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Title:
MODIFIED NUCLEOSIDE PHOSPHATES WITH HIGH THERMAL STABILITY
Document Type and Number:
WIPO Patent Application WO/2019/180112
Kind Code:
A1
Abstract:
The present invention provides for stable nucleotide reagents used for nucleic acid amplification by PCR and RT-PCR (Reverse Transcriptase-PCR) that comprises fluorinated nucleoside polyphosphates at the terminal phosphate, e.g. at the gamma-phosphate. The present invention also provides for methods for using the fluorinated nucleoside polyphosphates for detecting the presence or absence of a target nucleic acid sequence in a sample in an amplification reaction.

Inventors:
NIERTH, Alexander (Inc.4300 Hacienda Drive, Pleasanton ., 94588, US)
Application Number:
EP2019/057025
Publication Date:
September 26, 2019
Filing Date:
March 21, 2019
Export Citation:
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Assignee:
F. HOFFMANN-LA ROCHE AG (Grenzacherstrasse 124, 4070 Basel, 4070, CH)
ROCHE DIAGNOSTICS GMBH (Patent Department, Sandhofer Straße 116, Mannheim, 68305, DE)
ROCHE MOLECULAR SYSTEMS, INC. (4300 Hacienda Drive, Pleasanton, California, 94588, US)
International Classes:
C12Q1/686
Domestic Patent References:
WO2002012263A12002-02-14
WO1984003285A11984-08-30
WO1990001069A11990-02-08
WO1992008808A11992-05-29
WO1997046707A21997-12-11
WO1997046714A11997-12-11
WO1997046712A21997-12-11
WO2008046612A12008-04-24
Foreign References:
EP1970445A12008-09-17
US20120231462A12012-09-13
US5693502A1997-12-02
US4458066A1984-07-03
US4683202A1987-07-28
US4683195A1987-07-28
US4800159A1989-01-24
US4965188A1990-10-23
EP0439182A21991-07-31
US5130238A1992-07-14
US20180073056A12018-03-15
US20180073064A12018-03-15
US4666848A1987-05-19
US4889818A1989-12-26
US5455170A1995-10-03
Other References:
STUMBER M ET AL: "Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPgammaNH2 and GTPgammaF", EUROPEAN JOURNAL OF BIOCHEMISTRY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 269, no. 13, 1 January 2002 (2002-01-01), pages 3270 - 3278, XP003014224, ISSN: 0014-2956, DOI: 10.1046/J.1432-1033.2002.03003.X
SUSANNE ERMERT ET AL: "Phosphate-Modified Nucleotides for Monitoring Enzyme Activity", TOPICS IN CURRENT CHEMISTRY, vol. 375, no. 2, 1 April 2017 (2017-04-01), XP055584908, ISSN: 2365-0869, DOI: 10.1007/s41061-017-0117-8
ARZUMANOV A A ET AL: "Gamma-Phosphate-substituted 2'-deoxynucleoside 5'-triphosphates as substrates for DNA polymerases", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 271, no. 40, 4 October 1996 (1996-10-04), pages 24389 - 24394, XP002256215, ISSN: 0021-9258, DOI: 10.1074/JBC.271.40.24389
SMITH, E.S. ET AL.: "PCR Primer: A Laboratory Manual", 2003, COLD SPRING HARBOR LABORATORY PRESS, article "Amplification of RNA: High-temperature Reverse Transcription and DNA Amplification with a Magnesium-activated Thermostable DNA Polymerase", pages: 211 - 219
NARANG S. A. ET AL., METHODS IN ENZYMOLOGY, vol. 68, 1979, pages 90 - 98
BROWN E. L. ET AL., METHODS IN ENZYMOLOGY, vol. 68, 1979, pages 109 - 151
BEAUCAGE ET AL., TETRAHEDRON LETTERS, vol. 22, 1981, pages 1859
GAREGG ET AL., CHEM. SCR., vol. 25, 1985, pages 280 - 282
UHLMANN; PEYMAN, CHEMICAL REVIEWS, vol. 90, 1990, pages 543
VERMA S.; ECKSTEIN F., ANNU. REV. BIOCHEM., vol. 67, 1998, pages 99 - 134
WU D. Y.; WALLACE R. B., GENOMICS, vol. 4, 1989, pages 560 - 69
BARANY F., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 189 - 193
BARANY F., PCR METHODS AND APPLIC., vol. 1, 1991, pages 5 - 16
KWOH D.Y. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 1173 - 1177
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ABRAMSON R. D.; MYERS T. W., CURR OPIN BIOTECHNOL, vol. 4, 1993, pages 41 - 47
VAN LUU-THE ET AL.: "Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction", BIOTECHNIQUES, vol. 38, no. 2, February 2005 (2005-02-01), pages 287 - 293
VAN LUU-THE ET AL., BIOTECHNIQUES, vol. 38, no. 2, February 2005 (2005-02-01), pages 287 - 293
Attorney, Agent or Firm:
SCHWARZ, Ralf (Sandhofer Straße 116, Mannheim, 68305, DE)
Download PDF:
Claims:
CLAIMS:

1. A method of detecting the presence or absence of a target nucleic acid sequence in a sample comprising:

a) performing an amplifying step comprising contacting the sample with amplification reagents to produce an amplification product if the target nucleic acid sequence is present in the sample, and

b) detecting the amplification product,

wherein the amplification reagents comprise a modified nucleoside polyphosphate having a structure of:

where R = purine or pyrimidine base or an analog, and n = 1-4.

2. The method of claim 1 , wherein n = 1.

The method of claim 1 , wherein n = 2.

The method of any one of claims 1 to 3, wherein the amplification reagents further comprise a thermostable inorganic pyrophosphatase.

The method of any one of claims 1 to 4, wherein the amplification reagents further comprise a nucleic acid polymerase.

The method of claim 5, wherein the nucleic acid polymerase possesses reverse transcriptase activity.

7. A method of amplifying a target nucleic acid sequence using amplification reagents, wherein the amplification reagents comprise a nucleoside polyphosphate having a structure of:

where R = purine or pyrimidine base or an analog, and n = 1-4.

8. The method of claim 7, wherein n = 1.

9. The method of claim 7, wherein n = 2.

27

10. The method of any one of claims 7 to 9, wherein the amplification reagents further comprise a thermostable inorganic pyrophosphatase enzyme.

11. The method of any one of claims 7 to 10, wherein the amplification reagents further comprise a nucleic acid polymerase.

12. The method of claim 11, wherein the nucleic acid polymerase possesses reverse transcriptase activity.

13. A reaction mixture for amplification of a target nucleic acid sequence comprising a nucleic acid polymerase, buffer and a nucleoside polyphosphate having a structure of:

where R = purine or pyrimidine base or an analog, and n = 1-4.

14. The reaction mixture of claim 13, wherein n = 1.

15. The reaction mixture of claim 13, wherein n = 2.

16. The reaction mixture of any one of claims 13 to 15 further comprising a thermostable inorganic pyrophosphatase enzyme.

17. The reaction mixture of any one of claims 13 to 16, wherein the nucleic acid polymerase possesses reverse transcriptase activity.

18. A kit for amplification of a target nucleic acid sequence comprising a nucleic acid polymerase, buffer and a nucleoside polyphosphate having a structure of:

where R = purine or pyrimidine base or an analog, and n = 1-4.

19. The kit of claim 18, wherein n = 1.

20. The kit of claim 18, wherein n = 2.

21. The kit of any one of claims 18 to 20 further comprising a thermostable inorganic pyrophosphatase enzyme.

22. The kit of any one of claims 18 to 21, wherein the nucleic acid polymerase possesses reverse transcriptase activity.

Description:
MODIFIED NUCLEOSIDE PHOSPHATES WITH HIGH

THERM AT, STABILITY

FIELD OF THE INVENTION

The present invention provides stable nucleotide reagents, methods for their synthesis, methods for their use, and kits comprising them. The nucleotide reagents are useful in many recombinant DNA and RNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR).

