvested by centrifugation at the late exponential phase ofgrowth, washed with sterile lo/o saline, and resuspended in 100 pl of pure water.Cell suspensions were stored at -20'C until use.

2.2. 165 rDNA amplification165 rDNA fragments that corresponded to positions 8 to 1510 of the Escherichia

coli nwbering system were amplifred by PCR directly from crude cell lysates ac-

c.ording.to the previously described protocol [11]. The procedure for preparation ofcell lysates was slightly modified as follows. 40 pl of thawed cell suspensions were

mixed with 10 irl of proteinase K soiution (1 mg/ml) and 50 pl of BL buffer (40 mMTris, 17o Tween 20,0.5oA Nonidet P-40, 1 mM EDTA, pH 8.0), incubated at 60'Cfor 20 min and then at 95"C for 5 min, and centrifuged to remove unbroken cells

A. Hiraishi et al.lElsevier lournal, 19 (1994) 145-154

and large debris. The resultant supernatant was used directly for PCR. Previously,the volume of cell lysates for PCR use was determined on the basis of dry weight ofcells in the initial suspensions [11]. In the present study, we attempted to optimizethis using the absorbance of cell lysates at 260 nm (A26s), and met with best ampli-lrcation results when applying an aliquot equivalent to 5 pl with an A26s of 3.0. Forexample, when the sample had an A26s of 15, we use 1 pl of it to prime a 100 plamplification reaction. The reaction tubes were prepared in triplicate to yield PCRproducts sufficient for several sequencing reactions that cover the entire 165 rDNAmoiecule. PCR products were treated with chloroform-phenol, precipitated withammonium acetate and ethanol, and purified by electrophoresis on 17o agarosegel. Separated target fragments were cut off from the gel and withdrawn with a

Sepaglas BandPrep kit (Pharmacia LKB Biotechnology, Uppsala, Sweden) follow-ing the manufacturer's specihcation.

2.3. Oligodeoxynucleotide primersOligodeoxynucleotide PCR primers that were homologous or complementary to

positions 8 to 27 (5'-AGAGTTTGATCATGGCTCAG-3') and 1510 to 1492 (5'-GGTTACCTTGTTACGACTT-3') in E. coli 163 rRNA numbering [6] were synthe-sized on a Cyclon Plus DNA synthesizer (Milligen/Biosearch, Burlington, USA)using standard phosphoramidite chemistry and purified by reverse-phase high-per-formance liquid chromatography. For sequencing, eight oligodeoxynucleotide pri-rners labeled with fluorescein isothiocyanate at 5' terminus were obtained fromTakara Suzo Co. (Kyoto, Japan). All these primers were designed to anneal toconserved regions of eubacterial 165 rDNA [20]. Reverse sequencing primers, desig-

nated rlL,r2L,r3L,r4L, r5L, were complemetary to positions 536 to 518,821 to803, 1111 to 1093, 1406 to 1389, and 1509 to 1492, respectively Ull.Forward pri-mers were homologous to positions 9 to 27 (flL), 518 to fi6 (nD and 1094 to 1112

(f3L).

2.4. Sequencing reactionsLinear PCR sequencing (cycle sequencing) reactions with Tth DNA polymerase

were performed by using reagents from the Pharmacia AutoCycle Sequencing kit(Lot DD 1693101) according to the instruction by the manufacturer with some

modihcations. For a sequencing reaction, a mixture of 0.5 pmol of template (PCR-amplified, purified rDNA), 8 pmol of fluorescent primer, 4.3 pil of Reaction Buffer,3.4 pl of dimethylsulfoxide, and 2,rzl of diluted Tth polymerase (8U) (made up to 26

prl with pure water) was prepared as the master mix. Of this mix, 6 pl was added to 4prl each of the four termination mixes. The manufacturer specifies to prepare each

termination mix with a d/ddNTP ratio of l:1 (viv) from the sequencing kit. How-ever, the 'C' reaction with this formula limited the length of chain extension andresulted in emphasizing fluorescent peaks close to the primer compared to the otherthree reactions. We found that changing the d/ddCTP ratio to 1:0.6 aided in resol-ving longer sequences comparable to those obtained with the other three, and thusadopted this modification for all'C' reactions. Cycie reactions (total 25 cycles) wereperformed either in a Pharmacia GeneATAQ controller or in a GeneAmp PCR 9600

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148 A. Hiraishi et al.lElsevier Journal, 19 ( 1994) 145-154

System Thermal Cycler (Perkin-Elmer Cetus Instruments, Norwark, USA), withramping at the maximum speed. With the former controller, we used a cycle profileof 95"C-36 s, 55'C-36 s, and 72"C-84 s as recommended by the manufacturer, butmodified it for the latter (95"C-30 s, 55'C-52 s, and 72"C-90 s) as previously re-ported [11]. The reason for this is that the Perkin-Elmer Cetus apparatus gave un-satisfactory results with the recommended cycle profile, possibly due to differentfeatures of ramping and temperature transmission, but produced better resultswhen operated under the modified conditions. In both cases, the last cycle wasfollowed by a denaturation step at 95'C for 2 min. The reaction mixtures weretransferred onto ice, mixed with 7 prl of formamide/blue dextran stop solution, andstored at - 20"C until analysis.

