WO2001023611A2 - Procedes - Google Patents

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Publication number
WO2001023611A2
WO2001023611A2 PCT/GB2000/003780 GB0003780W WO0123611A2 WO 2001023611 A2 WO2001023611 A2 WO 2001023611A2 GB 0003780 W GB0003780 W GB 0003780W WO 0123611 A2 WO0123611 A2 WO 0123611A2
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WO
WIPO (PCT)
Prior art keywords
probe
polymerase
target polynucleotide
fluorescence
oligonucleotide
Prior art date
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PCT/GB2000/003780
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English (en)
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WO2001023611A3 (fr
Inventor
Alexander Vasilyevich Karpukhin
Natalya Nikolaevna Veiko
Original Assignee
Socrates Bio-Tech International Limited
Lee, Nicholas, John
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Socrates Bio-Tech International Limited, Lee, Nicholas, John filed Critical Socrates Bio-Tech International Limited
Priority to AU75411/00A priority Critical patent/AU7541100A/en
Publication of WO2001023611A2 publication Critical patent/WO2001023611A2/fr
Publication of WO2001023611A3 publication Critical patent/WO2001023611A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the present invention relates to methods for detecting the amplification of a target polynucleotide.
  • it relates to a method for detecting the amplification of a target polynucleotide by means of the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • PCR is widely used in all areas of biological science to amplify specific target nucleotide sequences.
  • a common method for the analysis of PCR products is gel electrophoresis.
  • this technique has the disadvantages that it is slow and laborious.
  • Methods for PCR product analysis based on the intercalation of dyes in DNA are improvements over gel electrophoresis in terms of speed and ease of use. Nevertheless, these methods have the disadvantage that non-specific amplification products are indistinguishable from the true amplicons.
  • the oligonucleotide probe is hybridised to the target polynucleotide downstream from a primer for a polymerase having a 5 '-3' exonuclease activity.
  • the polymerase extends the primer as part of the PCR reaction, the oligonucleotide is digested, releasing one of the "quencher” and “reporter” molecules and causing the distance between these molecules to become such that the fluorescent emission is no longer quenched by energy transfer.
  • a fluorescent signal is generated, providing real-time monitoring of amplification.
  • the chemistry of providing the reporter and quencher molecules on the oligonucleotide probe is difficult and contributes to the high cost of the method.
  • several factors detract from the performance of the method The first is the relatively poor quenching observed in the intact oligonucleotide probe. This contributes to a relatively high noise level and means that the signal generated by amplification can only be 6-8 times the base or noise level. This reduces the sensitivity of the technique.
  • the second factor is the efficiency of hybridisation of the probe, which is reduced by virtue of the modifications made by the fluorophores and, in particular, by virtue of there being two fluorophores, the quencher and the reporter.
  • the third factor is the efficiency with which polymerase digests the bound oligonucleotide probe, which is also reduced by the two fluorophores.
  • a method for detecting the amplification of a target polynucleotide comprising: extending an oligonucleotide primer annealed to the target polynucleotide with a polymerase having a 5 '-3' exonuclease activity, the target polynucleotide having annealed thereto an oligonucleotide probe which incorporates a modified nucleotide having a fluorescent characteristic which is modified by one or more neighbouring unmodified nucleotides; and detecting a change in fluorescence as the oligonucleotide probe is degraded by the exonuclease activity of the polymerase as the polymerase extends the primer and modification of the fluorescent characteristic of the modified nucleotide is reduced.
  • the method of the invention does not require a separate "quencher” molecule. This makes the oligonucleotide easier to produce and reduces the cost by comparison to the TaqMan method, which requires two fluorescent molecules attached to two nucleotides in the oligonucleotide probe.
  • the neighbouring unmodified nucleotides that modify the fluorescent characteristic of the modified nucleotide are preferably located in the oligonucleotide probe, i.e. they may be the nucleotides either side of the modified nucleotide or located nearby.
  • nucleotides in the target polynucleotide also to have a similar or additive modifying effect when the oligonucleotide probe is annealed to the target.
  • the probe has a single modified nucleotide. This reduces the cost of the probe. Moreover, because only one nucleotide is modified, the adverse influence on hybridisation of the probe to the target polynucleotide and cleavage by the polymerase is less than in the TaqMan method. With only a single modified nucleotide, it is also possible for the oligonucleotide probe to be practically entirely digested by the polymerase.
  • the fluorescence of the modified nucleotide is quenched by the one or more neighbouring unmodified nucleotides, because degradation of the oligonucleotide causes a corresponding - and easily measured - increase in fluorescence.
