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Processes Useful for Nucleic Acid Amplification and Sequencing, And (19) & (11) EP 0 971 039 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) C12P 19/34 (2006.01) 23.03.2011 Bulletin 2011/12 (21) Application number: 99112181.5 (22) Date of filing: 24.06.1999 (54) Processes useful for nucleic acid amplification and sequencing, and for the production of nucleic acid having decreased thermodynamic stability Verfahren zur Amplifikation und Sequenzierung von Nucleinsäuren und zur Herstellung von Nucleinsäuren mit verminderter thermodynamischer Stabilität Méthode utiles pour l’amplification et pour le séquençage d’acides nucléiques, et la production d’acides nucléiques présentant une stabilité thermodynamique réduite (84) Designated Contracting States: • AUER T ET AL: "SELECTIVE AMPLIFICATION OF DE FR GB IT RNA UTILIZING THE NUCLEOTIDE ANALOG DITPAND THERMUS THERMOPHILUS DNA (30) Priority: 24.06.1998 US 104067 POLYMERASE" NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. (43) Date of publication of application: 24, no. 24, 1996, pages 5021-5025, XP002916438 12.01.2000 Bulletin 2000/02 ISSN: 0305-1048 • GREIN T ET AL: "3 DEAZA AND 7- (73) Proprietor: ENZO DIAGNOSTICS, INC. DEAZAPURINES: DUPLEX STABILITY OF New York, NY 10022 (US) OLIGONUCLEOTIDES CONTAINING MODIFIED ADENNE OR GUANINE BASES" BIOORGANIC & (72) Inventors: MEDICINAL CHEMISTRY LETTERS, OXFORD, • Rabbani, Elazar GB, vol. 4, no. 8, 1994, pages 971-976, New York 10003 (US) XP000601094 ISSN: 0960-894X • Stavrianopolous, Jannis G. • SOWERS L C ET AL: "EQUILIBRIUM BETWEEN Bay Shore, New York 11706 (US) WOBBLE AND IONIZED BASED PAIR FORMED • Donegan, James J. BETWEEN FLUOROURACIL AND GUANINE IN Long Beach, New York 11561 (US) DNA AS STUDIED BY PROTON AND FLUORINE • Coleman, Jack NMR" JOURNAL OF BIOLOGICAL CHEMISTRY, East Northport, New York 11731 (US) vol. 263, no. 29, 1988, pages 14794-14801, • Walner Marleen XP002260887 ISSN: 0021-9258 Farmingdale, New York 11735 (US) • WILTON ET AL: "SNAPBACK SSCP ANALYSIS: ENGINEERED CONFORMATION CHANGES FOR (74) Representative: Vossius & Partner THE RAPID TYPING OF KNOWN MUTATIONS" Siebertstrasse 4 HUMAN MUTATION, WILEY-LISS, NEW YORK, 81675 München (DE) NY, US, vol. 11, 1 March 1998 (1998-03-01), pages 252-258, XP002094957 ISSN: 1059-7794 (56) References cited: • PATEL R ET AL: "Formation of chimeric DNA WO-A-92/00989 WO-A-98/06732 primer extension products by template switching WO-A-98/43991 WO-A1-97/04131 onto an annealed downstream oligonucleotide" DD-A- 265 429 US-A- 4 707 440 PROCEEDINGS OF THE NATIONAL ACADEMY US-A- 5 047 519 US-A- 5 260 433 OF SCIENCES OF USA, NATIONAL ACADEMY OF US-A- 5 270 184 US-A- 5 470 723 SCIENCE. WASHINGTON, US, vol. 93, April 1996 US-A- 5 476 928 (1996-04), pages 2969-2974, XP002158856 ISSN: 0027-8424 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 971 039 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 0 971 039 B1 • WALKER G T ET AL: "STRAND DISPLACEMENT • SAQI J ET AL: "BASE-MODIFIED AMPLIFICATION - AN ISOTHERMAL, IN VITRO OLIGODEOXYNUCLEOTIDES I. EFFECT OF 5- DNA AMPLIFICATION TECHNIQUE" NUCLEIC ALKYL, 5- (1-ALKENYL) AND 5- (1-ALKYNYL) ACIDS RESEARCH, OXFORD UNIVERSITY SUBSTITUTION OF THE PYRIMIDINES ON PRESS, SURREY, GB, vol. 20, no. 7, 1992, pages DUPLEX STABILITY AND HYDROPHOBICITY" 1691-1696, XP002019521 ISSN: 0305-1048 TETRAHEDRON LETTERS, ELSEVIER SCIENCE • KURFURST ET AL: PUBLISHERS, AMSTERDAM, NL, vol. 34, no. 13, "Oligo-.alpha.-deoxyribonucleotides with a 1993, pages 2191-2194, XP001029137 ISSN: modified nucleic base and covalently linked to 0040-4039 reactive agents" TETRAHEDRON, ELSEVIER • SANGHVI Y S: "HETEROCYCLIC BASE SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. MODIFICATIONS IN NUCLEIC ACIDS AND THEIR 49, no. 32, 6 August 1993 (1993-08-06), pages APPLICATIONS IN ANTISENSE 6975-6990, XP002117189 ISSN: 0040-4020 OLIGONUCLEOTIDES" ANTISENSE RESEARCH • NIELSEN P E ET AL: "PEPTIDE NUCLEIC ACIDS AND APPLICATIONS, CRC PRESS, GB, 1993, (PNA): OLIGONUCLEOTIDE ANALOGS WITH A pages 273-288, XP002921486 POLYAMIDE BACKBONE" REACTIVE • HONEYMAN KAITE ET AL: "Development of a DERIVATIVES OF OLIGONUCLEOTIDES IN snapback method of single-strand conformation ANTISENSE RESEARCH AND APPLICATIONS, polymorphism analysis for genotyping Golden CRC PRESS, BOCA RATON, FL,, US, 1993, pages Retrievers for the X-linked muscular dystrophy 363-373, XP000943964 allele." AMERICAN JOURNAL OF VETERINARY RESEARCH, vol. 60, no. 6, June 1999 (1999-06), pages 734-737, XP001096004 ISSN: 0002-9645 2 EP 0 971 039 B1 Description FIELD OF THE INVENTION 5 [0001] This invention relates to the field of recombinant nucleic acid technology, and more particularly, to processes for nucleic acid amplification. BACKGROUND OF THE INVENTION 10 [0002] The first system described for the successful in vitro exponential amplification of target nucleic acids is the Polymerase Chain Reaction (PCR) (Saiki et al., 1985 Science 230; 1350-1354). PCR has been widely used for allele determination, forensic identification, gene analysis, diagnostics, cloning, direct sequencing and other applications. Subsequently, Reverse Transcriptase (RT) was used to transform an RNA molecule into a DNA copy allowing the use of RNA molecules as substrates for PCR amplification by DNA polymerase. In addition, conditions have been described 15 that allow certain DNA polymerases to perform reverse transcription by themselves (Myers, T.W. and Gelfand, D.H. [1991] Biochem. 