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Title:
REVERSIBLY PROTECTED NUCLEOTIDE REAGENTS WITH HIGH THERMAL STABILITY
Document Type and Number:
WIPO Patent Application WO/2018/122157
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 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.

Inventors:
GUPTA AMAR (US)
Application Number:
EP2017/084337
Publication Date:
July 05, 2018
Filing Date:
December 22, 2017
Export Citation:
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Assignee:
ROCHE DIAGNOSTICS GMBH (DE)
HOFFMANN LA ROCHE (CH)
ROCHE MOLECULAR SYSTEMS INC (US)
International Classes:
C07H19/00; C12Q1/6844
Domestic Patent References:
WO2003020891A22003-03-13
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:
US20090269759A12009-10-29
US20150111759A12015-04-23
US5693502A1997-12-02
US4458066A1984-07-03
US4683202A1987-07-28
US4683195A1987-07-28
US4800159A1989-01-24
US4965188A1990-10-23
EP0439182A21991-07-31
US5130238A1992-07-14
US4666848A1987-05-19
US4889818A1989-12-26
US5455170A1995-10-03
Other References:
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 AND 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
GUATELLI J.C. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 1874 - 1878
WHELEN A. C.; PERSING D. H., ANNU. REV. MICROBIOL., vol. 50, 1996, pages 349 - 373
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 et al. (DE)
Download PDF:
Claims:
CLAIMS

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 B = purine or pyrimidine base or an analog, L= linker or nothing, Z= label or nothing, R= OH or O , Ri and R2 = H or OH, n =1-7, X= O, S, or NH, Y= an organic moiety that can be removed chemically or enzymatically.

2. The method of claim 1 , wherein X=0 and Y= an organic moiety that can be removed chemically by a reducing agent. 3. The method of claim 2, wherein the reducing agent is selected from the group consisting of a phosphine derivative, sodium sulfite, (Na2S03), β-Mercaptoethanol and dithiothreitol.

4. The method of claim 3, wherein the phosphine derivative is tris-(2-carboyxy ethyl) phosphine (TCEP).

5. The method of any one of claims 1 to 4, wherein Y is HO-CH2-CH2-S-S-CH2-CH2-. 6. The method of any one of claims 1 to 5, wherein X=0 and Y= an organic moiety that can be removed enzymatically by an esterase.

7. The method of claim 6, wherein the esterase is a carboxylesterase. 8. The method of any one of claims 6 to 7, wherein Y is selected from the group consisting of CH3C(0)-S-CH2-CH2- and CH2CH2C(0)OCH2- .

9. 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 B = purine or pyrimidine base or an analog, L= linker or nothing, Z= label or nothing, R= OH or O , Ri and R2 = H or OH, n =1-7, X= O, S, or NH, Y= an organic moiety that can be removed chemically or enzymatically.

10. The method of claim 9, wherein X=0 and Y= an organic moiety that can be removed chemically by a reducing agent.

11. The method of claim 10, wherein the reducing agent is selected from the group consisting of a phosphine derivative, sodium sulfite, (Na2S03), β-Mercaptoethanol and dithiothreitol.

12. The method of claim 11 , wherein the phosphine derivative is tris-(2-carboyxyethyl) phosphine (TCEP).

13. The method of any one of claims 10 to 12, wherein Y is HO-CH2-CH2-S-S-CH2-

14. The method of claim 9, wherein X=0 and Y= an organic moiety that can be removed enzymatically by an esterase.

15. The method of claim 14, wherein the esterase is a carboxylesterase.

16. The method of claim 14, wherein Y is selected from the group consisting of CH3C(0)-S-CH2-CH2- and CH2CH2C(0)OCH2- .

17. A modified nucleosi lyphosphate having a structure of:

where B = purine or pyrimidine base or an analog, L= linker or nothing, Z= label or nothing, R= OH or O , Ri and R2 = H or OH, n =1-7, X= O, S, or NH, and Y is selected from the group consisting of HO-CH2-CH2-S-S-CH2-CH2- , CH3C(0)-S- CH2-CH2-, and CH2CH2C(0)OCH2- .

The modified nucleoside polyphosphate of claim 17, wherein Y is HO-CH2-CH2-S- S-CH2-CH2-.