BACKGROUND OF THE INVENTION

Nucleic acid amplification reagents are typically comprised of temperature sensitive components, and therefore must often be stored and shipped at temperature well below ambient temperature. This is particularly the case with deoxyribonucleoside triphosphates or their ribonucleoside triphosphate analogs. These reagents are prone to degradation via loss of consecutive phosphate groups from the termini, resulting in the formation of nucleoside diphosphates and monophosphates, both of which are no longer substrates of nucleic acid polymerases. Furthermore, the buildup of diphosphates can inhibit enzyme activity. The stability of nucleoside polyphosphates can be improved by esterifying the terminal phosphates. For example, dNTP analogs containing a methylester at the terminal phosphate were completely stable under conditions of heat stress that were sufficient to completely degrade normal dNTPs. However, esterification of the terminal phosphate can have a negative effect on the ability of these nucleotides to serve as effective substrates for certain polymerase enzymes. A need exists for having dNTP analogs that are both thermally stable and efficient substrates of polymerase enzymes.

SUMMARY OF THE INVENTION

The present invention provides for stable nucleotide reagents used for nucleic acid amplification by PCR and RT-PCR (Reverse Transcriptase-PCR) that comprises modified nucleoside polyphosphates. The present invention also provides for methods for using the modified nucleoside polyphosphates for detecting the presence or absence of a target nucleic acid sequence in a sample in an amplification reaction.

Therefore, in one aspect, the present invention involves a method of detecting the presence or absence of a target nucleic acid sequence in a sample comprising performing an amplifying step comprising contacting the sample with amplification reagents to produce an amplification product if the target nucleic acid sequence is present in the sample, and detecting the amplification product, wherein the amplification reagents comprise a modified nucleoside polyphosphate having a structure of:

where R = purine or pyrimidine base or an analog, and n = 1-4. In one embodiment, n = 1. In another embodiment, n = 2. In yet another embodiment, n = 3-4. In some embodiments, the amplifying step is repeated at least once. In some embodiments, the amplifying step is repeated at least about 20 times, but may be repeated as many as at least 40, at least 60, or at least 100 times. In some embodiments, the amplification product is detected once after amplification occurred. In some embodiments, the amplification product is detected during and/or after the amplifying step. In some embodiments, the amplification product is detected at a defined time point during and/or after 3, 5, 10, 20, 30, 40, or 50 amplifying steps or during and/or after each amplifying step. In some embodiments, the amplification reagents comprise a thermostable inorganic pyrophosphatase enzyme. In some embodiments, the amplification reagents comprise a nucleic acid polymerase. In some embodiments, the nucleic acid polymerase possesses reverse transcriptase activity.

In another aspect, the present invention involves a method of amplifying a target nucleic acid sequence using amplification reagents wherein the amplification reagents comprise a modified nucleoside polyphosphate having a structure of:

where R = purine or pyrimidine base or an analog, and n = 1-4. In one embodiment, n = 1. In another embodiment, n = 2. In yet another embodiment, n = 3-4. In some embodiments, the amplifying step is repeated at least once. In some embodiments, the amplifying step is repeated at least about 20 times, but may be repeated as many as at least 40, at least 60, or at least 100 times. In some embodiments, the amplification product is detected once after amplification occurred. In some embodiments, the amplification product is detected during and/or after the amplifying step. In some embodiments, the amplification product is detected at a defined time point during and/or after 3, 5, 10, 20, 30, 40, or 50 amplifying steps or during and/or after each amplifying step. In some embodiments, the amplification reagents comprise a thermostable inorganic pyrophosphatase enzyme. In some embodiments, the amplification reagents comprise a nucleic acid polymerase. In some embodiments, the nucleic acid polymerase possesses reverse transcriptase activity.

In another aspect, the present invention involves a composition comprising a modified nucleoside polyphosphate having a structure of:

where R = purine or pyrimidine base or an analog, and n = 1-4. In one embodiment, n = 1. In another embodiment, n = 2. In yet another embodiment, n = 3-4.

In another aspect, the present invention involves either reaction mixtures or kits comprising a nucleic acid polymerase, buffer, and a modified nucleoside polyphosphate having a structure

where R = purine or pyrimidine base or an analog, and n = 1-4. In one embodiment, n = 1. In another embodiment, n = 2. In yet another embodiment, n = 3-4. In some embodiments, the reaction mixtures of kits comprise a thermostable inorganic pyrophosphatase enzyme. In some embodiments, the nucleic acid polymerase possesess reverse transcriptase activity. In some embodiments, the kit components are included in the kit as separate components, in separate vials or containers. In some embodiments, one or more of the kit components is included in the kit in the same vial or container.

The embodiments and advantages of the invention are described in more detail in the Detailed Description of the Invention and in the Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of fluorinated deoxyribonucleoside polyphosphate analogs that were synthesized.

FIG. 2 is a schematic for the chemical synthesis of 2’ -deoxyribonucleoside 5’-0-(Y-fluoro- triphosphate) (dNTP-yF) sodium salt. FIG. 3 is a schematic for the chemical synthesis of 2’-deoxyribonucleoside 5’-0-(5-fluoro- tetraphosphate) (dN4P-5F) sodium salt.

FIG. 4 shows the thermostability of natural dUTP and dUTP-yF at 65°C.

FIG. 5 shows the growth curves of quantitative PCR amplification reactions from a DNA target, using Z05 polymerase and rTh pyrophosphatase enzymes.

FIG. 6 shows the growth curves of quantitative PCR amplification reactions from a DNA target, using Z05 polymerase and TIP pyrophosphatase enzymes.

FIG. 7 shows the growth curves of reverse transcriptase-quantitative PCR amplification reactions from an armored HIV-l RNA target, using C21 polymerase and rTh pyrophosphatase enzymes.

FIG. 8 shows the growth curves of reverse transcriptase-quantitative PCR amplification reactions from an armored HIV-l RNA target, using C21 polymerase and TIP pyrophosphatase enzymes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for stable nucleotide reagents used for nucleic acid amplification by PCR and RT-PCR (Reverse Transcriptase-PCR) that comprises fluorinated deoxyribonucleoside polyphosphates. The present invention also provides for methods for using the fluorinated deoxyribonucleoside polyphosphates for detecting the presence or absence of a target nucleic acid sequence in a sample in an amplification reaction and for amplifying a target nucleic acid sequence in a sample. The increased stability of the fluorinated deoxyribonucleoside polyphosphates allows long-term storage in complex aqueous mixtures, such as mastermix reagents used in PCR-based diagnostic tests.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although essentially any methods and materials similar to those described herein can be used in the practice or testing of the present invention, only exemplary methods and materials are described. For purposes of the present invention, the following terms are defined below.

The terms“a,”“an,” and“the” include plural referents, unless the context clearly indicates otherwise.

The term “ambient temperature” refers to the temperature of the surrounding and is synonymous with“room temperature” when referring to the temperature of a temperature- controlled indoor building. Typically, ambient temperature refers to a temperature range of between l5°C and 25°C although slightly cooler or warmer temperatures may still be considered within the range of ambient temperature.

The term“aptamer” refers to a single-stranded DNA that recognizes and binds to DNA polymerase, and efficiently inhibits the polymerase activity as described in U.S. Pat. No. 5,693,502. Use of aptamer and dUTP/UNG in RT-PCR is also discussed, for example, in Smith, E.S. et al, (Amplification of RNA: High-temperature Reverse Transcription and DNA Amplification with a Magnesium-activated Thermostable DNA Polymerase, in PCR Primer: A Laboratory Manual, 2nd Edition, Dieffenbach, C.W. and Dveksler, G.S., Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 211-219, (2003)).

