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Methods of Amplifying and Sequencing Nucleic Acids (19) & (11) EP 2 159 285 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 03.03.2010 Bulletin 2010/09 C12N 15/10 (2006.01) C12Q 1/68 (2006.01) (21) Application number: 09166658.6 (22) Date of filing: 28.01.2004 (84) Designated Contracting States: (72) Inventors: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR • Leamon, John H. HU IE IT LI LU MC NL PT RO SE SI SK TR Guilford, CT 06437 (US) Designated Extension States: • Lohman, Kenton AL LT LV MK Guilford, CT 06437 (US) • Rothberg, Jonathan M. (30) Priority: 29.01.2003 US 443471 P Guilford, CT 06437 (US) 23.04.2003 US 465071 P • Weiner, Michael P. 06.06.2003 US 476602 P Guilford, CT 06437 (US) 06.06.2003 US 476504 P 06.06.2003 US 476313 P (74) Representative: Aston, Heidi Frances 06.06.2003 US 476592 P Mintz, Levin, Cohn, Ferris, Glovsky and 25.08.2003 US 497985 P Popeo IP, LLP Alder Castle (62) Document number(s) of the earlier application(s) in 10 Noble Street accordance with Art. 76 EPC: London EC2V 7JX (GB) 04785758.6 / 1 590 477 Remarks: (71) Applicant: 454 Life Sciences Corporation This application was filed on 28-07-2009 as a Branford CT 06405 (US) divisional application to the application mentioned under INID code 62. (54) Methods of amplifying and sequencing nucleic acids (57) An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is pro- vided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and perform- ing multiple sequencing reaction on the amplified DNA with only one primer hybridization step. EP 2 159 285 A2 Printed by Jouve, 75001 PARIS (FR) EP 2 159 285 A2 Description FIELD OF THE INVENTION 5 [0001] This invention relates to a method and apparatus for determining the base sequences of DNA. More particularly, this invention relates to methods and an apparatus with which the base sequences of a genome can be amplified and determined automatically or semiautomatically. BACKGROUND OF THE INVENTION 10 [0002] Development of rapid and sensitive nucleic acid sequencing methods utilizing automated DNA sequencers has revolutionized modem molecular biology. Analysis of entire genomes of plants, fungi, animals, bacteria, and viruses is now possible with a concerted effort by a series of machines and a team of technicians. However, the goal of rapid and automated or semiautomatic sequencing of a genome in a short time has not been possible. There continues to be 15 technical problems for accurate sample preparation, amplification and sequencing. [0003] One technical problem which hinders sequence analysis of genomes has been the inability of the investigator to rapidly prepare numerous nucleic acid sample encompassing a complete genome in a short period of time. [0004] Another technical problem is the inability to representatively amplified a genome to a level that is compatible with the sensitivity of current sequencing methods. Modem economically feasible sequencing machines, while sensitive, 20 still require in excess of one million copies of a DNA fragment for sequencing. Current methods for providing high copies of DNA sequencing involves variations of cloning or in vitro amplification which cannot amplify the number of individual clones (600,000 or more, and tens of millions for a human genome) necessary for sequencing a whole genome eco- nomically. [0005] Yet another technical problem in the limitation of current sequencing methods which can perform, at most, one 25 sequencing reaction per hybridization of oligonucleotide primer. The hybridization of sequencing primers is often the rate limiting step constricting the throughput of DNA sequencers. [0006] In most cases, Polymerase Chain Reaction (PCR; Saiki, R.K., et al., Science 1985, 230, 1350-1354; Mullis, K., et al., Cold Spring Harb. Symp. Quant. Biol. 1986, 51 Pt 1, 263-273) plays an integral part in obtaining DNA sequence information, amplifying minute amounts of specific DNA to obtain concentrations sufficient for sequencing. Yet, scaling 30 current PCR technology to meet the increasing demands of modem genetics is neither cost effective nor efficient, especially when the requirements for full genome sequencing are considered. [0007] Efforts to maximize time and cost efficiencies have typically focused on two areas: decreasing the reaction volume required for amplifications and increasing the number of simultaneous amplifications performed. Miniaturization confers the advantage of lowered sample and reagent utilization, decreased amplification times and increased throughput 35 scalability. [0008] While conventional thermocyclers require relatively long cycling times due to thermal mass restrictions (Woolley, A.T., et al., Anal. Chem. 1996, 68, 4081-4086), smaller reaction volumes can be cycled more rapidly. Continuous flow PCR devices have utilized etched microchannels in conjunction with fixed temperature zones to reduce reaction volumes to sub-microliter sample levels (Lagally, E.T., et al., Analytical Chemistry 2001, 73, 565-570; Schneegas, I., et al., Lab 40 on a Chip - The Royal Society of Chemistry 2001, 1, 42-49). [0009] Glass microcapillaries, heated by air (Kalinina, O., et al., Nucleic Acids Res. 1997, 25, 1999-2004) or infrared light (Oda, R.P., et al., Anal. Chem. 1998, 70, 4361-4368; Huhmer, A.F. and Landers, J.P., Anal. Chem. 2000, 72, 5507-5512), have also served as efficient vessels for nanoliter scale reactions. Similar reaction volumes have been attained with microfabricated silicon thermocyclers (Bums, M.A., et al., Proc. Natl. Acad. Sci. USA 1996, 93, 5556-5561). 