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Wo 2007/086935 A2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date PCT (10) International Publication Number 2 August 2007 (02.08.2007) WO 2007/086935 A2 (51) International Patent Classification: Not classified (US). MYERS, Eugene, W. [-/US]; 20 Commercial Street, Branford, CT 06405 (US). SIMPSON, John, (21) International Application Number: W. [US/US]; 23 Woodland Road, Suite B4, Madison, PCT/US2006/030235 CT 06443 (US). VOLKMER, Greg, A. [-/US]; 20 Commercial Street, Branford, CT 06405 (US). (22) International Filing Date: 1 August 2006 (01.08.2006) (74) Agent: HOPKINS, Brian, P.; Mintz, Levin, Cohn, Ferris, (25) Filing Language: English Glovsky And Popeo, Rc, Chrysler Center, 666 Third Av enue, New York, NY 10017 (US). (26) Publication Language: English (81) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of national protection available): AE, AG, AL, AM, 11/195,254 1 August 2005 (01.08.2005) US AT,AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI, (71) Applicant (for all designated States except US): 454 LIFE GB, GD, GE, GH, GM, HN, HR, HU, ID, IL, IN, IS, JP, SCIENCES CORPORATION [US/US]; 20 Commercial KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, Street, Branford, CT 06405 (US). LU, LV,LY,MA, MD, MG, MK, MN, MW, MX, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, (72) Inventors; and SD, SE, SG, SK, SL, SM, SY, TJ, TM, TN, TR, TT, TZ, (75) Inventors/Applicants (for US only): MCDADE, Keith, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW E. [US/US]; 20 Christian Hill Road, Higganum, CT 06441 (US). FIERRO, Joseph, M. [-/US]; 20 Commercial (84) Designated States (unless otherwise indicated, for every Street, Branford, CT 06405 (US). KNIGHT, James, R. kind of regional protection available): ARIPO (BW, GH, [-/US]; 20 Commercial Street, Branford, CT 06405 GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (US). CHARUMILIND, Jerry [-/US]; 20 Commercial ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), Street, Branford, CT 06405 (US). LEAMON, John, H. European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, [US/US]; 152 Nortontown Road, Guilford, CT 06437 FR, GB, GR, HU, IE, IS, IT, LT, LU, LV,MC, NL, PL, PT, [Continued on next page] (54) Title: METHODS OF AMPLIFYING SEQUENCING NUCLEIC ACIDS (57) Abstract: An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step. RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, For two-letter codes and other abbreviations, refer to the "Guid- GN, GQ, GW, ML, MR, NE, SN, TD, TG). ance Notes on Codes and Abbreviations" appearing at the begin- Published ning of each regular issue of the PCT Gazette. — without international search report and to be republished upon receipt of that report METHODS OF AMPLIFYING AND SEQUENCING NUCLEIC ACIDS FIELD OF THE INVENTION 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 Development of rapid and sensitive nucleic acid sequencing methods utilizing automated DNA sequencers has revolutionized modern 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 technical problems for accurate sample preparation, amplification and sequencing. One technical problem which hinders sequence analysis of genomes has been the inability of the investigator to rapidly prepare numerous nucleic acid samples encompassing a complete genome in a short period of time. Another technical problem is the inability to representatively amplify a genome to a level that is compatible with the sensitivity of current sequencing methods. Modern economically feasible sequencing machines, while sensitive, 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 economically. Yet another technical problem is the limitation of current sequencing methods which can perform, at most, one sequencing reaction per hybridization of oligonucleotide primer. The hybridization of sequencing primers is often the rate limiting step constricting the throughput of DNA sequencers. In most cases, Polymerase Chain Reaction (PCR; Saiki, R.K., et ah, Science 1985, 230, 1350-1354; Mullis, K., et al, Cold Spring Harb. Symp. Quant. Biol. 19S6, 5 1 Pt 1, 263- 273) plays an integral part in obtaining DNA sequence information and amplifying minute amounts of specific DNA to obtain concentrations sufficient for sequencing. Yet, scaling current PCR technology to meet the increasing demands of modern genetics is neither cost effective nor efficient, especially when the requirements for full genome sequencing are considered. 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 scalability. 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, L, et al, Lab on a Chip - The Royal Society of Chemistry 2001, 1, 42-49). 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 (Burns, M.A., et al., Proc. Natl. Acad. Sd. USA 1996, 93, 5556-5561). 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). 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 µl. 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 pi reaction pits etched in a single silicon wafer. Unfortunately, recovery and utilization of the amplicon from these methods have proven problematic, requiring evaporation through selectively permeable membranes. 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. 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, 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. BRIEF SUMMARY OF THE INVENTION 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. 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.
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