Automated PCR/sequence template purification - Springer Link

Automated PCR/Sequence Template Purification RESEARCH PROTOCOL

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Automated PCR/Sequence Template Purification S. C. Clarke* and M. A. Diggle Abstract A commercially available filtration method is described for the purification of polymerase chain reaction (PCR) templates and sequence-labeled products. The methodology is described for the automation of this application and its use on a high-throughput liquid handling robot and capillary-based automated DNA sequencer. The application provides good-quality DNA, is relatively cheap, and can be used in either 96- or 384-well format. Index Entries: DNA sequencing; polymerase chain reaction; automation; purification.

1. Introduction Recent advances in DNA sequencing technology have resulted in reduced costs, improved quality, and higher throughput (1–3). Wholegenome sequences are now commonplace (4), and the discovery of new genes and gene functions has increased (5). DNA sequencing is also increasingly used in clinical laboratories, particularly in the fields of human genetics and medical microbiology (2,6). In the latter, DNA sequencing has found its place in bacterial identification and typing. In particular, multilocus sequence typing (MLST) has found a niche very quickly because of its high level of discrimination, reproducibility, and electronic portability (7,8). MLST has now been validated for a number of important bacterial pathogens including Neisseria meningitidis and Streptococcus pneumoniae (7,9). However, the quality of DNA for such methods must be high and is an essential component of the methods success. Many methods are available for the purification or clean-up of PCR products, plasmids, or sequence-labeled products and include polyethylene glycol precipitation (10), sodium acetate/ethanol precipitation, and commercial spin columns or filters (11,12). However, these methods work with varying success, can be laborious, and may be expensive for high-throughput use. There is a need

for purification methods that provide good quality DNA, are relatively cheap, and can be used for high-throughput applications. Two new commercially available products have been released onto the market that are filter-based and purify DNA by size exclusion. These are available in either 96- or 384-well microtiter plate format. The plate format and methodology lend themselves to automation on liquid handling robots. Here we describe, for the first time, the use of these products for the purification of PCR and sequence templates on an automated liquid handling robot for subsequent use on a 96-capillary automated DNA sequencer. The method was applied to the bacterial typing method, MLST, which has gained a large amount of support as a reproducible typing method, which can be used for a number of pathogenic bacteria.

2. Materials 1. 2. 3. 4. 5. 6. 7. 8.

Vacuum manifold system. Liquid handling robot. Automated DNA sequencer. Dye terminator cycle sequencing kit. Millipore Multiscreen™ 96- or 384-PCR plates. Millipore Multiscreen™ 96- or 384-SEQ plates. Sterile distilled water (18 MΩ purity). DNA sequencing analysis software.

*Author

to whom all correspondence and reprint requests should be addressed: Scottish Meningococcus and Pneumococcus Reference Laboratory, House on the Hill, Department of Microbiology, Stobhill Hospital, Glasgow, G21 3UW, UK. Phone/fax: 44 141 201 3836. Email: [email protected]. Molecular Biotechnology 2002 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2002/21:3/221–224/$11.00

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222 3. Methods 3.1. PCR Product Purification 1. Place a Millipore MultiScreen™ 96- or 384PCR plate (Millipore, Watford, UK) on a vacuum manifold; this is done automatically onto the integrated vacuum manifold when using the Roboseq liquid handling robot. 2. Add the entire volume (range 25–50 µL) of each post-PCR reaction, up to a maximum volume of 100 µL, to the appropriate well of the Multiscreen plate. Purify the PCR product by applying a vacuum at 350 millibars pressure for 15 min or until the wells are dry. To ensure full purification, apply the vacuum for 1 min after the last well is dry. 3. Resuspend the PCR products by adding 20 µL of 18 MΩ distilled water and repeat pipet a volume of 15 µL 100 times slowly if performing manually or at 50% pipeting speed on a liquid handling robot. Redistribute the purified PCR products into four 96-well microtiter plates.

3.2. Labeled Sequence Product Purification 1. After performing cycle sequencing labeling reaction, according to local protocols, place a Millipore MultiScreen™ 96- or 384-SEQ plate on a vacuum manifold; this is done automatically onto the integrated vacuum manifold when using the Roboseq liquid handling robot. 2. Adjust the volume to 20 µL with 0.3 mM EDTA (pH 8.0) prepared with 18 MΩ distilled water. Transfer the entire volume of each post-PCR reaction, usual range 25–50 µL depending on the gene being sequenced, up to a maximum volume of 100 µL, onto the appropriate well of the Multiscreen plate. Purify the product by applying a vacuum of 850 millibars pressure for 5 min or until the wells were dry. 3. Resuspend the purified labeled sequence products by adding 20 µL of 18 MΩ distilled water and repeat pipeting a volume of 15 µL 20 times, slowly if performing manually, or at 50% pipeting speed if using a liquid handling robot. Redistribute the purified labeled sequence products into plates ready for DNA sequencing. When using the Multiscreen™ 384-plates ready for sequencing on a MegaBACE sequen-


Clarke and Diggle cer, the products are redistributed into four 96-well microtiter plates.

