pp Indian Journal of Microbiology Vol. 36, March 1996, 9-72. O Association of Microbiologists of India

A Rapid Method for Sequencing of rRNA (s) Amplified by Polymerase Chain Reaction using an Automated DNA Sequencer PP Dwivedi’, BKC Patel*’, GN Rees’ and B Ollivier3 ’ Faculty of Science and Technology, Griffith University, Nathan Campus, Brisbane, Queensland - 41 11, Australia. ‘Microbiology Research Unit, Faculty of Applied Science, University of Canberra, Belconnen, ACT-2616, Australia. Laboratoire de Microbiologie ORSTOM, Universite de Provence, 13331, Marseille cedex3, France.

A method for DNA sequencing of ribosomal RNA (rRNA) , amplified by polymerase chain reaction (PCR),using internal primers, designed on the basis of conserved regions of rRNA genes for determining a near complete sequence (99%) of the gene using an automated DNA sequencer (Applied Biosystem Incorporation, USA) is described. The procedure is extremely rapid as cloning of the gene is not required for sequence determination. In addition time consuming steps such as ethanol precipitation and hazardous steps such as phenollchloroform extractions are excluded from the protocol for the purification of extension products after Taq cycle sequencing using the ABI dye of terminator chemistry. The method has been successfully used for sequencing the 16s and 18S.rRNA genes of microbes which includes six members of domain , one of domain and one belonging to Eukarya domain,

Key words : Polymerase chain reaction (PCR), DNAsequencing, ribosomal RNA (rRNA) genes.

Comparisons of 16s and 18s ribosomal RNA Biosystem Incorporation (ABI), USA. This method (rRNA) sequences have been useful for infering avoids cloning and subsequent screening of re- quantitative evolutionary relationship amongst combinants for 16s or 18s rRNA genes. The numerous, diverse, cellular life forms (I,2). For procedure also avoids the use of phenol and such studies ribosomal RNA gene(s), are ampli- chloroform extraction for separation of fied from the of any organism using the unincorporated dye terminators from extension polymerase chain reaction (3), cloned and the products after Taq cycle sequencing reactions. sequence determined (4-6). To facili- The method described in present communication tate cloning of rRNA gene(s), restriction enzyme is very useful for rapidly obtaining the desired sites (viz. SAI 1 and Bam HI) which occur rareJy sequence of rRNA gene(s) as compared to other in the rRNA genes are engineered into the ampli- methods used to date. fication primers. However, such infrequent sites, may be present within the gene and in such Materials and Methods cases, incomplete sequence information is ob- Designing of amplification and sequencing tained. Escherichia coli, Desulfoacinum infernum primers and Halofhermofhrix oreinii (7),have been re- Amplification and sequencing primers, used for ported to have Sal I restriction enzyme site in studying rRNA genes, have been described in their 16s rRNA gene. If this problem is encoun- Table 1. Of these, two bacterial and one archael tered, then blunt end ligation or TA-cloning is the primer’ have an engineered polylinker region only way of cloning and obtaining the near com- which contains rare restriction enzyme site use- plete nucleotide sequence of the 16s or 18s ful for cloning of the 16s rRNA genes. A further rRNA gene (8). ten internal sequencing primers were designed We have designed and used such amplifica- on the basis of conserved regions of the 16s and tion primers for amplifying the 16s rRNA and 18s 18s rRNA gene sequences available in data base rRNA genes of microorganisms. In addition we and from data available in the literature. The have also designed oligonucleotide sequencing positions of the primers have been numbered on primers for the conserved regions of these genes the basis of corresponding nucleotide numbers of and have developed a method using these prim- the 16s rRNA gene of Escherichia coli and the ers for DNA sequencing of such genes using an 18s rRNA gene of Candida albicans (9). automated DNA sequencer from Applied *Corresponding Author o lndian J Microbio/, March, 1996 V