“Recombinant”, as used herein, refers to an amino acid sequence or a nucleotide sequence that has been intentionally modified by recombinant methods. By the term“recombinant nucleic acid” herein is meant a nucleic acid, originally formed in vitro, in general, by the manipulation of a nucleic acid by restriction endonucleases, in a form not normally found in nature. Thus an isolated, mutant DNA, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell, it will replicate non-recombinantly, /. e. , using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention. A“recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.

A nucleic acid is“operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

The term“host cell” refers to both single-cellular prokaryote and eukaryote organisms ( e.g ., bacteria, yeast, and actinomycetes) and single cells from higher order plants or animals when being grown in cell culture.

The term“vector” refers to a piece of DNA, typically double-stranded, which may have inserted into it a piece of foreign DNA. The vector or may be, for example, of plasmid origin. Vectors contain“replicon” polynucleotide sequences that facilitate the autonomous replication of the vector in a host cell. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell, which, for example, replicates the vector molecule, encodes a selectable or screenable marker, or encodes a transgene. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of or coincidental with the host chromosomal DNA, and several copies of the vector and its inserted DNA can be generated. In addition, the vector can also contain the necessary elements that permit transcription of the inserted DNA into an mRNA molecule or otherwise cause replication of the inserted DNA into multiple copies of RNA. Some expression vectors additionally contain sequence elements adjacent to the inserted DNA that increase the half-life of the expressed mRNA and/or allow translation of the mRNA into a protein molecule. Many molecules of mRNA and polypeptide encoded by the inserted DNA can thus be rapidly synthesized.

“Amplification reagents” are chemical or biochemical components that enable the amplification of nucleic acids. Such reagents comprise, but are not limited to, nucleic acid polymerases, buffers, mononucleotides such as nucleoside triphosphates, oligonucleotides e.g. as oligonucleotide primers, salts and their respective solutions, detection probes, dyes, and more.

As is known in the art, a“nucleoside” is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines.

“Nucleotides” are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2’-, 3’- or 5’-hydroxyl moiety of the sugar. A nucleotide is the monomeric unit of an“oligonucleotide”, which can be more generally denoted as an“oligomeric compound”, or a“polynucleotide”, more generally denoted as a“polymeric compound”. Another general expression for the aforementioned is deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

An“oligomeric compound” is a compound consisting of“monomeric units” which may be nucleotides alone or non-natural compounds (see below), more specifically modified nucleotides (or nucleotide analogs) or non-nucleotide compounds, alone or combinations thereof.

“Oligonucleotides” and “modified oligonucleotides” (or “oligonucleotide analogs”) are subgroups of oligomeric compounds. The term“oligonucleotide” refers to components formed from a plurality of nucleotides as their monomeric units. The phosphate groups are commonly referred to as forming the intemucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3’ to 5’ phosphodiester linkage. Oligonucleotides and modified oligonucleotides (see below) useful for the invention may be synthesized as principally described in the art and known to the expert in the field. Methods for preparing oligomeric compounds of specific sequences are known in the art, and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods may include, for example, the phosphotriester method described by Narang S. A. et al, Methods in Enzymology 68 (1979) 90-98, the phosphodiester method disclosed by Brown E. L. et al, Methods in Enzymology 68 (1979) 109-151, the phosphoramidite method disclosed in Beaucage et al, Tetrahedron Letters 22 (1981) 1859, the H-phosphonate method disclosed in Garegg et al, Chem. Scr. 25 (1985) 280-282 and the solid support method disclosed in US 4,458,066.

In the process described above, the oligonucleotides may be chemically modified, i.e. the primer and/ or the probe comprise a modified nucleotide or a non-nucleotide compound. The probe or the primer is then a modified oligonucleotide.

“Modified nucleotides” (or“nucleotide analogs”) differ from a natural nucleotide by some modification but still consist of a base, a pentofuranosyl sugar, a phosphate portion, base-like, pentofuranosyl sugar-like and phosphate-like portion or combinations thereof. For example, a label may be attached to the base portion of a nucleotide whereby a modified nucleotide is obtained. A natural base in a nucleotide may also be replaced by e.g. a 7-deazapurine whereby a modified nucleotide is obtained as well.

The term “methylated dNTP” or “methylated-dNTPs” refers to any deoxyribonucleoside triphosphate or dNTP having one or more added methyl groups to the triphosphate moiety. Examples include methylated dATP, methylated dCTP, methylated dGTP, methylated dTTP, and methylated dUTP. One or more methyl groups may esterify one or more of any of the a-, b-, or g-phosphate groups found in dNTPs. In a particular example, the methylated-dNTPs are such that the terminal g-phosphate group of each dNTP is modified with a methyl group, which yields a more stable dNTP component for use in amplification reactions, such as PCR, as compared with conventional unmodified dNTPs.

The term“fluorinated deoxyribonucleoside polyphosphate” refers to any deoxyribonucleoside polyphosphate that was fluorinated at one of the phosphate groups. Examples include dATP- yF, dCTP-yF, dGTP-yF, dTTP-yF, and dUTP-yF. Further examples include dA4P-6F, dC4P-6F, dG4P-6F, dT4P-6F, and dU4P-6F. The fluorine atom may replace one oxygen at one of any of the a-, b-, g-, d-, e-, z-phosphate groups found in deoxyribonucleoside polyphosphates. In a particular example, the fluorinated polyphosphates are such that the terminal phosphate group of each deoxyribonucleoside polyphosphate is fluorinated, which yields a more stable polyphosphate component for use in amplification reactions, such as PCR, as compared with conventional unmodified dNTPs.

The terms “thermostable inorganic pyrophosphatase” and “thermostable inorganic pyrophosphatase enzyme” are used interchangeably in this text and refer to an enzyme that catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate: P2O7 4 + H2O -> 2HPO4 2 and is able to retain activity at temperatures used in PCR conditions (e.g. over 90°C). An example of a thermostable inorganic pyrophosphatase is one that is derived from the thermophile organism Thermococcus litoralis which is commercially available from New England BioLabs (Ipswich, MA).

A“modified oligonucleotide” (or“oligonucleotide analog”), belonging to another specific subgroup of oligomeric compounds, possesses one or more nucleotides and one or more modified nucleotides as monomeric units. Thus, the term“modified oligonucleotide” (or “oligonucleotide analog”) refers to structures that function in a manner substantially similar to oligonucleotides and can be used interchangeably in the context of the present invention. From a synthetical point of view, a modified oligonucleotide (or an oligonucleotide analog) can for example be made by chemical modification of oligonucleotides by appropriate modification of the phosphate backbone, ribose unit or the nucleotide bases (Uhlmann and Peyman, Chemical Reviews 90 (1990) 543; Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134). Representative modifications include phosphorothioate, phosphorodithioate, methylphos- phonate, phosphotriester or phosphoramidate inter-nucleoside linkages in place of phosphodiester intemucleoside linkages; deaza- or azapurines and -pyrimidines in place of natural purine and pyrimidine bases, pyrimidine bases having substituent groups at the 5 or 6 position; purine bases having altered substituent groups at the 2, 6 or 8 positions or 7 position as 7-deazapurines; bases carrying alkyl-, alkenyl-, alkinyl or aryl-moieties, e.g. lower alkyl groups such as methyl, ethyl, propyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or aryl groups like phenyl, benzyl, naphtyl; sugars having substituent groups at, for example, their 2’ position; or carbocyclic or acyclic sugar analogs. Other modifications are known to those skilled in the art. Such modified oligonucleotides (or oligonucleotide analogs) are best described as being functionally interchangeable with, yet structurally different from, natural oligonucleotides. In more detail, exemplary modifications are disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134 or WO 02/12263. In addition, modification can be made wherein nucleoside units are joined via groups that substitute for the intemucleoside phosphate or sugar phosphate linkages. Such linkages include those disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134. When other than phosphate linkages are utilized to link the nucleoside units, such structures have also been described as“oligonucleosides”.