2.5. Automated electrophoresisImmediately prior to electrophoresis, the reaction mixtures were redenatured by

heating at 95'C for 3 min, cooled quickly in ice-water, and then loaded onto the gel(3 pl for each well). Automated electrophoresis and analysis of DNA sequence reac-tions were performed with a Pharmacia A.L.F. DNA sequencer. The standard se-

quencing gels contained 60/o acrylamide gels and 7 M urea in I x TBE solution andwere 0.5 mm thick. The Hydrolink gels contained 6Yo Long Ranger solution (ATBiochem, Malvern, USA) and 7 M urea in 1 x TBE and had 0.35 rnm thickness.Running conditions for the sequencer were as follows (those for Hydrolink gels aregiven in parentheses when modihed): voltage, 1500 V (1300 V); power 34 W (45 W);current, 38 mA; gel temperature, 45"C (50"C); laser power 3 mW; sampling interval,2.00 s (1.25 s); running time, 6 h.

oE 600(EIlo

$ aoo

800

200

I

a'/

Electrophoresis time (h)

Fig. 1. Relationships between electrophoresis time and sequencing speed on Hydrolink gels (r) and stan-

dard polyacrylamide gels (l) in automated 165 rDNA sequencing. Arrows indicate the average numberof nucleotide bases resolved by the automated reader.

A. Hiraishi et al.f Elsevier Journal, 19 ( 1994) 145-154

3. Results and discussion

We investigated the performance of Hydrolink gels compared with standardpolyacrylamide gels for 165 rDNA sequencing, using the PCR products from C.testosteroni, E. coli, and P. aeruginosa and four internal sequencing primers (f2L,

r2L, r3L, and r4L). There were no marked differences in the sequence resolu-tion among the used rDNAs or the sequencing primers. Fig. 1 shows the compara-tive performance of the two gel systems for cycle sequencing products. Automatedelectrophoresis on Hydrolink gels allowed high-speed mobility of fragments andresulted in a marked increase in readable sequences. Upon this electrophoresis, anaverage of 540 bases of nucleotides was resolved by automated reading in a singlesequencing run. The resolved sequence was extended up to around 600 bases, insome cases 700 bases, by manual correction of the errors and ambiguities and bymanual confirmation of bases that were presented but not identified positively bythe automated reader. Compared to the standard polyacrylamide electrophoresis,the Hydrolink system gave 30o/o more sequence information. In concurrent studieswe have found that 0.35 mm thick polyacrylamide gels also allow high sequencingspeeds but have less positive effects on sequence resolution. Fig. 2 shows an exampleof a processed data output of a sequencing run on Hydrolink gels when the 165

Fig. 2. Direct automated sequencing with primer r3L of the 165 rDNA from Rhodopseudomonas sp. strainMB314. Processed data output from the sequencer is shown.

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r50 A. Hiraishi er al.lElsevier-Journal, 19 (1994) 145-]54

Table IAccuracl, and ambiguities in automated sequencing on polyacrylarnide and Hydrolink gels

Base range 6% Acrylamide (0.5 rnm thickness) 6% Hydrolink (0.35 rnm thickness)

Accuracy (7o) Ambiguities (%) Accuracy (%) Anibiguities (%)

0-100I 00-200200-300300+00400-s00500 600

600 700

99.0

99.6

99.2

98.7

96.1

96.1

91.8

99.899.8

99.0

96.3

90.6

1.8

t.2236.5

19 0

4.0i.91.1

3.0

7.0

8.415.6

rDNA of Rhodopseudoruonas sp. M8314 rvas sequenced rvith primer r3L. In thiscase, approximately 700 bases of nucleotides were resolved by both automated andmanual readings, covering a stretch of positions 1080 to 320 of the E. coli numberingsystem.

As reported previously [1], the reactions with primers flL and r5L. rvhich arehomologous to the forward and reverse PCR prirners, respectively. reduced thequality and length of readable sequences with frequent occurrence of ambiguities,resultirlg in the resolution of less than 200 bases in a single sequencing run. This ma),be due in part to less stable template-primer annealing in the termini of the PCR-generated rDNAs. Thus, the end primer approach to cycle sequencing of the PCRproducts seems less effective to obtain sequence information on their terminal re-gions. On the other hand, the sequencing with the internal primers r1L and f3Lserved this purpose. Because of high sequencing speeds with the Hydrolink system,the analysis of the reactions rvith these primers covered all sequences (400 to 500

bases) between the primer-annealing site and the 5'-terminus of both strands rvithin3.5 h of electrophoresis (see Fig. 3).