  • the modified nucleotide can be positioned anywhere within the probe, although it is preferred that the modified nucleotide be located more proximal to the 5' end of the probe (for example, within the first five to ten or so nucleotides) as this improves cleavage by the polymerase. It is believed that positioning the modified nucleotide at the 5' end of the probe reduces the chances of the oligonucleotide dissociating from the target sequence when it is partially cleaved.
  • the modified nucleotide may be a ribonucleotide or a deoxyribonucleotide, depending on whether the oligonucleotide probe is an RNA or a DNA molecule respectively, which in turn depends on whether the target sequence is an RNA or a DNA molecule respectively.
  • Modified nucleotides that have a fluorescent characteristic that is modified by one or more neighbouring unmodified nucleotides are well known to those skilled in the art. As is discussed in US Patent No. 5763167, it is known that fluorescence in nucleotide analogues results from a molecular rigidity of the heterocycle structure and fluorescence is not an exclusive or inherent property for any particular class of structural analogues.
  • Fluorescent analogues of nucleosides include certain pyrazolo pyrimidines, substituted N-nucleosides, azanucleosides, deazanucleosides and ethenonucleosides. It is preferred that the modified nucleotide is an ethenonucleoside such as ethenodeoxyadenosine, ethenodeoxycytidine or ethenodeoxyguanosine, or their corresponding ribonucleosides.
  • the modified nucleotide may alternatively be a nucleotide substituted with a lower alkyl moiety such as a methyl group. An example of such a substituted nucleotide is 7-methyldeoxyguanosine.
  • modified nucleotides can be incorporated into an oligonucleotide probe using only small modifications to standard synthesis techniques, making the probe of the present invention easier to synthesise than probes used in the TaqMan method.
  • 1,N 6 etheno deoxy and ribo adenosine and 3,N 4 deoxy and ribo cytidine can be synthesised and incorporated into an oligonucleotide according to the methods described and referred to in Srivatava et al, Nuc. Acids Res. 22(7): 1296-1304 (1994).
  • the synthesis of 1,N 6 ethenoadenosine is described in Secrist et al, Biochemistry, 11: 3499-3506 (1972).
  • ethenodeoxy cytidine can be synthesised and incorporated into an oligonucleotide as described in Zhang at al, Chem. Res. Toxicol. 8: 148-156, (1995) and Misra et al, Carcinogenesis , 15: 1647- 1652, (1994), ethenodeoxy uanosine can be synthesised and incorporated into an oligonucleotide as described in Kusmierek et al, Chem. Res. Toxicol. 5: 634-638, (1992) and Kusmierek et al, Chem. Res. Toxicol.
  • a preferred modified nucleotide is 1,N 6 ethenodeoxy adenosine.
  • the etheno adduct of adenosine adds a new ring to the nucleotide which causes the formation of a new system, and it is believed that excited electronic transitions in this system are responsible for a fluorescent emission (Secrist et al, Biochemistry, 11: 3499-3506 (1972)). Only one structure, the neutral form, is the origin of fluorescence (Secrist et al, Biochemistry, 11: 3499-3506 (1972), Spencer, Eur. J. Biochem, 45: 425-429 (1974)).
  • the fluorescence of ethenoadenosine is influenced by, for example, pH, the fluorescent intensity at pH 2.2 being less than 10% of the intensity at pH 7.0 (Secrist et al, Biochemistry, 11: 3499-3506 (1972)).
  • the inclusion of ethenoadenosine in a polynucleotide causes quenching of the fluorescence of the ethenoadenosine, with optical evidence suggesting that this quenching is due to the planar etheno modified bases being involved in base stacking interactions (Steiner et al, Biochim. Biophys Acta, 294: 24-37 (1973); Tolman et al, Biochemistry 13: 4869-4878 (1974)).
  • the fluorescent emission of ethenoadenosine is greatly quenched in both dynamic (time dependent) and static (time-independent) processes by all neighbouring nucleosides (Tolman et al, Biochemistry 13: 4869-4878 (1974)).
  • the fluorescent properties of 1,N 6 ethenodeoxyadenosine are broadly the same as those of 1,N 6 ethenoadenosine (Giedroc, Biochem. 30: 8230-8242 (1991), Leonard CRC Crit. Rev. in Biochem 15: 125-199 (1984)).
  • Etheno modification of a substrate may change the enzymological properties of that substrate (Tolman et al, Biochemistry 13: 4869-4878 (1974), Deibel et al, Anal. Biochem. 144: 336-346 (1985)).
  • Tolman et al disclose that etheno adducts of nucleic acids are more resistant or even completely resistant to the action of some nucleases.