Description:
REVERSIBLY PROTECTED NUCLEOTIDE REAGENTS WITH HIGH THERMAL STABILITY

FIELD OF THE INVENTION

The present invention provides stable nucleotide reagents, methods for their preparation, methods for their use, and kits comprising them. The nucleotide reagents are useful in many recombinant DNA 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 deoxynucleoside 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 active as substrates of nucleic acid polymerases. The stability of nucleoside polyphosphates can be improved quite dramatically by esterifying the terminal phosphates. For example, g-methyl-dNTP analogs 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 nucleotide triphsosphates that are both thermally stable and are inactive or have reduced activity initially as substrates but are readily activated by simple chemical or enzymatic steps.

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 B = purine or pyrimidine base or an analog, L= linker or nothing, Z= label or nothing, R= OH or O " , Ri and R 2 = H or OH, n =1-7, X= O, S, or NH, Y= an organic moiety that can be removed chemically or enzymatically. In one embodiment, X=0 and Y= an organic moiety that can be removed chemically by a reducing agent. In another embodiment, the reducing agent is selected from the group consisting of a phosphine derivative, sodium sulfite, (Na 2 S0 3 ), β- Mercaptoethanol and dithiothreitol. In another embodiment, the phosphine derivative is tris-(2- carboyxyethyl) phosphine (TCEP). In another embodiment, Y is HO-CH 2 -CH 2 -S-S-CH 2 -CH 2 -. In one embodiment, X=0 and Y= an organic moiety that can be removed enzymatically by an esterase. In another embodiment, the esterase is a carboxylesterase. In yet another embodiment, Y is selected from the group consisting of CH 3 C(0)-S-CH 2 -CH 2 - and CH 2 CH 2 C(0)OCH 2 - . 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 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 ha ing a structure of:

where B = purine or pyrimidine base or an analog, L= linker or nothing, Z= label or nothing, R= OH or O " , Ri and R 2 = H or OH, n =1-7, X= O, S, or NH, Y= an organic moiety that can be removed chemically or enzymatically. In one embodiment, X=0 and Y= an organic moiety that can be removed chemically by a reducing agent. In another embodiment, the reducing agent is selected from the group consisting of a phosphine derivative, sodium sulfite, (Na 2 S0 3 ), β- Mercaptoethanol and dithiothreitol. In another embodiment, the phosphine derivative is tris-(2- carboyxyethyl) phosphine (TCEP). In another embodiment, Y is HO-CH 2 -CH 2 -S-S-CH 2 -CH 2 -. In one embodiment, X=0 and Y= an organic moiety that can be removed enzymatically by an esterase. In another embodiment, the esterase is a carboxylesterase. In yet another embodiment, Y is selected from the group consisting of CH 3 C(0)-S-CH 2 -CH 2 - and CH 2 CH 2 C(0)OCH 2 - . In another aspect, the present invention involves a composition comprising a modified nucleoside polyphosphate ha ing a structure of:

where B = purine or pyrimidine base or an analog, L= linker or nothing, Z= label or nothing, R= OH or O , Ri and R 2 = H or OH, n =1-7, X= O, S, or NH, and Y is selected from the group consisting of HO-CH 2 -CH 2 -S-S-CH 2 -CH 2 - , CH 3 C(0)-S-CH 2 -CH 2 -, and CH 2 CH 2 C(0)OCH 2 - . In one embodiment, Y is HO-CH 2 -CH 2 -S-S-CH 2 -CH 2 -.

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 an example of a chemical activation reaction using the modified nucleoside polyphosphate, Hydroxyethyl disulfide-dNTP (HEDS-dNTP).

FIG. 2 shows an example of an enzymatic activation reaction using the modified nucleoside polyphosphate, Acetyl-thioethyl-dNTP (ATE-dNTP).

FIG. 3 is a schematic for the prepration of dATP-2-hydroxyethyldisulfide ester (HEDS-dATP). FIG. 4 is a HPLC chromatogram that shows the conversion of HEDS-dATP by TCEP after 5 min, 10 min, 20 min, 30 min and 60 min treatment at 60°C. Peak 1 represents an intermediate product; peak 2 represents the starting material; peak 3 represents the product after activation by TCEP.

FIG. 5 shows the growth curves of PCR amplification reactions from an HIV-1M template at 200 copies/reaction using HEDS-dNTPs that have been converted to dNTPs by treatment with lmM TCEP for 30 minutes at 60°C. FIG. 6 shows the growth curves of PCR amplification reactions from an HIV-IM template at 200 copies/reaction using HEDS-dNTPs that have been converted to dNTPs by treatment with 5mM sodium sulfite, (Na 2 S0 3 ).