Table I shows comparative data on percentage accuracy and ambiguities ol nu-cleotide bases in a sequencing run on both electrophoresis systems when using pri-mers f2L, f3L, rZL, r3L, and r4L. Although the Hydrolink gel system shorved highperformance especially in resolving long sequences olmore than 300 bases, it did notahvays enhance the accuracy, rather increased sequencing ambiguitres. The highelectrophoretic velocity on Hydrolink gels resulted in compressing the interval offluorescent signal peaks, whereby some peaks were poorly separated even in a shortrange of sequences from the primer peak. For example, this was the case in initial i0bases beyond the primer due to much higher mobility of short fragments (see Fig. 1)

and in multiple neighboring bases occurring in a single sequencing track. This is themain reason for the presence of increased ambiguities in the Hydrolink systetn.Nevertheless, this problem may not be critical, because most ambiguities are cor-rected by nanual reading on line u,ith overlapping sequences from a nested set ofthe reactions. Consequently, no or fe,uv undetermined positions (0 to 4 bases) weregenerated through the analysis of the overall 165 rDNA sequences from the four

A. Hiraishi et al.f Elseviet'Journal, 19 (1994) 145 154

Fig. 3, Direct automated sequencing u,ith primer rl L of the 165 rDNA from Rhodopseudornonas sp. strainMB3l4, Raw data output for the reaction products before (upper) and after (lower) 3 days of freeze-storage are shown.

organisms. Although the obtained sequence information contained some errors,these were not polymerase-mediated but produced entirely through post-run auto-mated analysis. We confirmed high fidelity of Tth polymerase in the cycle sequencereactions by bidirectional sequencing of the PCR products and by comparing ourresults with the sequence information available frorn the EMBL/GenBank Nucleo-tide Sequence data bases.

During the course of this study we had an interesting observation concerning theeffect of freeze-storage of the cycle reaction products on sequence resolution. In thecase of radioisotopic sequencing, long-term storage of the reaction products prior toelectrophoresis causes the appearance of higher background and the reduction ofsequence quality in general, while that of the fluorescent products had an unex-pected effect of enhancing fluorescent signals and sequence resolution (Fig. 3). Thiseffect was pronounced in particular on the products obtained rvith the Perkin-ElmerCetus apparatus and observed even in 8 week-freezed samples. The reason for this isnot known with certainty, but one can assume that freeze-storing stimulates thedenaturation of the double-stranded products in the presence of formamide added

I51

t52 A. Hiraishi et al.lElsevier Journal, 19 (1994) 145-154

<_--_-14L

<--' 1389-1406RL

<---- ,093-1111rzL803-821<_--

11L518-536 3',5'

3'

8-27

1492-1510

1?[,,,,Fig. 4. Diagrammatic representation of the position of sequencing primers effective for direct automatedsequencing of the PCR-amplified 163 rDNA. Striped regions shows the PCR primer-annealing sites.

Numbers refer to the position in the E. coll numbering system. Arrows with broken lines indicate thesequencing direction and range with each primer.

as stop solution, so that the reannealing of the heat-separated complementary DNAstrands (template-product) is weakened when applying to the gel. Unlike radioiso-tope-labeled sequencing reactions, the fluorescent products eliminate the need toconsider the reduction of labeling activity during storage. By taking advantage ofthis feature, we have added the freeze-storage step (overnight or longer time) to theprocedure for automated 165 IDNA sequencing.

The results of the present study have shown that the automated Hydrolink gel

electrophoresis aliows high sequencing speeds and improved sequence resolutioncompared to the standard polyacrylamide gel system. In this study, we tested fivereverse primers and three forward primers for sequencing of the 1.5 kb rDNA frag-ment. Considering the high perfonnance of the automated Hydrolink system, theuse of the three universal primers [3] plus one forward primer, RL, may be suffi-cient to obtain sequence data covering the entire 165 rDNA molecule. As shown inFig. 4, however, we are routinely using four reverse primers (rLL, r2L, r3L, and r4L)in addition to the forward primer to generate overlapping sequence informationwhich is desirable for correcting the ambiguity and ensuring the accuracy. Since thecycle sequencing method using Tth polymerase gives highly reproducible results, fullsequencing of both strands of the PCR products may be unnecessary unless other-wise ambiguity problems still remain. The running time of electrophoresis can alsobe shortened to 4 h to read at least 500 bases of nucleotides per clone, which is

sufficient to obtain complete sequences of the amplihed 165 rDNA by using thefive primers. This strategy makes it possible to perform the sequencing run twice ina day and to obtain complete 165 rDNA sequence data for several strains within 2days, using an automated DNA sequencer with the one dye detection system.