45 [0010] In many cases, these miniaturizations have reduced total PCR reaction times to less than 30 minutes for modified electric heating elements (Kopp, M.U., et al., Science 1998, 280, 1046-1048; Chiou, J., Matsudaira, P., Sonin, A. and Ehrlich, D., Anal. Chem. 2001, 73, 2018-2021) and hot air cyclers (Kalinina, O., et al., Nucleic Acids Res. 1997, 25, 1999-2004), and to 240 seconds for some infrared controlled reactions (Giordano, B.C., et al., Anal. Biochem. 2001, 291, 124-132). 50 [0011] Certain technologies employ increased throughput and miniaturization simultaneously; as in the 1536-well system design by Sasaki et al. (Sasaki, N., et al., DNA Res. 1997, 4, 387-391), which maintained reaction volumes under 1 Pl. As another example, Nagai et al. (Nagai, H., et al., Biosens. Bioelectron. 2001, 16, 1015-1019; Nagai, H., et al., Anal. Chem. 2001, 73, 1043-1047) reported amplification of a single test fragment in ten thousand 86 pl reaction pits etched in a single silicon wafer. Unfortunately, recovery and utilization of the amplicon from these methods have 55 proven problematic, requiring evaporation through selectively permeable membranes. [0012] Despite these remarkable improvements in reactions volumes and cycle times, none of the previous strategies have provided the massively parallel amplification required to dramatically increase throughput to levels required for analysis of the entire human genome. DNA sequencers continue to be slower and more expensive than would be desired. 2 EP 2 159 285 A2 In the pure research setting it is perhaps acceptable if a sequencer is slow and expensive. But when it is desired to use DNA sequencers in a clinical diagnostic setting such inefficient sequencing methods are prohibitive even for a well financed institution.The large-scale parallel sequencing of thousands of clonally amplified targets would greatly facilitate large-scale, whole genome library analysis without the time consuming sample preparation process and expensive, 5 error-prone cloning processes. Successful high capacity, solid-phase, clonal DNA amplification can be used for numerous applications. Accordingly, it is clear that there exists a need for preparation of a genome or large template nucleic acids for sequencing, for amplification of the nucleic acid template, and for the sequencing of the amplified template nucleic acids without the constraint of one sequencing reaction per hybridization. Furthermore, there is a need for a system to connect these various technologies into a viable automatic or semiautomatic sequencing machine. 10 BRIEF SUMMARY OF THE INVENTION [0013] This invention describes an integrated system, comprising novel methods and novel apparatus for (1) nucleic acid sample preparation, (2) nucleic acid amplification, and (3) DNA sequencing. 15 [0014] The invention provides a novel method for preparing a library of multiple DNA sequences, particularly derived from large template DNA or whole (or partial) genome DNA. Sequences of single stranded DNA are prepared from a sample of large template DNA or whole or partial DNA genomes through fragmentation, polishing, adaptor ligation, nick repair, and isolation of single stranded DNA. The method provides for generating a ssDNA library linked to solid supports comprising: (a) generating a library of ssDNA templates; (b) attaching the ssDNA templates to solid supports; and (c) 20 isolating the solid supports on which one ssDNA template is attached. [0015] The invention also provides for a method of amplifying each individual member of a DNA library in a single reaction tube, by, e.g., encapsulating a plurality of DNA samples individually in a microcapsule of an emulsion, performing amplification of the plurality of encapsulated nucleic acid samples simultaneously, and releasing said amplified plurality of DNA from the microcapsules for subsequent reactions. In one embodiment, single copies of the nucleic acid template 25 species are hybridized to DNA capture beads, suspended in complete amplification solution and emulsified into micro- reactors (typically 100 to 200 microns in diameter), after which amplification (e.g., PCR) is used to clonally increase copy number of the initial template species to more than 1,000,000 copies of a single nucleic acid sequence, preferably between 2 and 20 million copies of a single nucleic acid. The amplification reaction, for example, may be performed simultaneously with at least 3,000 microreactors per microliter of reaction mix, and may be performed with over 300,000 30 microreactors in a single 100Pl volume test tube (e.g., a PCR reaction tube).
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