Amplification and purification of PCR unincorporated dye terminators using Sephadex product G-50 gravity columns prepared in a home made 16s and 18s rRNA gene (s)were amplified using 24 column-unit which is a modified version of various amounts (20 ng to 500 pg) of partially previously described stationary column-unit (12). purified genomic DNA (IO), 5 pI of 10 x Taq DNA For this, the columns were packed with 600 pI of polymerase buffer (500 mM KCI, 100 mM Tris- Sephadex G-50,prepared in STE buffer (50 mM HCI pH 9, 1.75 mM magnesium chloride, 1% NaCI, 10 mM Tris-HCI pH 7.4, 1 mM EDTA) and Triton X-IOO), 0.2 mM dNTPs and 50 p mole of allowed to settle for 5 min. The oil free extension each amplification primer (depending on the type products were loaded on to the column and al- of microbe under study, see Table-I). The total lowed to flow under gravity as described earlier PCR volume was made upto 50 pI with sterile (12). The unincorporated dye terminators were distilled water and overlaid with 40 p1 of sterile adsorbed in Sephdex G-50 column and mineral oil. fluorolabelled DNA was eluted with 100 pl of The samples were placed in a thermal cycler sterile water. The eluted fluorolabelled DNA was (Corbett Research Limited, Australia) for ampli- dried in a speedyvac concentrator to complete fication of the rRNA genes. The denaturation dryness and finally resuspended in a suitable step was performed by incubating the samples at volume of formamide and loaded on a sequenc- 95OC for 7 min. After denaturation,

Table 1. Sequencing and amplificationprimers for rRNA genes

Name of Corresponding Sequence (5'-3') Primers used Primer sequence in E. coli for AD, BD &ïn C. albicansfor ED BD* AD* ED* ~~~ FF-1 1-21 5'-AACCTGGTTGATCCTGCCAGT-3' nt nt . + + FR-1 1772-1795 5'-CCGAATTCGTCGACAACUCCGGUUGAUCCUGCCGG(A)AG(C)-3'5'-GATCCTTCTGCAGGTTCACCTAC-3' nt nt ARFD-I 8-28 nt + nt Fd-I 8-27 5'-CCG AATTCGTCGACAACAGACTTTGATCCTGGCTCAG-3' + F-1 339-357 5'-CTCCTACGGGAGGCAGCAG-3' + + F-2 783-803 5'-CAGGATTAGATACCCTGGTAG-3' ++ F-3 907-926 5'-AAACTCAAAGGAATTGACGG-3' + F-4 1390-1405 5'-TGTACACACCGCCCGT-3' * ++ 4. + Rd-I 1542-1525 S'-CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3' + + R-1 357-342 5'-CTGCTGCCTCCCGTAG-3' +++ R-2 536-519 5'-CCAGGGTATCTAATCCTG-3'5'-GTATTACCGCGGCTGCTG-3' R-3 800-783 + nt - R-4 '926-907 5'-CCGTCAATTCCTTTGAGTTT-3' +++ R-5 1115-1 100 5'-GGGGTTGCGCTCGTTG-3' + R-6 1513-1494 5'-TACGGTTACCTTGTTACGAC-3' +++ Fdl and Rdl :Amplification primers for bacteria domian (BD). ARFD-1and R-6 :Amplification primers for Archae domain (AD). Bold Letters : Polylinker region engineered with restrictionenzyme sites. ARFD-1 & FD-1(EcoR1 &Sal I), Rd-I (Xma I, BamHl and Hind 111). FI - F4 : Forward sequencing primers. RI - R-6: reverse sequencing primers. + : Primers worked forsequencing. - : Primers did not work forsequencing. nt : Not tested for sequencing. * : See table-2for organisms for this study. The purification of extension products of cycle compared to spin columns since these are more sequencing away from the unincorporated dye convenient in handling many samples in parallel. terminators is normally carried out using phenol/ In addition, the results obtained by gravity G-50 chloroform extraction method or spin columns in columns are more consistent than spin columns. AB1 protocols. However, we have routinely sepa- Using this method 24 samples are handled very rated the extension products by using a modified efficiently, but the number can be increased by Sephadex G-50 gravity columns (12). This method adding more columns to the multi-column unit. is extremely quick because it does not use etha- The sequence data obtained using the proto- nol precipitation. Ethanol precipitation is required col described above have allowed us to read 400 after phenol/chloroform extraction and is not only to 450 and analysis of the data has a tedious process but may result in significant shown that accuracy often exceeds 98%. Howev- loss of extension products leading to poor quality er, sometimes signal intensities are insufficient sequence data. The gravity fed Sephadex G-50 in 400-600 bp region for reliable base callings. multi-columns are more advantageous to use as Comparable sequence data are also obtainable