30; 7661-7666). Finally, Rose et al. (U.S. patent #5,508,178) have described the use of inverted repeat sequences as choices for PCR primer sequences, allowing the use of a single primer to initiate polymerization from each end of a target nucleic acid to create a PCR amplicon that in single-stranded form can be drawn as a "pan-handles" with self complementary sequences at each end. In order to utilize targets that lack inverted repeats, this group has also 20 described various methods to introduce sequences into a PCR amplicon, such that the final product would have self- complementary sequences at each end (U.S. patents #5,439,793, #5,595,891, and #5,612,199). [0003] Both the original PCR amplification system and various improved PCR systems suffer from the limitation of a necessity for expensive dedicated thermocyclers to provide the multiple temperature conditions that are intrinsic to the PCR method. This necessity is derived from the problem that the extension of a primer creates a product that has a 25 stronger association with a template than the primer used to create it. As such, in a system like PCR, temperatures that allow binding of a primer are too low to allow separation of the extended product from its template and temperatures that are elevated enough to allow the separation of the extended product are too high to allow another priming event. The second priming event can not take place until after the first extended strand is separated from its template. As such, in PCR amplification, primer binding to template and the sequential release of the extended primers from the template 30 have to be carried out at separate distinct temperatures and require a thermocycler to provide repeated sequences of distinct thermal steps. The existence of discrete cycles with different conditions also necessitates an optimization of temperature for each individual temperature step as well as an appropriate timing for each step. Similar problems also apply when ligation is used in the LCR reaction (Backman, K. et al. European Patent Application Publication No. 0 320 308, Landegren, U., et al., 1988 Science 241; 1077, Wu, D. and Wallace, R.B. 1989 Genomics 4; 560, Barany, F. 1991 35 Proc. Nat. Acad. Sci. USA 88; 189) where the temperature required for binding individual probes is less than the tem- perature required to release them after they have been stabilized by a ligation event. [0004] Others have recognized these limitations and tried to overcome them by providing means to accomplish multiple cycles under isothermal conditions. Examples of this are 3SR (Kwoh, D.Y. et al., Proc. Nat. Acad. Sci. USA 86; 1173-1177) and NASBA (Kievits, T. et al., 1991 J. Virol. Methods 35; 273-286). 40 [0005] Each of the preceding systems has the limitation of a necessity for the introduction of an RNA promoter into the structure of the nucleic acid being amplified. Consequently, there is also a limitation that these systems are dependent upon a cycling reaction between DNA and RNA forms of the sequence of interest. A dependency upon the production of an RNA intermediate introduces a limitation of susceptibility to RNases, enzymes that are ubiquitous in the environment and are frequently present in biologically derived specimens. In addition, the nature of the design of these amplification 45 systems has the further limitation that they require the presence of four distinct enzymatic activities: DNA polymerase, Reverse Transcriptase, RNase H and RNA polymerase. In the TMA reaction, these activities are provided by the Reverse Transcriptase and RNA polymerase enzyme whereas in 3SR and NASBA they are provided by Reverse Transcriptase, RNase H and RNA polymerase enzymes. Each of these activities is required for the system to be functional, and as such there is a necessity for the manufacturer to test and titrate each function individually, thereby increasing the cost 50 compared to systems that utilize a single enzyme activity. In addition, at a minimum, at least two different enzymes have to be used to provide all the necessary functions, thus rendering these systems more expensive than those that utilize a single enzyme.
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