FIG. 7 shows the Ct values of PCR reactions having HIV-IM, HIV-2 and IC as templates and using either normal dNTPs or HEDS-dNTPs that have been stored for 22 days at 37°C.

FIG. 8 shows the Ct values of PCR reactions having HIV-IM, HIV-2 and IC as templates and using either normal dNTPs or HEDS-dNTPs that have been stored for 22 days at 45°C.

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 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 and for amplifying a target nucleic acid sequence in a sample. 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 15°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 polymerase nucleic acid, 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, i.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 mR A 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 internucleoside 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.

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, methyl phosphonate, phosphotriester or phosphoramidate inter-nucleoside linkages in place of phosphodiester internucleoside 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 internucleoside 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 deoxynucleoside 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.

"Labels", 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, CAL635, Coumarin343, Quasar705, Cyan500, CY5.5, LC-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-, terra-, 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(=0 2 ~), ester (~C(=0)~0~), 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 105°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 sec 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 sec to about 1 min (e.g., about 20 sec to about 50 sec; about 30 sec to about 40 sec). 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 sec to about 5 min, or about 15 sec to 2 min, or about 20 sec to about 1 min, or about 25 sec to about 35 sec. 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^ slope) -l) 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 DABCYL 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 LC-Red 640, LC-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., LightCycler™ 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 turn-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 LightCycler® technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LightCycler® instrument is a rapid thermal cycler combined with a microvolume 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.

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 as described above may be 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 5 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, nucleotide triphosphates (NTPs) or deoxynucleotide triphosphates (dNTPs), probe, 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 minutes, 20 minutes, 15 minutes, 12.5 minutes, 10 minutes, 5 minutes, or 1 minute.

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. spsl7 DNA polymerase

i) a Thermus sp. Z05 DNA polymerase

j) a Thermotoga neapolitana DNA polymerase

k) a Termosipho africanus DNA polymerase

1) 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-Xb 7 -Xb 8 -P-N-L-Q-N; wherein Xb 7 is an amino acid selected from S or T and wherein Xbg 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 Xbs 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, Mn 2+ can be the divalent cation and is typically included as a salt, for example, manganese chloride (MnCl 2 ), manganese acetate (Mn(OAc) 2 ), or manganese sulfate (MnS0 4 ). If MnCl 2 is included in a reaction containing 50 mM Tricine buffer, for example, the MnCl 2 is generally present at a concentration of 0.5-7.0 mM; 2.5-3.5 mM is generally present when 200 μΜ 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, ΔΖ05 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 deoxynucleoside 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 quite dramatically by esterifying the terminal phosphates. For example, γ-methyl-dNTP analogs 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 nucleotides described in this invention are both thermally stable, and either inactive or have reduced activity as substrates, but are readily activated when desired by a simple chemical or enzymatic step. A general structure exemplifying this concept is shown below:

Where,

B=purine or pyrimidine base or analog

L=linker or nothing

Z=label or nothing

R=OH or O

R1, R2=H or OH

n=l-7

X= O, S, or NH

Y=an organic moiety, that can be removed chemically or enzymatically Preferred embodiments:

Y=HO-CH 2 -CH 2 -S-S-CH 2 -CH 2 -, CH3C(O)-S-CH2CH2- or CH 2 CH 2 C(0)OCH 2 Chemical Activation

The protecting group is chosen such that an initial reaction, for example a reaction with reducing agent, produces a reactive intermediate which further decomposes to give the desired active nucleotide. The reducing agent may be any reagent that is capable of reducing a disulfide. Some examples of such reducing agents are β-Mercaptoethanol, dithiothreitol and TCEP (tris-(2-carboyxy ethyl) phosphine).

In the method of the present invention, the reaction components are configured in such a way that the dNTP analogs are kept separate from the reducing agent until the reaction mixture is prepared and the sample is added. An example of a chemical activation reaction using the modified nucleoside polyphosphate, Hydroxyethyl disulfide-dNTP (HEDS-dNTP) is shown in FIG. l .