A“nucleic acid” as well as the“target nucleic acid” is a polymeric compound of nucleotides as known to the expert skilled in the art.“Target nucleic acid” is used herein to denote a nucleic acid in a sample which should be analyzed, i.e. the presence, non-presence and/or amount thereof in a sample should be determined.

The term“primer” is used herein as known to the expert skilled in the art and refers to oligomeric compounds, primarily to oligonucleotides, but also to modified oligonucleotides that are able to prime DNA synthesis by a template-dependent DNA polymerase, i.e. the 3’ -end of the e.g. primer provides a free 3’-OH group whereto further nucleotides may be attached by a template-dependent DNA polymerase establishing 3’- to 5’-phosphodiester linkage whereby deoxyribonucleoside triphosphates are used and whereby pyrophosphate is released.

A“probe” also denotes a natural or modified oligonucleotide. As known in the art, a probe serves the purpose to detect an analyte or amplificate. In the case of the process described above, probes can be used to detect the amplificates of the target nucleic acids. For this purpose, probes typically carry labels.

“Fabels”, often referred to as“reporter groups”, are generally groups that make a nucleic acid, in particular oligonucleotides or modified oligonucleotides, as well as any nucleic acids bound thereto distinguishable from the remainder of the sample (nucleic acids having attached a label can also be termed labeled nucleic acid binding compounds, labeled probes or just probes). Exemplary labels are fluorescent labels, which are e.g. fluorescent dyes such as a fluorescein dye, a rhodamine dye, a cyanine dye, and a coumarin dye. Exemplary fluorescent dyes are FAM, HEX, JA270, CAF635, Coumarin343, Quasar705, Cyan500, CY5.5, EC-Red 640, LC- Red 705.

In some embodiments, the label may be attached to the purine or pyrimidine base or an analog via a“linker” or a“linker arm”. The length of a linker arm is the distance in Angstroms (A) from the nucleotide base to the fluorescent moiety. In general, a linker arm is from about 10 A to about 25 A. The linker arm may be of the kind described in WO 84/03285. WO 84/03285 also discloses methods for attaching linker arms to a particular nucleotide base, and also for attaching fluorescent moieties to a linker arm. Exemplarily, an acceptor fluorescent moiety, such as an LC Red 640-NHS-ester, can be combined with C6-Phosphoramidites (available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.)) to produce LC Red 640- Phosphoramidite. Frequently used linkers to couple a donor fluorescent moiety such as fluorescein to an oligonucleotide include thiourea linkers (FITC-derived, for example, fluorescein-CPG’s from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3’-amino-CPGs that require coupling of a fluorescein-NHS-ester after oligonucleotide synthesis. Moreover, other non-nucleoside linkers can be, for example, aliphatic, aromatic, aryl, cyclic, chiral, achiral, a peptide, a carbohydrate, a lipid, a fatty acid, tri-, tetra-, penta-, hexa-polyethylene glycol (HEG) or poly-polyethylene glycol (PEG), or a heterocyclic moiety. Other conventional non-nucleoside linkers employ homobifunctional and heterobifunctional crosslinking reagents. Homobifunctional reagents carry two identical functional groups, whereas heterobifunctional reagents contain two dissimilar functional groups to link the biologies to the bioadhesive. A vast majority of the heterobifunctional cross- linking agents contain a primary amine-reactive group and a thiol-reactive group. Covalent crosslinking agents are selected from reagents capable of forming disulfide (S— S), glycol (— CH(OH)— CH(OH)— ), azo (— N=N— ), sulfone (— S(=02— ), ester (— C(=0)~ O— ), or amide (— C( = 0)— N— ) bridges.

Any primer and/or probe may be chemically modified, i.e. the primer and/ or the probe comprise a modified nucleotide or a non-nucleotide compound. The probe or the primer is then a modified oligonucleotide.

A method of nucleic acid amplification is the Polymerase Chain Reaction (PCR) which is disclosed, among other references, in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188. PCR typically employs two or more oligonucleotide primers that bind to a selected nucleic acid template (e.g. DNA or RNA). Primers useful for nucleic acid analysis include oligonucleotides capable of acting as a point of initiation of nucleic acid synthesis within the nucleic acid sequences of the target nucleic acids. A primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically. The primer can be single- stranded for maximum efficiency in amplification, but the primer can be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double stranded nucleic acids is by heating. A“thermostable polymerase” is a polymerase enzyme that is heat stable, i.e., it is an enzyme that catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded template nucleic acids. Generally, the synthesis is initiated at the 3’ end of each primer and proceeds in the 5’ to 3’ direction along the template strand. Thermostable polymerases have e.g. been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable also can be employed in PCR assays provided the enzyme is replenished.

If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One method of separating the nucleic acid strands involves heating the nucleic acid until it is predominately denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditions necessary for denaturing template nucleic acid will depend, e.g. , on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90°C to about l05°C for a time depending on features of the reaction such as temperature and the nucleic acid length. Denaturation is typically performed for about 5 s to 9 min In order to not expose the respective polymerase like e.g. the Z05 DNA Polymerase to such high temperatures for too long and thus risking a loss of functional enzyme, it can be preferred to use short denaturation steps.

If the double-stranded template nucleic acid is denatured by heat, the reaction mixture is allowed to cool to a temperature that promotes annealing of each primer to its target sequence on the target nucleic acids.

The temperature for annealing can be from about 35°C to about 70°C, or about 45°C to about 65°C; or about 50°C to about 60°C, or about 55°C to about 58°C. Annealing times can be from about 10 s to about 1 min (e.g., about 20 s to about 50 s; about 30 s to about 40 s). In this context, it can be advantageous to use different annealing temperatures in order to increase the inclusivity of the respective assay. In brief, this means that at relatively low annealing temperatures, primers may also bind to targets having single mismatches, so variants of certain sequences can also be amplified. This can be desirable if e.g. a certain organism has known or unknown genetic variants which should also be detected. On the other hand, relatively high annealing temperatures bear the advantage of providing higher specificity, since towards higher temperatures the probability of primer binding to not exactly matching target sequences continuously decreases. In order to benefit from both phenomena, in some embodiments of the invention the process described above comprises annealing at different temperatures, for example first at a lower, then at a higher temperature. If, e.g. , a first incubation takes place at 55°C for about 5 cycles, non-exactly matching target sequences may be (pre-)amplified. This can be followed e.g. by about 45 cycles at 58°C, providing for higher specificity throughout the major part of the experiment. This way, potentially important genetic variants are not missed, while the specificity remains relatively high.

The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient for extension to occur from the annealed primer to generate products complementary to the nucleic acid to be analyzed. The temperature should be sufficient to synthesize an extension product from each primer that is annealed to a nucleic acid template, but should not be so high as to denature an extension product from its complementary template (e.g., the temperature for extension generally ranges from about 40° to 80°C (e.g., about 50°C to about 70°C; about 65°C). Extension times can be from about 10 s to about 5 min, or about 15 s to 2 min, or about 20 s to about 1 min, or about 25 s to about 35 s. The newly synthesized strands form a double-stranded molecule that can be used in the succeeding steps of the reaction. The steps of strand separation, annealing, and elongation can be repeated as often as needed to produce the desired quantity of amplification products corresponding to the target nucleic acids. The limiting factors in the reaction are the amounts of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) can be repeated at least once. For use in detection, the number of cycling steps will depend, e.g., on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficient for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated as many as 40, 60, or even 100 times.

PCR can be carried out in which the steps of annealing and extension are performed in the same step (one-step PCR) or, as described above, in separate steps (two-step PCR). Performing annealing and extension together and thus under the same physical and chemical conditions, with a suitable enzyme such as, for example, the Z05 DNA polymerase, bears the advantage of saving the time for an additional step in each cycle, and also abolishing the need for an additional temperature adjustment between annealing and extension. Thus, the one-step PCR reduces the overall complexity of the respective assay. In general, shorter times for the overall amplification can be preferred, as the time-to-result is reduced and leads to a possible earlier diagnosis.