4. Notes 1. Specimens. The authors used a random selection of Neisseria meningitidis and Streptococcus pneumoniae strains, received by the Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) between July and August 2001. All strains were initially isolated from hospitals throughout Scotland. 2. Genotypic characterization. All strains were processed for MLST as previously described (13), except that the DNA purification methods were changed as described below. Also, the procedures were performed on a Roboseq liquid handling robot (MWG Biotech, Milton Keynes, UK) and a MegaBACE 1000 96-capillary automated DNA sequencer (Amersham Pharmacia Biotech, Little Chalfont, UK). åDyenamic ET dye terminator chemistry (Amersham Pharmacia Biotech, Little Chalfont, UK) was used. This allowed the automation of all of the procedures required for the DNA amplification of the MLST genes and subsequent sequence-labeling from meningococcal and pneumococcal isolates. 3. Millipore MultiScreen™ 96- or 384-PCR plate. This removes all residual primers and dNTPs that could interfere with sequence labeling or DNA sequencing (Fig. 1). 4. Millipore MultiScreen™ 96- or 384-SEQ plate. This removes all unincorporated dye terminators and residual primers that could interfere with DNA sequencing (Fig. 1).

Acknowledgments Funding for the liquid handling robot and automated DNA sequencer was generously provided by the Meningitis Association (Scotland) and National Services Division (Scottish Executive). References 1. Schwartz, I. (2000) Microbial genomics: from sequence to function. Emerg. Infect. Dis. 6, 493–495. 2. Boxer, M. (2000) Molecular techniques: divide or share. J. Clin. Pathol. 53, 19–21. 3. Olive, D. M. and Bean, P. (1999) Principles and applications of methods for DNA-based typing of

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Fig. 1. Sequence data quality. PCR and sequence product purification was performed using Millipore Multiscreen™ plates and provided good template quality. (A) Agarose gel analysis. Lanes 1 and 9 are 100bp ladders. Lanes 2–8 are unpurified PCR products while lanes 10–16 are purified PCR products. (B) Sequence analysis on the MegaBACE 1000 using the Cimarron software after sequence product purification. The quality line (shown below the nucleotide sequence) indicates the quality of the sequence.


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microbial organisms. J. Clin. Microbiol. 37, 1661–1669. Weinstock, G. M., Smajs, D., Hardham, J. and Norris, S. J. (2000) From microbial genome sequence to applications. Res. Microbiol. 151, 151–158. Weinstock, G. M. (2000) Genomics and bacterial pathogenesis. Emerg. Infect. Dis. 6, 496–504. Pitt, T. L. and Saunders, N. A. (2000) Molecular bacteriology: a diagnostic tool for the millennium. J. Clin. Pathol. 53, 71–75. Maiden, M. C., Bygraves, J. A., Feil, E., et al. (1998) Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA 95, 3140–3145. Enright, M. C. and Spratt, B. G. (1999) Multilocus sequence typing. Trends Microbiol. 7, 482–487. Enright, M. C. and Spratt, B. G. (1998) A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144 ( Pt 11), 3049–3060.


Clarke and Diggle 10. Lis, J. T. and Schleif, R. (1975) Size fractionation of double-stranded DNA by precipitation with polyethylene glycol. Nucleic Acids Res. 02, 383–389. 11. Baker, M. P., Mitchell, A., Bridge, C., et al. (2001) Isolation of genomic DNA from blood using a novel filter-based DNA purification technology. Biotechniques 31, 142–145. 12. Andersson, B., Lu, J., Edwards, K. E., Muzny, D. M. and Gibbs, R. A. (1996) Method for 96-well M13 DNA template preparations for large-scale sequencing. Biotechniques 20, 1022–1027. 13. Clarke, S. C., Diggle, M. A. and Edwards, G. F. (2001) Semiautomation of Multilocus Sequence Typing for the Characterization of Clinical Isolates of Neisseria meningitidis. J. Clin. Microbiol. 39, 3066–3071. 14. Clarke, S. C., Diggle, M. A., Reid, J. A., Thom, L. and Edwards, G. F. (2001) Introduction of an automated service for the laboratory confirmation of meningococcal disease in Scotland. J. Clin. Pathol. 54, 556–557.

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