Enzymatic Activation

The protecting group is chosen such that an initial enzymatic reaction, for example a reaction with an esterase, produces a reactive intermediate which further decomposes to give the desired active nucleotide. In the method of this invention, the reaction components are configured in such a way that the dNTP analogs are kept separate from the activating enzyme until the reaction mixture is prepared the sample is added. An example of an enzymatic activation reaction using the modified nucleoside polyphosphate, Acetyl-thioethyl-dNTP (ATE-dNTP) is shown in FIG. 2.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made. For example, all the compositions and methods described above can be used in various combinations. 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: Preparation of dATP-2-hydroxyethyldisulfide ester

The prepration of dATP-2-hydroxyethyldisulfide ester (HEDS-dATP) is shown in FIG. 3. Briefly, 500 μΜ of dATP was suspended in 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 4.5 and incubated with 0.82 mM 2,2 , -disulfanediyl-bis(ethan-l-ol), followed by incubation in ice for 15 minutes. Then, increasing amounts of EDC (N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) were gradually added to the mixture reaching a final concentration of 2.61 mM. The reaction mixture was incubated at room temperature for 7 days and the products were analyzed by ultra-high performance liquid chromatography (UPLC). Approximately 90% yield of the product, HEDS-dATP, was produced using this protocol. Example 2: Activation of HEDS-dATP by tris-(2-carboyxyethyl) phosphine (TCEP)

The reducing agent tris-(2-carboyxyethyl) phosphine (TCEP) was used to reduce HEDS-dATP to dATP by heat treatment at 60°C. FIG. 4 depicts the HPLC chromatograms that show the conversion of HEDS-dATP after 0 min, 10 min, 20 min, 30 min and 60 min treatment of ImM TCEP at 60°C. Peak 1 represents an intermediate product. Peak 2 represents the starting material, HEDS-dATP. Peak 3 represents the product, dATP.

Example 3: Amplification reactions using HEDS-dNTPs

PCR reaction mixtures were prepared on a 96-well plate with the following final concentrations: 60 mM Tricine(pH 8.3), 120 mM potassium acetate, 3% glycerol, 5.4% DMSO, 0.015% Tween 20, target HIV-1M template (200 copies/reaction), 600 nM each of forward and revers primer, 100 nM TaqMan ® probe, 900 units/mL Z05D DNA polymerase (with 5" nuclease activity), 200 units/mL UNG, 44 μΜ EDTA, and 3.3 mM manganese acetate. In one reaction set, 400 μΜ each of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and 800 μΜ of deoxyuridine triphosphate (dUTP) were added and in another reaction set, 400 μΜ each of HEDS-dATP, HEDS-dGTP (dGTP-2-hydroxyethyldisulfide ester), HEDS-dCTP (dCTP-2-hydroxyethyl- disulfide ester), and 800 μΜ of HEDS-dUTP (dUTP- 2-hydroxyethyldisulfide ester) were added. FIGs. 5 and 6 show the growth curves of PCR amplification reactions using HEDS-dNTPs that were converted to dNTPs using ImM TCEP (FIG. 5) and using 5mM sodium sulfite, Na 2 S0 3 (FIG. 6). While HEDS-dNTPs that were activated using TCEP showed growth curves with approximately ICt delay relative to standard dNTPs, growth curves from HEDS-dNTPs activated using sodium sulfite were virtually identical to those using standard dNTPs.

Example 4: Stability studies using HEDS-dNTPs

Nucleoside triphosphates in the form of either standard dNTPs or HEDS-dNTPs were stored for 22 days in either 37°C or 45°C. These stored nucleoside trisphosphates were then utilized in a PCR reaction with the assay conditions as described in Example 3 with ImM TCEP as the reducing agent for the activation of the HEDS-dNTPs and using HIV-1M (200 copies per reaction), HIV-2 (100 copies per reaction) or an internal control sequence (IC, 1000 copies per reaction) as templates. The efficiency of PCR amplification using these stored reagents are represented as average Ct values (threshold cycle number) and are represented as bars in FIG. 7 (for reagents incubated at 37°C) and FIG. 8 (for reagents incubated at 45°C). The results demonstrated that in storage at 37°C, standard dNTPs exhibited higher Ct values (ranging from 1.9 to 3.9) than HEDS-dNTPs, signifying less efficient amplification most likely due to some degree of degradation of the standard dNTPs. For storage at 45°C, two of the three reaction using standard dNTPs did not generate any growth curves, due to almost complete degradation while all three reactions using HEDS-dNTPs exhibited Ct values that did not differ significantly from HEDS-dNTPs stored at 37°C (comparing the dark gray bars between FIG. 7 and FIG. 8).