Other nucleic acid amplification methods to be used comprise the Ligase Chain Reaction (LCR; Wu D. Y. and Wallace R. B., Genomics 4 (1989) 560-69; and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991)189-193); Polymerase Ligase Chain Reaction (Barany F., PCR Methods and Applic. 1 (1991) 5-16); Gap-LCR (WO 90/01069); Repair Chain Reaction (EP 0439182 A2), 3SR (Kwoh D.Y. et al, Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli J.C. et al, Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; WO 92/08808), and NASBA (US 5,130,238). Further, there are strand displacement amplification (SDA), transcription mediated amplification (TMA), and Qb-amplification (for a review see e.g. Whelen A. C. and Persing D. H., Annu. Rev. Microbiol. 50(1996) 349-373; Abramson R. D. and Myers T. W., Curr Opin Biotechnol 4 (1993) 41-47).

The term“Cp value” or“crossing point” value refers to a value that allows quantification of input target nucleic acids. The Cp value can be determined according to the second-derivative maximum method (Van Luu-The et al, “Improved real-time RT-PCR method for high- throughput measurements using second derivative calculation and double correction,” BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). In the second derivative method, a Cp corresponds to the first peak of a second derivative curve. This peak corresponds to the beginning of a log-linear phase. The second derivative method calculates a second derivative value of the real-time fluorescence intensity curve, and only one value is obtained. The original Cp method is based on a locally defined, differentiable approximation of the intensity values, e.g., by a polynomial function. Then the third derivative is computed. The Cp value is the smallest root of the third derivative. The Cp can also be determined using the fit point method, in which the Cp is determined by the intersection of a parallel to the threshold line in the log- linear region (Van Luu-The et al, BioTechniques, Vol. 38, No. 2, February 2005, pp. 287- 293). The Cp value provided by the LightCycler instrument offered by Roche by calculation according to the second-derivative maximum method.

The term“PCR efficiency” refers to an indication of cycle to cycle amplification efficiency. PCR efficiency is calculated for each condition using the equation: % PCR efficiency = (10 1 x 100, wherein the slope was calculated by linear regression with the log copy number plotted on the y-axis and Cp plotted on the x-axis. PCR efficiency can be measured using a perfectly matched or mismatched primer template. The term“FRET” or“fluorescent resonance energy transfer” or“Foerster resonance energy transfer” refers to a transfer of energy between at least two chromophores, a donor chromophore and an acceptor chromophore (referred to as a quencher). The donor typically transfers the energy to the acceptor when the donor is excited by light radiation with a suitable wavelength. The acceptor typically re-emits the transferred energy in the form of light radiation with a different wavelength. When the acceptor is a “dark” quencher, it dissipates the transferred energy in a form other than light. Whether a particular fluorophore acts as a donor or an acceptor depends on the properties of the other member of the FRET pair. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYF and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry™ Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

The methods set out above can be based on Fluorescence Resonance Energy Transfer (FRET) between a donor fluorescent moiety and an acceptor fluorescent moiety. A representative donor fluorescent moiety is fluorescein, and representative corresponding acceptor fluorescent moieties include EC-Red 640, EC-Red 705, Cy5, and Cy5.5. Typically, detection includes exciting the sample at a wavelength absorbed by the donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by the corresponding acceptor fluorescent moiety. In the process according to the invention, detection can be followed by quantitating the FRET. For example, detection is performed after each cycling step. For example, detection is performed in real time. By using commercially available real-time PCR instrumentation ( e.g ., FightCycler™ or TaqMan®), PCR amplification and detection of the amplification product can be combined in a single closed cuvette with dramatically reduced cycling time. Since detection occurs concurrently with amplification, the real-time PCR methods obviate the need for manipulation of the amplification product, and diminish the risk of cross-contamination between amplification products. Real-time PCR greatly reduces tum-around time and is an attractive alternative to conventional PCR techniques in the clinical laboratory.

The following patent applications describe real-time PCR as used in the FightCycler® technology: WO 97/46707, WO 97/46714 and WO 97/46712. The FightCycler® instrument is a rapid thermal cycler combined with a micro volume fluorometer utilizing high quality optics. This rapid thermocycling technique uses thin glass cuvettes as reaction vessels. Heating and cooling of the reaction chamber are controlled by alternating heated and ambient air. Due to the low mass of air and the high ratio of surface area to volume of the cuvettes, very rapid temperature exchange rates can be achieved within the thermal chamber.

TaqMan® technology utilizes a single-stranded hybridization probe labeled with two fluorescent moieties. When a first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to a second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is generally a quencher molecule. Typical fluorescent dyes used in this format are for example, among others, FAM, HEX, CY5, JA270, Cyan and CY5.5. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product) and is degraded by the 5’ to 3’ exonuclease activity of the Taq or another suitable polymerase as known by the skilled artisan, such as a mutant Z05 polymerase, during the subsequent elongation phase. As a result, the excited fluorescent moiety and the quencher moiety become spatially separated from one another. As a consequence, upon excitation of the first fluorescent moiety in the absence of the quencher, the fluorescence emission from the first fluorescent moiety can be detected.

In both detection formats described above, the intensity of the emitted signal can be correlated with the number of original target nucleic acid molecules.

Recently, methods using“tagged” TaqMan® probes for performing multiplexed PCR assays have been described in U.S. Patent Publication No. 2018/0073056 and U.S. Patent Publication No. 2018/0073064.

As an alternative to FRET, an amplification product can be detected using a double-stranded DNA binding dye such as a fluorescent DNA binding dye ( e.g ., SYBRGREEN I® or SYBRGOLD® (Molecular Probes)). Upon interaction with the double-stranded nucleic acid, such fluorescent DNA binding dyes emit a fluorescence signal after excitation with light at a suitable wavelength. A double-stranded DNA binding dye such as a nucleic acid intercalating dye also can be used. When double-stranded DNA binding dyes are used, a melting curve analysis is usually performed for confirmation of the presence of the amplification product. Molecular beacons in conjunction with FRET can also be used to detect the presence of an amplification product using the real-time PCR methods of the invention. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and the fluorescent labels are typically located at each end of the probe. Molecular beacon technology uses a probe oligonucleotide having sequences that permit secondary structure formation (e.g. a hairpin). As a result of secondary structure formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution. After hybridization to the amplification products, the secondary structure of the probe is disrupted and the fluorescent moieties become separated from one another such that after excitation with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.

Thus, a method according to the invention is the method described above using FRET, wherein said probes comprise a nucleic acid sequence that permits secondary structure formation, wherein said secondary structure formation results in spatial proximity between said first and second fluorescent moiety.

Efficient FRET can only take place when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety.

Thus, in an embodiment, said donor and acceptor fluorescent moieties are within no more than five nucleotides of each other on said probe. In a further embodiment, said acceptor fluorescent moiety is a quencher.

As described above, in the TaqMan® format, during the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product) and is degraded by the 5’- to 3’ -exonuclease activity of the Taq or another suitable polymerase as known by the skilled artisan, such as a mutant Z05 polymerase, during the subsequent elongation phase. Thus, in an embodiment, in the process described above, amplification employs a polymerase enzyme having 5’- to 3’ -exonuclease activity.

It is further advantageous to carefully select the length of the amplicon that is yielded as a result of the process described above. Generally, relatively short amplicons increase the efficiency of the amplification reaction. Thus, an aspect of the invention is the process described above, wherein the amplified fragments comprise up to 450 bases, up to 300 bases, up to 200 bases, or up to 150 bases.