Table 2.16s or 18s rRNAgenes studied from various domains

Name of Organisms studied Source Reference domain Eukarya Neocallimastrix (strain aei) Australia Denman unpublished resu!ts. Archae Thermococcus sp (strain Rt3) New Zealand Jones et. al. -do- Bacteria Clostridium índicus sp. nov. India . Chrisotomas et. al. -do- (strain Indi 84) Bacteria Desulfotomaculum thermosapovorans France Fardeau et. al. -510- sp. nov. (strain MLF) Bacteria Desulfoacinum infernum gen. nov. Australia Rees et. ai. -do- sp. nov. (strain B aG1) Bacteria Thermophilic spirochete New Zealand Jonsson et. al. -do- (strain R118BI) Bacteria Desulfotomaculum sp (strain T93B) Norway Unpublished results. -do- Bacteria Lacfobacter fhermoamylovorans France Combet et. a/. -do- gen. nov. sp. nov. (strain DKP) 12 lndian J Microbiol, March, 1996 from the 16s rRNA genes cloned in p-bluescript References (-) vector (sequence data not shown). We have I. Olsen GJ, Lane DJ, Giovannoni SJ & Pace NR,Ann Rev been successful in sequencing various new rRNA Microbiol, 40 (1986) 337. WoeseCR, MicrobiolRev, gene(s) from Bacterial, Archael and Eukaryotic 2. 51(1987)221. 3. Saiki RK, Gelfand DH, StoffelS,Scharf SJ, HiguchiR, organisms but the data are yet to be published Horn GT, Multis KB & Erlich HA, Science, 239 (1988) (Table 2). 487. The restriction enzyme site(s) described in 4. Weisburg WG, Barns SM, Pelletier DA & Lane DJ, J polylinker region of amplification primers (Table Bacteriol, 173(1991)697. . 5. Eisen JA, Smith SW 8 Cavanaugh CM, JBacteriol, 174 1) have been used earlier in our laboratory for the (1992) 3416. RA, directional cloning of rRNA genes (17-20) but the 6. Patel BKC,Saul DS, Reeves Williams LC, Cavanagh occurence of rare restriction enzyme sites in the JE, Nichols PD & Bergquist PL, FEMS Microbiol Lett, 16s rRNA genes of the microbes, e.g. 115(1994)313. 7. Cayo1 JL, Ollivier B, Patel BKC, PrensierG, Guezennec ~ Desulfoacinum infernum (Table 2) and J & Gracia JL, Int J System Eacteriol, (1 (7), 44 994) 534. Ha loth erm o fh fix oreinii p rovid es in co mp Ie t e 8. Sambrook J, Fritsch EF & Maniatis T, MolecularClon- sequence data which if available may be useful ing :A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory for identification of probe sites or group specific Press, USA, 1989. A 9. Neefs JM, Van de Peer Y,De Rijk P, Goris & De signature sequences. We do not clone 16s or Wachter R, NucleicacidRes, 19(1991) 1987. I 18s rRNA genes for their sequence determina- 10. MarmurJ, J MolBiol, 3 (1961) 208. tion, but instead routinely sequence rRNA genes 11. Whitehouse,EH &SpearsT, Eiotechniques, 11 (1991) CRS from their PCR-product by the method described No.145. 12. Rosenthal A & Charnock-Jones DS, J DNA Sequencing in this paper. The method is also useful from a and Mapping, 3 (1992) 61. .d. health safety point of view and to inexperienced 13. Annonym, Applied Biosystem Mode1373A : Foster City, users, as it avoids the use of phenol and chloro- California, USA. form during sequencing manipulations. This pro- 14. Bottger EC, FEMS Microbiol Lett, 65 (1989) 171. 6 15. Lane DJ, Pace BI Olsen GJ, Stahl DA, Sogin ML & Pace cedure has been used for sequencing of 16S/18S NR, Proc Nat1 Acad Sci USA, 82 (1994) 6955. genes from the limited range of organisms from 16. Lane DJ, In NucleicAcid Techniquesin EacterialSystem- domains bacteria, archae and eukarya described atics (Stackebrandt E & Goodfellow M eds.)John Wiley andsons, in Table-2. The sequencing of the genes from USA, (1991)~115. 17. Love CA, Patel BKC, Ludwig W & Stackebrandt E, other phylalsubphyla is underway. FEMS Microbiol Lett, 107 (1993) 317. 18. Love CA, Patel BKC, Nichols PD & Stackebrandt E, Acknowledgements Syst Appl /Microbio/,16 (1993) 244. 19. Redburn AC d Patel BKC, FEMS Microbiol Lett, 113 The financial assistance for these studies from (1993) 81. Australian Research Council and Queensland 20. Redburn AC and Patel BKC, FEMS Microbiol Lett, 115 Water Resource Commission is gratefully ac- (1994) 33. knowledged. We thank Brenda Cheung for her assistance in running the sequencing gels on ABI seqencer.