  • the present inventors have found that etheno modified oligonucleotides are a good substrate for nucleases such as Taq polymerase, Tfl (Thermus flavus) polymerase (Gut et al, Virol.
  • a probe including ethenodeoxyadenosine has superior hybridisation and cleavage properties.
  • Ethenodeoxyadenosine is also highly quenched in an oligonucleotide, meaning that the ratio of fluorescent signal to background (a measure of the quenching) is as high as approximately 20-25. This is significantly more than the ratio of 6-8 achieved in the TaqMan technique and results in a far more sensitive method of detecting amplification.
  • oligonucleotide as used herein includes linear oligomers of natural or modified monomers or linkages, including nucleosides such as deoxyribonucleosides, ribonucleosides, and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to monomer interactions, such as Watson-Crick type base pairing or the like.
  • monomers are linked by phosphodiester bonds or analogues thereof to form oligonucleotides, provided that they can be digested by the a polymerase having a 5 '-3' exonuclease activity.
  • nucleoside as used herein includes the natural nucleosides, including 2'-deoxy and 2' hydroxyl forms e.g. as described in Kornberg and Baker, DNA Replication, 2 nd Edn. (Freeman, San Francisco, 1992). It also includes analogues of the natural nucleosides, including synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g.
  • Such analogues include synthetic nucleosides designed to enhance binding properties, reduce degeneracy, increase specificity and the like.
  • Oligonucleotide probes of the present invention can be synthesised by a number of approaches, e.g. Ozaki et al, Nuc. Acids Res. 20: 5205-5214 (1992); Agrawal et al, Nuc. Acids Res. 18: 5419-5423 (1990) or the like.
  • the oligonucleotide probes are synthesised on an automated DNA synthesiser, e.g. an Applied Biosystems, Inc, Foster City, California model 392 or 394 DNA/RNA synthesiser using standard chemistries such as phosphoramidite chemistry (Beaucage and Iyer, Tetrahedron 48: 2223-2311 (1992), US Patent Nos.
  • the length of the oligonucleotide probe is in the range of 15 to 60 nucleotides, and more preferably 18 to 30 nucleotides.
  • the precise length and sequence of the oligonucleotide probe depends at least in part on the nature of the target polynucleotide to which it binds and will be varied to achieve appropriate annealing and melting properties for a particular target polynucleotide.
  • the binding location of the probe may be varied according to the nature of the target polynucleotide. For amplification of relatively short target polynucleotides (for example, up to 0.5 kb), the binding location of the probe may not be important.
  • the probe may be located near to a primer. This is because the completion of primed DNA synthesis is carried out at an increased temperature (for example up to 72 °C). Thus, locating the probe near a primer allows it to be cleaved relatively early in the process, reducing the chances of dissociation at the increased temperature.
  • the 3 ' terminal nucleotide of the probe is blocked or otherwise rendered incapable of extension by a nucleic acid polymerase.
  • Methods for achieving this are well known to the skilled person and include attaching a 3' phosphate group to the 3' end of the probe (Lee et al, Nucl. Acid. Res. 21: 3761-3766 (1993), Livak et al, PCR Methods Appl. 4: 357-362 (1995), Morris et al, J. Clin. Microbiol. 34: 2933- 2936 (1996)).
  • the oligonucleotide primers may be made in the same way as the oligonucleotide probes mentioned above and may have the same variations in backbone and so on, providing that hybridisation to the target polynucleotide is not compromised.
  • the precise length and sequence of the primers will depend on the target polynucleotide to be amplified.
  • the length of the primers is in the range 15 to 60 nucleotides and more preferably in the range 18 to 30 nucleotides.
  • the method of the present invention is preferably used to monitor amplification of a target polynucleotide by PCR (see for example Innis et al, editors, PCR Protocols, (Academic Press, New York, 1989; Sambrook et al, Molecular Cloning, Second Edition, (Cold Spring Harbour Laboratory, New York 1989)).
  • the binding site of the oligonucleotide probe is located between the PCR primers used to amplify the target polynucleotide.
  • PCR is carried out using Taq DNA polymerase, e.g. AmplitaqTM (Perkin-Elmer, Norwalk, Conn.).
  • Taq polymerase can also be obtained from MBI Fermentas, Perkin Elmer, Boehringer Mannheim and
  • thermostable, DNA polymerase may also be used in the method of the present invention, such as Tfl (Thermus flavus) polymerase (Gut et al, Virol. Methods 77(1): 37-46 (1999)). It is also preferred if the annealing temperature of the PCR is approximately 5-10 °C below the melting temperature of the oligonucleotide probe, i.e. the temperature at which the oligonucleotide probe disassociates with the target polynucleotide.