A“sequence” is the primary structure of a nucleic acid, i.e. the specific arrangement of the single nucleobases of which the respective nucleic acids consists. It has to be understood that the term“sequence” does not denote a specific type of nucleic acid such as RNA or DNA, but applies to both as well as to other types of nucleic acids such as e.g. PNA or others. Where nucleobases correspond to each other, particularly in the case of uracil (present in RNA) and thymine (present in DNA), these bases can be considered equivalent between RNA and DNA sequences, as well-known in the pertinent art. Clinically relevant nucleic acids are often DNA which can be derived e.g. from DNA viruses like e.g. Hepatitis B Virus (HBV), Cytomegalovirus (CMV) and others, or bacteria like e.g. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and others. In such cases, it can be advantageous to use an internal control nucleic acid consisting of DNA, in order to reflect the target nucleic acids properties. The terms“cell”,“cell line”, and“cell culture” can be used interchangeably and all such designations include progeny. Thus, the words“transformants” or “transformed cells” include the primary transformed cell and cultures derived from that cell without regard to the number of transfers. All progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included in the definition of transformants. The cells can be prokaryotic or eukaryotic.

The term“control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for procaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, positive retroregulatory elements (see U.S. Pat. No. 4,666,848), and possibly other sequences. Eucaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

The term“operably linked” refers to the positioning of the coding sequence such that control sequences will function to drive expression of the protein encoded by the coding sequence. Thus, a coding sequence“operably linked” to control sequences refers to a configuration wherein the coding sequences can be expressed under the direction of a control sequence.

The terms“restriction endonucleases” and“restriction enzymes” refer to enzymes, typically bacterial in origin, which cut double-stranded DNA at or near a specific nucleotide sequence. Families of amino acid residues having similar side chains are defined herein. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine, glycine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The term“reagent solution” is any solution containing at least one reagent needed or used for PCR purposes. Most typical ingredients are polymerase, nucleotide, primer, ions, magnesium, salts, pH buffering agents, nucleoside triphosphates (NTPs) or deoxyribonucleoside triphosphates (dNTPs), one or more nucleic acid probes, fluorescent dye (may be attached to probe), nucleic acid binding agent, a nucleic acid template. The reagent may also be other polymerase reaction additive, which has an influence on the polymerase reaction or its monitoring.

The term“mastermix” refers to a mixture of all or most of the ingredients or factors necessary for PCR to occur, and in some cases, all except for the template and primers which are sample and amplicon specific. Commercially available mastermixes are usually concentrated solutions. A mastermix may contain all the reagents common to multiple samples, but it may also be constructed for one sample only. Using mastermixes helps to reduce pipetting errors and variations between samples due to differences between pipetted volumes.

The term“thermostable polymerase” refers to an enzyme that is stable to heat, is heat resistant and retains sufficient activity to effect subsequent primer extension reactions after being subjected to the elevated temperatures for the time necessary to denature double-stranded nucleic acids. Heating conditions necessary for nucleic acid denaturation are well known in the art and are exemplified in U.S. Patent Nos. 4,965,188 and 4,889,818. As used herein, a thermostable polymerase is suitable for use in a temperature cycling reaction such as PCR. The examples of thermostable nucleic acid polymerases include Thermus aquaticus Taq DNA polymerase, Thermus sp. Z05 polymerase, Thermus flavus polymerase, Thermotoga maritima polymerases, such as TMA-25 and TMA-30 polymerases, Tth DNA polymerase, and the like.

A“polymerase with reverse transcriptase activity” is a nucleic acid polymerase capable of synthesizing DNA based on an RNA template. It is also capable of replicating a single or double-stranded DNA once the RNA has been reverse transcribed into a single strand cDNA. In an embodiment of the invention, the polymerase with reverse transcriptase activity is thermostable.

In the amplification of an RNA molecule by a DNA polymerase, the first extension reaction is reverse transcription using an RNA template, and a DNA strand is produced. The second extension reaction, using the DNA template, produces a double-stranded DNA molecule. Thus, synthesis of a complementary DNA strand from an RNA template by a DNA polymerase provides the starting material for amplification.

Thermostable DNA polymerases can be used in a coupled, one-enzyme reverse transcription/amplification reaction. The term“homogeneous”, in this context, refers to a two- step single addition reaction for reverse transcription and amplification of an RNA target. By homogeneous it is meant that following the reverse transcription (RT) step, there is no need to open the reaction vessel or otherwise adjust reaction components prior to the amplification step. In a non-homogeneous RT-PCR reaction, following reverse transcription and prior to amplification one or more of the reaction components such as the amplification reagents are e.g. adjusted, added, or diluted, for which the reaction vessel has to be opened, or at least its contents have to be manipulated. Both homogeneous and non-homogeneous embodiments are comprised by the scope of the invention.

Reverse transcription is an important step in an RT-PCR. It is, for example, known in the art that RNA templates show a tendency towards the formation of secondary structures that may hamper primer binding and/or elongation of the cDNA strand by the respective reverse transcriptase. Thus, relatively high temperatures for an RT reaction are advantageous with respect to efficiency of the transcription. On the other hand, raising the incubation temperature also implies higher specificity, i.e. the RT primers will not anneal to sequences that exhibit mismatches to the expected sequence or sequences. Particularly in the case of multiple different target RNAs, it can be desirable to also transcribe and subsequently amplify and detect sequences with single mismatches, e.g. in the case of the possible presence of unknown or rare substrains or subspecies of organisms in the fluid sample.

In order to benefit from both advantages described above, i.e. the reduction of secondary structures and the reverse transcription of templates with mismatches, the RT incubation can be carried out at more than one different temperature.

Therefore, an aspect of the invention is the process described above, wherein said incubation of the polymerase with reverse transcriptase activity is carried out at different temperatures from 30°C to 75°C, or from 45°C to 70°C, or from 55°C to 65°C.

As a further important aspect of reverse transcription, long RT steps can damage the DNA templates that may be present in the fluid sample. If the fluid sample contains both RNA and DNA species, it is thus favorable to keep the duration of the RT steps as short as possible, but at the same time ensuring the synthesis of sufficient amounts of cDNA for the subsequent amplification and optional detection of amplificates.

Thus, an aspect of the invention is the process described above, wherein the period of time for incubation of the polymerase with reverse transcriptase activity is up to 30 min, 20 min, 15 min, 12.5 min, 10 min, 5 min, or 1 min.

A further aspect of the invention is the process described above, wherein the polymerase with reverse transcriptase activity and comprising a mutation is selected from the group consisting of a) a CS5 DNA polymerase

b) a CS6 DNA polymerase

c) a Thermotoga maritima DNA polymerase

d) a Thermus aquaticus DNA polymerase

e) a Thermus thermophilus DNA polymerase

f) a Thermus flavus DNA polymerase

g) a Thermus filiformis DNA polymerase

h) a Thermus sp. spsl 7 DNA polymerase

i) a Thermus sp. Z05 DNA polymerase

j) a Thermotoga neapolitana DNA polymerase

k) a Termosipho africanus DNA polymerase

l) a Thermus caldophilus DNA polymerase

Particularly suitable for these requirements are enzymes carrying a mutation in the polymerase domain that enhances their reverse transcription efficiency in terms of a faster extension rate. Therefore, in the process described above, wherein the polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved nucleic acid extension rate and/or an improved reverse transcriptase activity relative to the respective wildtype polymerase.

In an embodiment, in the process described above, the polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved reverse transcriptase activity relative to the respective wildtype polymerase.

Polymerases carrying point mutations that render them particularly useful are disclosed in WO 2008/046612. In particular, polymerases to be used can be mutated DNA polymerases comprising at least the following motif in the polymerase domain:

T-G-R-L-S-S-Xb7-Xb8-P-N-L-Q-N; wherein Xb7 is an amino acid selected from S or T and wherein Xb8 is an amino acid selected from G, T, R, K, or L, wherein the polymerase comprises 3’-5’ exonuclease activity and has an improved nucleic acid extension rate and/or an improved reverse transcription efficiency relative to the wildtype DNA polymerase, wherein in said wildtype DNA polymerase Xb8 is an amino acid selected from D, E or N.