A. Hiraishi et al.lElseviet' Journal, 19 ( 1994) 145-154

The convenience of the present sequencing strategy is enhanced by cornbinationwith direct PCR amplification of 165 rDNA from crude lysates, which can be

achieved through proteinase K digestion as reported here and elsewhere [11], heatextraction 16,21), or mechanic disruption [7]. Concurrent studies from our labora-tory have shown that a cornbination of ultrasonic disruption with protease treat-ment provides most reproducible results and works well lor a rvide variety of pro-karyotic organisms including gram-positive bacteria (Hiraishi, Shin, Takeuchi, andMori, unpublished) and methanogens (Hiraishi, Kamagata, and Nakamura, unpub-lished).

In summary, the present protocol for automated sequencing, involving Hydrolinkgel electrophoresis, is quick to perform and is useful for resolving over 500 nucleo-tides per clone in a sequencing run. We believe this protocol to facilitate 165 rDNA-based phylogenetic studies in bacterial systernatics.

4. References

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[3] Lane, D.J., Pace, B., Olsen, G.J., Stahl, D.A., Sogin, M.L and Pace, N.R. (1985) Rapiddetermination ol l65 ribosomal RNA sequences lor phylogenetic analyses. Proc. Natl. Acad. Sci.

usA 82, 6955 -69s9.

[4] Bottger, E.C. (1989) Rapid determination of bacterial libosomal RNA sequences by direct sequencing

of enzymatically amplified DNA. FEMS Microbiol. Lett. 65, 111-116.

[5] Wisotzkey, J.D., Jurtshuk, P. and Fox, G.E. (1990) PCR amplification of 165 rDNA lrorn lyophilizedcell cultures lacilitates studies in molecular systematics. Curr. Microbiol. 21. 325-321 .

[6] Weisburg, W.G., Barns. S.M., Pelletier, D.A. and Lane, D.J. (1991) 165 Ribosornal DNAamplification lor phylogenetic study. J. Bacteriol. 173, 697-103.

[7] Frothingham, R., Allen, R.L. and Wilson, K.H. (1991) Rapid 165 ribosomal DNA sequencing from a

single colony without DNA extraction or purification. BioTechniques 11, 4244.[8] Both, B., Krupp, G. and Stackebrandt, E, (1991) Direct sequencing of double-strarrded polymerase

chain reaction-arnplified 165 rDNA. Anal. Biochem. 199:216218.

[9] Dorsch, M. and Stackebrandt , E. (1992) Some rnodifications in the procedure of direct sequencing ofPCR amplilted 165 rDNA. J. Microbiol, Methods 16,271-279.

[0] Embley, T.M. (1991) The linear PCR reaction: a sirnple and robust method lor sequencing arnplifledrRNA genes. Lett. Appl. Microbiol. 13,171-174.

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[14] Smith, L.M., Sanders, J.2., 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. Nature321, 61+-679.

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[6] Carothers. A.M., Urlaub, G., Mucha, J., Grunberger, D. and Chasin, L.M. (1990) Point mutation

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analysis of a mammalian gene: rapid preparation of total RNA, PCR amplification of cDNA, andIa4 sequencing by a novel method. BioTechniques 7, 494499.

[17] Kusukarva, N., Uemori. T., Asada, K. and Kato, I. (1990) Rapid and reliable protocol for directsequencing of material amplified by the polymerase chain reaction. BioTechniques 9, 66-72.

[1E] Voss, H., Wiemann, S., Wirkner, U, Schwager, C.U., Zimmermann, J. Stegemann, J. Erfle, H.,Hewitt, N.H., Rupp, T. and Ansorge, W. (1992) Automated DNA sequencing system resolving 1,000

bases with fluorescein-15-dATP as internal label. Methods Mol. Cell. Biol. 3, 153-155.

[9] Ansorge, W., Voss, H., Wiemann, S., Schwager, C.U., Sproat, B., Zimmermann, J. Stegemann, J,

Erfle, H., Hewitt, N.H. and Rupp, T. (1992) High-throughput automated DNA sequencing facilitywith fluorescent labels at the European Molecular Biology Laboratory. Electrophoresis 13, 616-619.

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[21] Fuerst, J.A., Hawkins, J.A., Holmes, A., SIy, L.I., Moore, C.J. and Stackebrandt, E. (1993)Porphyrobacter neustonensrs gen. nov., sp. nov., an aerobic bacteriochlorophyll-synthesizing buddingbacterium from fresh water. Int. J. Syst. Bacteriol, 43,125-134.