  • Alternative amplification methods include RT-PCR, in which the target polynucleotide is RNA (Yajima et al, Clin. Chem, 44(12): 2441-2445 (1998); Martell et al, J. Clin. MicrobioL , 37(2): 327-332 (1999); Gut et al, Virol. Methods
  • the invention also relates to a kit for practising the method of the first aspect.
  • the invention provides a kit for detecting the amplification of a target polynucleotide by means of a polymerase having a 5 '-3' exonuclease activity extending a primer annealed to the target polynucleotide, the kit comprising: an oligonucleotide probe for annealing to the target polynucleotide, the probe incorporating a modified nucleotide having a fluorescent characteristic which is modified by one or more neighbouring unmodified nucleotides.
  • the kit may further comprise the polymerase and an appropriate primer or set of primers.
  • it may comprise additional reagents that are necessary for performing the invention, such as dNTP mixtures, buffers, molecular size standards, wax beads and the like.
  • the reagents may be provided in pre-measured amounts so as to simplify the performance of the method.
  • the kit may also contain detailed instructions for carrying out the method.
  • the invention provides the use of an oligonucleotide probe which incorporates a modified nucleotide having a fluorescent characteristic which is modified by one or more neighbouring unmodified nucleotides for detecting the amplification of a target polynucleotide, the amplification preferably being carried out by a polymerase having 5 '-3' exonuclease activity.
  • a forward primer 1 and a probe 2 having a fluorescent nucleotide 3 are annealed to the upper (sense) strand 4 of a target polynucleotide.
  • the fluorescent nucleotide 3 is located in the middle of the target polynucleotide, although this is not essential.
  • a reverse (antisense) primer 5 is also annealed to the lower antisense strand 6.
  • Example 1 the etheno adduct of a nucleotide is used as a fluorescent molecule in an oligonucleotide probe, which probe is used to detect the amplification of a target sequence of 170 bp from 16S rDNA of My coplasma hominis.
  • sequences of the primers used for the amplification are known from Grau et al, Molecular and Cellular Probes, 8; 139-148 (1994) and are shown in Table 1.
  • a sequence of 25 nucleotides within the primers was selected as a probe ("eA" indicates ethenodeoxyadenosine).
  • the primers were synthesised using the solid phase phosphoramidite method (Beaucage et al, Tetrahedron Lett, 22: 1859-1862, (1981); McBride et ⁇ /, Tetrahedron Lett, 24: 245-248, (1983)) on a "Biosearch 8700" synthesiser.
  • the primers were synthesised using the conventional procedure in which protected deoxynucleotide phosporamidites (obtained from Glen Research) are used.
  • the final base and phosphate deprotection, and cleavage from the support were achieved by treatment with concentrated ammonium hydroxide, and the oligonucleotide was isolated using preparative denaturing PAAG.
  • Ethenodeoxyadenosine phosphoramidite was synthesised in accordance with Srivastava et al, Nucleic Acids Research, 22:1296-1304, (1994), and was incorporated into the oligonucleotide probe as a label using certain modifications to the standard DNA synthesis protocol. Fast base deprotecting groups and a short treatment with ammonia were used to ensure no degradation of the etheno moiety (Srivastava et al, Nucleic Acids Research, 22:1296-1304, 1994). The probe was purified by HPLC using standard protocols.
  • Oligonucleotide probe Mhpl was dissolved in the reaction buffer at a concentration 1 ng/ ⁇ l and the fluorescence was measured to be 3 ⁇ 1 fluorescence units. After this, the phosphodiesterase (5-10 ⁇ l) was added to 400 ⁇ l of oligonucleotide solution and incubated at 37 °C for 5 hours. After this digestion, the fluorescence was measured to be 78 ⁇ 2 fluorescence units. Thus, the fluorescence of the ethenodeoxyadenosine in the oligonucleotide probe was quenched to about 3.8% of the level of ethenodeoxyadenosine in solution, i.e. the fluorescence of ethenodeoxyadenosine in solution is about 26 times that of ethenodeoxyadenosine in the oligonucleotide.
  • PCR amplifications of the 170 bp Mycoplasma hominis DNA fragment using the primers of Table 1 were performed using 50 ⁇ l reaction volumes that contained 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl, 2 units Taq polymerase (Perkin Elmer) and 200 ⁇ M each of dATP, dCTP, dGTP and dTTP.
  • the amplification reaction contained 0.4 ⁇ M of each primer, 80 ng of target DNA, and 50 ng of labelled oligonucleotide probe.
  • the thermal regimen was 96 °C (4 min), 35 cycles of 96 °C (45 sec), 55 °C (45 sec), 72 °C (45 sec), and hold at 72 °C for 7 min.