One example is mutants of the thermostable DNA polymerase from Thermus species Z05 (described e.g. in US 5,455,170), said variations comprising mutations in the polymerase domain as compared with the respective wildtype enzyme Z05. An embodiment for the method according to the invention is a mutant Z05 DNA polymerase wherein the amino acid at position 580 is selected from the group consisting of G, T, R, K and L.

For reverse transcription using a thermostable polymerase, a divalent cation such as Mn 2+ or Mg 2+ is typically included as a salt, for example, manganese chloride (MnCk), or manganese acetate (Mn(OAc)2), or manganese sulfate (MnS0 4 ) or magnesium chloride (MgCk), magnesium acetate (Mg(OAc)2), or magnesium sulfate (MgS0 4 ). If MnCk is included in a reaction containing 50 mM Tricine buffer, for example, the MnCk is generally present at a concentration of 0.5-7.0 mM; 2.5-3.5mM is generally present when 200 mM of each dGTP, dATP, dUTP, and, dCTP are utilized.

A“modified” thermostable polymerase refers to a polymerase in which at least one monomer differs from the reference sequence, such as a native or wild-type form of the polymerase or another modified form of the polymerase. Exemplary modifications include monomer insertions, deletions, and substitutions. Modified polymerases also include chimeric polymerases that have identifiable component sequences (e.g., structural or functional domains, etc.) derived from two or more parents. Also included within the definition of modified polymerases are those comprising chemical modifications of the reference sequence. The examples of modified thermostable polymerases include G46E E678G CS5 DNA polymerase, G46E L329A E678G CS5 DNA polymerase, G46E L329A D640G S671F CS5 DNA polymerase, G46E L329A D640G S671F E678G CS5 DNA polymerase, a G46E E678G CS6 DNA polymerase, Z05 DNA polymerase, DZ05 polymerase, AZ05-Gold polymerase, AZ05R polymerase, E615G Taq DNA polymerase, E678G TMA-25 polymerase, E678G TMA-30 polymerase, and the like.

The term“thermoactive polymerase” refers to an enzyme that is active at the elevated temperatures necessary to ensure specific priming and primer extension (e.g., 55-80°C).

The terms “peptide,” “polypeptide,” and “protein” are used interchangeably. The terms “nucleic acid” and“polynucleotide” are used interchangeably. Amino acid sequences are written from amino terminus to carboxy terminus, unless otherwise indicated. Single-stranded nucleic acid sequences are written 5’ to 3’, unless otherwise indicated. The top strand of a double-stranded nucleic acid sequence is written 5’ to 3’, and the bottom strand is written 3’ to 5’, unless otherwise indicated.

Nucleic acid amplification reagents are typically comprised of temperature sensitive components, and therefore must often be stored and shipped at temperatures well below ambient. This is particularly the case with deoxyribonucleoside triphosphates or their ribonucleoside analogs. These reagents are prone to degradation via loss of consecutive phosphate groups from the terminii, resulting in the formation of nucleoside diphosphates and monophosphates, both of which are no longer active as polymerase substrates.

The stability of nucleoside polyphosphates can be improved by esterifying the terminal phosphates. For example, dNTP analogs containing a methylester at the terminal phosphate were completely stable under conditions of heats stress that were sufficient to completely degrade normal dNTPs. However, esterification of the terminal phosphate can have a negative effect on the ability of nucleotides to serve as effective substrates for certain polymerase enzymes.

The present invention presents a simple and elegant solution to address this problem. The standard dexoynucleoside triphosphates are replaced by analogs whereby the terminal phosphate contains a fluorine moiety as shown below:

where R = purine or pyrimidine base or an analog, and n = 1-4.

The following examples are given to illustrate embodiments of the present invention as it is presently preferred to practice. It will be understood that the examples are illustrative, and that the invention is not be considered as restricted except as indicated in the appended claims.

EXAMPLES

Example 1: Chemical synthesis of 2’-deoxyribonucleoside 5’-0-(y-fluorotriphosphate) and 2’-deoxyribonucleoside 5’-0-(6-fluorotetraphosphate)

The chemical structures of 2’-deoxyribonucleoside 5’-O-fy-fluoro triphosphate) (dNTP-yF) and 2’-deoxyribonucleoside 5’-0-(5-fluorotetraphosphate) (dN4P-5F) are shown in FIG. 1 and schematics of their synthesis as sodium salts are shown in FIG. 2 and FIG. 3, respectively.

Abbreviations: DEPC = diethyl pyrocarbonate, dATP = 2’-deoxyadenosine 5’ -triphosphate, dCTP = 2’-deoxycytidine 5’ -triphosphate, dGTP = 2’-deoxyguanosine 5’ -triphosphate, DMF = NJT -dimethylformamide, DMSO = dimethyl sulfoxide, dUTP = 2’-deoxyuridine 5'- triphosphate, MeCN = acetonitrile, qPCR = real-time polymerase chain reaction, RT = room temperature, RT-qPCR = reverse transcription polymerase chain reaction, TBAF = tetrabutylammonium fluoride, THF = tetrahydrofuran, UPLC-MS = ultra-performance liquid chromatography coupled to a mass spectrometer.

A. Synthesis of 2’-deoxyadenosine 5’-0-(y-fhiorotriphosphate), dATP-yF

The sodium salt of dATP (1.5 mmol) was converted to the triethylammonium salt and lyophilized to dryness. The compound was dissolved in dry MeCN and transferred to the reaction flask. Subsequently, the solvent was evaporated and the residue was dried under high vacuum. Under argon the triethylammonium triphosphate was dissolved in an anhydrous mixture of DMF (7.7 mL) and tributylamine (968 pL), followed by dropwise addition of 1- fluoro-2, 4-dinitrobenzene (562 pL). An immediate coloration of the reaction mixture to yellow was observed, which turned to dark orange within few minutes. The reaction solution was stirred for further 30 min at RT, followed by the addition of anhydrous TBAF (4.48 mL, 1.0 M in THF). After stirring overnight at RT, UPLC-MS analysis indicated full consumption of the starting material. An inverse quench of the reaction was performed by dropwise addition of the reaction solution to a stirring solution of sodium iodide in dry acetone (20 mL, 0.5 M). The precipitate was isolated by centrifugation (4 min at 4000 rpm) and the supernatant was discarded. The pellet was washed with dry acetone by repeated re-suspension and centrifugation. The raw product was dried and purified with reversed-phase liquid chromatography. The combined product fractions were concentrated by solid phase extraction (Ci s) and converted to the sodium salt by precipitation with sodium iodide in dry acetone (0.5 M). The final colorless product was obtained at 73% final yield (>99% purity).

B. Synthesis of 2’-deoxycytosine 5’-0-(y-fhiorotriphosphate), dCTP-yF

Synthesis was performed analogous to Example 1A, by using dCTP (1.11 mmol), tributylamine (720 pL), DMF (5.8 mL), TBAF (3.33 mL, 1.0 M in THF), and l-fluoro-2, 4- dinitrobenzene (418 pL). Before introducing l-fluoro-2, 4-dinitrobenzene, dry DMSO (1.0 mL) was added to obtain a clear solution of the starting material. The sodium salt of purified product was isolated at 55% final yield (>99% purity).