  • the amplified 170 bp DNA fragment was tested by gel electrophoresis and by hybridisation of a biotin-labelled DNA probe. A correlation between the fluorescent signal intensity, the intensity of PCR specific product on the electrophoresis gel and the hybridisation signal was observed.
  • Example 2
  • Example 1 The process (probe, primers, procedure, etc) of Example 1 was used to assay for the presence of Mycoplasma hominis in 15 clinical samples. These samples were prepared for PCR using the standard phenol-chloroform extraction procedures
  • Example 3 Example 1 was repeated using varying amounts of probe in the sample. In addition, further amplifications were carried out as in Example 1 , except that the label was placed in an alternative position in the probe. The sequence of this probe used was:
  • Probe Probe Quantity (ng) Signal (fluorescence, Control - PCR without relative units) target (fluorescence, relative units)
  • Salmonella primer 1 GCTGCGCGCGAACGGCGAAG
  • HPV-16 primer 1 CCTTATTGGTTACAACGAGCAC
  • HPV-16 primer 2 GCGTCCTAAAGGAAACTGATCTA
  • HPV- 16 probe HPVp
  • Salp was designed to anneal to the same strand as that to which Salmonella probe 1 anneals
  • HPVp was designed to anneal to the same strand as that to which HPV- 16 probe 1 anneals.
  • the Salmonella primers were used to amplify DNA from Salmonella typhimurium, and the HPV-16 primers were used to amplify cloned DNA (Proc. Natl. Acad. Sci. USA, 80: 3812-3815 (1993)). In each case, DNA from the respective samples was purified by standard procedures (Maniatis et al, Molecular Cloning. A Laboratory Manual, Cold Spring Harbour Laboratory (1982)).
  • PCR was carried out as in Example 1, except that 2 mM MgCl 2 was added to the buffer and the thermal regimen for the Salmonella amplification was 95 °C (5 min); 35 cycles of 95 °C (45 sec), 63 °C (2 min), and that for the HPV-16 amplification was according to Swan et al, J. Clin. Microbiol 35: 886-891 (1997).

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Abstract

La présente invention concerne un procédé de détection de l'amplification d'un polynucléotide cible. Dans le procédé, le polynucléotide cible comporte une sonde oligonucléotidique annelée qui comprend un oligonucléotide modifié présentant une caractéristique fluorescente qui est modifiée par un ou plusieurs nucléotides non modifiés voisins. Une amorce oligonucléotidique annelée au polynucléotide cible est allongée avec une polymérase ayant une activité exonucléase 5'-3' et le changement de la fluorescence est détecté lorsque la sonde oligonucléotidique est décomposée par l'activité exonucléase de la polymérase quand la polymérase allonge l'amorce et que la modification de la caractéristique fluorescente du nucléotide modifié est réduite.
PCT/GB2000/003780 1999-09-30 2000-10-02 Procedes WO2001023611A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU75411/00A AU7541100A (en) 1999-09-30 2000-10-02 Methods

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GB9923144.1 1999-09-30
GBGB9923144.1A GB9923144D0 (en) 1999-09-30 1999-09-30 Methods

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WO2001023611A2 true WO2001023611A2 (fr) 2001-04-05
WO2001023611A3 WO2001023611A3 (fr) 2001-11-01

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5487972A (en) * 1990-08-06 1996-01-30 Hoffmann-La Roche Inc. Nucleic acid detection by the 5'-3'exonuclease activity of polymerases acting on adjacently hybridized oligonucleotides
US5763167A (en) * 1992-02-12 1998-06-09 Chromagen Applications of fluorescent N-nucleosides and fluorescent structural analogs of N-nucleosides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5487972A (en) * 1990-08-06 1996-01-30 Hoffmann-La Roche Inc. Nucleic acid detection by the 5'-3'exonuclease activity of polymerases acting on adjacently hybridized oligonucleotides
US5763167A (en) * 1992-02-12 1998-06-09 Chromagen Applications of fluorescent N-nucleosides and fluorescent structural analogs of N-nucleosides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TOLMAN G L ET AL: "CHLORO ACETALDEHYDE MODIFIED DI NUCLEOSIDE PHOSPHATES DYNAMIC FLUORESCENCE QUENCHING AND QUENCHING DUE TO INTRA MOLECULAR COMPLEXATION" BIOCHEMISTRY, vol. 13, no. 24, 1974, pages 4869-4878, XP000993063 ISSN: 0006-2960 cited in the application *

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AU7541100A (en) 2001-04-30
WO2001023611A3 (fr) 2001-11-01

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