C. Synthesis of 2’-deoxyguanosine 5’-0-(y-fluorotriphosphate), dGTP-yF

Synthesis was performed analogous to Example 1A, by using dGTP (1.09 mmol), tributylamine (706 pL), DMF (5.6 mL), TBAF (3.3 mL, 1.0 M in THF), and l-fluoro-2, 4- dinitrobenzene (410 pL). Before introducing l-fluoro-2, 4-dinitrobenzene, dry DMSO (3.0 mL) was added to obtain a clear solution of the starting material. The sodium salt of purified product was isolated at 27% final yield (>99% purity). D. Synthesis of 2’-deoxyuridine 5’-0-(y-fluorotriphosphate), dUTP-yF

Synthesis was performed analogous to Example 1A, by using dUTP (1.0 mmol), tributylamine (649 pL), DMF (5.2 mL), TBAF (3.0 mF, 1.0 M in THF), and l-fluoro-2, 4-dinitrobenzene (377 pF). Before introducing l-fluoro-2, 4-dinitrobenzene, dry DMSO (3.0 mF) was added to the reaction mixture to obtain a clear solution of the starting material. The sodium salt of purified product was isolated at 42% final yield (>99% purity).

E. Synthesis of 2’-deoxyadenosine 5’-0-(6-fluorotetraphosphate), dA4P-6F

Sodium fluorophosphate (3.909 mmol) was converted to the triethylammonium salt by cation exchange. Evaporation of the solvent gave a colorless compound, which was dried under high vacuum. Under argon the fluorophosphate was dissolved in an anhydrous mixture of DMSO (8.2 mF) and MeCN (5.0 mF). Imidazole (1.33 g) was added and the solution was stirred for 10 min, followed by the addition of triphenylphosphine (2.563 g) and 2-aminophenyl disulfide (1.456 g). The reaction mixture was heated to 40°C until all components were fully dissolved and kept stirring overnight at RT. The progress of the reaction was followed by UPFC-MS until no additional product was formed. The reaction was stopped by dropwise addition to a stirring solution of cold sodium iodide in dry acetone (30 mF, 0.5 M). The formed precipitate was isolated by centrifugation (4 min at 4000 rpm). The pellet was washed repeatedly with dry acetone. The combined supernatants were cooled overnight at 4°C, which led to further precipitation of colorless product. The combined precipitates of sodium fluorophosphate imidazolide were dried under high vacuum and used without further purification.

The sodium salt of dATP (0.25 mmol) was converted to the triethylammonium salt and lyophilized to dryness. The residue was dissolved in dry MeCN, transferred to a reaction flask, freed of solvent in the rotary evaporator, and dried under high vacuum. Under argon the triphosphate was dissolved in anhydrous DMF (1.68 mF). In a second flask the fluorophosphate imidazolide (1.125 mmol) was dissolved in anhydrous DMF (1.68 mF). Subsequently, 1.0 mF of the imidazolide solution was transferred dropwise to the dATP solution. ZnCk (273 mg) was added and the mixture was stirred for 2.0 h at RT. Afterwards the remaining imidazolide solution was transferred to the reaction mixture and the progress of the reaction was followed by UPFC-MS until no additional product was formed. The reaction was stopped by adding 4.0 mF of an aqueous EDTA solution (0.5 M, pH 8.0). Vigorous stirring was continued for 15 min, after which the reaction mixture was added dropwise to a stirring solution of sodium iodide in dry acetone (0.5 M). The colorless precipitate was isolated by centrifugation (4 min at 4000 rpm) and the supernatant was discarded. The raw product was dried and purified with reversed-phase liquid chromatography. The combined product fractions were concentrated by solid phase extraction (Cis) and converted to the sodium salt by precipitation with sodium iodide in dry acetone (0.5 M). The final colorless product was obtained at 63% reaction yield (>98% purity).

F. Synthesis of 2’-deoxycytosine 5’-0-(6-fluorotetraphosphate), dC4P-6F

Synthesis was performed analogous to example 1E by using dCTP (0.25 mmol) as starting material. The sodium salt of purified product was isolated at 21% reaction yield (>98% purity).

G. Synthesis of 2’-deoxyguanosine 5’-0-(6-fluorotetraphosphate), dG4P-6F

Synthesis was performed analogous to Example 1E by using dGTP (0.25 mmol) as starting material, except that l-methyl-2-pyrrolidone (1.5 mL) was added to the reaction mixture before addition of ZnCk. The sodium salt of purified product was isolated at 25% reaction yield (>98% purity).

H. Synthesis of 2’-deoxyuridine 5’-0-(6-fluorotetraphosphate), dU4P-6F

Synthesis performed analogous to Example 1E by using dUTP (0.25 mmol) as starting material. The sodium salt of purified product was isolated at 68% reaction yield (>99% purity).

Example 2: Stability studies with dNTP-yF

An aqueous mixture was prepared, containing 300 mM fluoropolyphosphate, 40% DMSO and

I.0 M potassium acetate. As reference another mixture with 300 mM dNTP instead of the fluoropolyphosphate was prepared. Both solutions (300 pL) were incubated at 65°C in a Thermoshaker. At several time points samples (20 pL) of each solution were taken and analyzed by UPLC-MS. The peak areas of the absorption trace at 260 nm were determined to calculate the relative percentages of starting material and degradation products.

Results: Under the conditions employed regular dNTP quickly degraded to the mono- and diphosphates as confirmed by UPLC-MS. In contrast fluoropolyphosphates were stable and no degradation products were observed at 65°C for 140 h (FIG. 4).

Example 3: Amplification reactions with a DNA target

All quantitative PCR (qPCR) components were prepared with DEPC treated water. qPCR mixtures with a total volume of 50 pL were prepared by combining three components termed qPCR master mixture (20 pL), buffer mixture (20 pL), and dNTP mixture (10 pL). The qPCR master mixture contained tricine buffer (pH 8.2), potassium acetate, glycerol, DMSO, detergent, target DNA (400 cp/reaction), polymerase aptamer, Cy5.5 labeled probe DNA, forward and reverse primer DNA, SYBR™ green dye, and polymerase enzyme. The buffer mixture contained either manganese acetate (8.3 mM) alone or a blend with magnesium acetate (2.49 mM). The dNTP mixture contained dATP, dCTP, dGTP (2.0 mM each), and dUTP (4.0 mM). Another solution containing fluoropolyphosphates instead of the natural dNTPs was prepared at equal concentrations. qPCR mixtures were prepared in the absence and presence of a thermostable inorganic pyrophosphatase, which was either the Thermostable Inorganic Pyrosphatase (TIP) from New England BioLabs, or the Thermostable Pyrophosphatase (rTh) from Roche Molecular Systems, Inc.. Each qPCR mixture was prepared as triplicate in the wells of a 96-well plate. The plate was sealed and subjected to qPCR amplification cycles and subsequent DNA melting with a LightCycler™ 480 System. The analysis of qPCR growth curves was performed from fluorescence data collected in the Cy5.5 channel.

Results: qPCR assays of target DNA with dN4P-6F show equal or earlier Ct’s (ACt = -1.5) compared to regular dNTPs, demonstrating that polymerase enzymes accept fluorinated polyphosphate analogs as substrates with equal or better efficiency (FIG. 5 and FIG. 6). Moreover, the fluoropolyphosphate cleavage products are efficient substrates for thermostable inorganic pyrophosphatase enzymes. This demonstrates that qPCR assays with dN4P-6F substrates are compatible with unmodified and benzylated primers.

Example 4: Amplification reactions with an armored RNA target

qPCR experiments were performed analogous to Example 3 with the following changes. The target DNA was replaced by RNA of an equivalent sequence. A reverse transcription (RT) step was added before regular thermocycling.

Results: qPCR-RT of armored target RNA with dN4P-6F shows equal or earlier Ct’s (ACt = - 1.2) compared to regular dNTPs, demonstrating that polymerase enzymes accept fluorinated polyphosphate analogs as substrates in reverse transcription reactions with equal or better efficiency (FIG. 7 and FIG. 8). Moreover, the fluoropolyphosphate cleavage products are efficient substrates for thermostable inorganic pyrophosphatase enzymes. This demonstrates that reverse transcription qPCR assays with dN4P-6F substrates are compatible with unmodified and benzylated primers.