EP2373811A1 - Utilisation de cyclodextrines pour améliorer la spécificité, la sensibilité et le rendement de réactions d'amplification d'acide nucléique - Google Patents

Utilisation de cyclodextrines pour améliorer la spécificité, la sensibilité et le rendement de réactions d'amplification d'acide nucléique

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Publication number
EP2373811A1
EP2373811A1 EP09796681A EP09796681A EP2373811A1 EP 2373811 A1 EP2373811 A1 EP 2373811A1 EP 09796681 A EP09796681 A EP 09796681A EP 09796681 A EP09796681 A EP 09796681A EP 2373811 A1 EP2373811 A1 EP 2373811A1
Authority
EP
European Patent Office
Prior art keywords
cyclodextrin
nucleic acid
amplification
reaction mixture
target nucleic
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP09796681A
Other languages
German (de)
English (en)
Inventor
Marie-Claire Beckers
Philippe Cronet
Adeline Vitale
Eric Collette
Arzu GÜLLÜKAYA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kaneka Eurogentec SA
Original Assignee
Eurogentec SA
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.)
Filing date
Publication date
Application filed by Eurogentec SA filed Critical Eurogentec SA
Priority to EP09796681A priority Critical patent/EP2373811A1/fr
Priority claimed from PCT/EP2009/067110 external-priority patent/WO2010066908A1/fr
Publication of EP2373811A1 publication Critical patent/EP2373811A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • Pre-mixed dNTP is for example a solution in water of 5 mM of each 2'-deoxy- adenosine-5 '-triphosphate, 2 '-deoxy-cytidine-5 '-triphosphate, 2'-deoxy-guanosine-5'- triphosphate, 2'-deoxy-thymidine-5 '-triphosphate.
  • step b) DNA polymerase, a reaction buffer, dNTPs and primers; b) Contacting the sample containing the target nucleic acid or suspected of containing the target nucleic acid with an amplification reaction mixture containing at least one component from step a); c) Performing the amplification reaction on the final reaction mixture obtained in step b).
  • the concentration of the cyclodextrin in the reaction mixture, consisting of the sample and of the amplification reaction mixture is preferably comprised between 0.1 and 5OmM, more preferably between 0.5 and 5OmM.
  • the cyclodextrin is selected from the group consisting of ⁇ -cyclodextrins, ⁇ -cyclodextrins, ⁇ -cyclodextrin and derivatives thereof. Even more preferably, the cyclodextrin is selected from the group consisting of monopropanediamino- beta-cyclodextrin, 6-O-alpha-D-Maltosyl-beta cyclodextrin, hydroxyethyl-beta- cyclodextrin hydroxypropyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin.
  • PCR is the standard technique for nucleic acid amplification but variants of the PCR methods may also be used in the methods according to the invention.
  • composition may further comprise a sample containing a target nucleic acid or suspected of containing a target nucleic acid.
  • compositions are part of a kit for amplifying a target nucleic acid sequence in a sample.
  • the different reagents are provided in separate containers or as pre-mixes comprising several components.
  • the components may be provided as concentrated stock solutions which have to be mixed and diluted before the nucleic acid amplification.
  • the components may also be provided in a dehydrated, lyophilised or any other dry solid form intended for re-suspension in water or in an appropriate buffer.
  • thermostable DNA polymerase kept in the storage buffer containing cyclodextrin, is usually diluted before the amplification of the nucleic acid is performed.
  • kits according to the invention comprise in the same container a cyclodextrin and at least one dNTP.
  • the container comprises a cyclodextrin and a balanced concentrated pre-mix of dATP, dCTP, dGTP and dTTP.
  • This dNTP pre-mix containing a cyclodextrin is diluted before amplification of the nucleic acid.
  • the container comprises a cyclodextrin and a mixture of dATP, dCTP, dGTP and dUTP.
  • kits comprise in the same container a labelled probe hybridizing to the target nucleic acid and a cyclodextrin.
  • the kits of the present invention provide for the amplification of both DNA and
  • the cyclodextrin may be provided as a concentrated stock solution in a separate container or as a mixture with other components or reagents.
  • the final concentration of the cyclodextrin in the final reaction mixture is preferably comprised between 0.1 and 5OmM, more preferably between 0.5 and 5OmM and even more preferably between 0.1, 0.5, 1, 2, 4, 5 mM to 10, 15, 20, 25, 30, 40 and 5OmM.
  • the kit according to the invention may contain the cyclodextrin in solution or in a dry solid form for re-suspension in water or in an appropriate buffer.
  • the cyclodextrin may be provided as a pre-mix or concentrated stock solution with other components.
  • the kit may also comprise instructions for preparation of the amplification reaction mixture having the appropriate concentration in cyclodextrin.
  • the cyclodextrin may be directly added to the other reagents for preparation of the amplification reaction mixture.
  • the cyclodextrin may be used to pre-treat one of the components of the reaction such as for example the thermostable DNA polymerase, the dNTPs or the oligonucleotide primers.
  • the cyclodextrin may be added to the sample containing or suspected of containing the target nucleic acid.
  • thermostable DNA polymerase is used in the amplification reaction mixture which provides good specificity, yield and sensitivity.
  • the amount of thermostable DNA polymerase in the final reaction mixture is comprised between 0.01, 0.02, 0.03, 0.04 units/ ⁇ l to 0.05 , 0.075, 0.1 and 0.2 units/ ⁇ l.
  • the kits of the present invention typically may not comprise all the components required for the amplification of nucleic acids. Some components such as the primers and the sample may be provided by the final user of the kit.
  • kits of the present invention may also comprise an internal positive control (IPC).
  • the internal positive control comprises primers and/or a specific probe and a control DNA template.
  • This IPC may also contain cyclodextrin.
  • Figure 9 PCR without initial heating step: Amplification with Taq PO8, Taq PO8 +6mM cyclodextrin and Hot GoldStar
  • Example 1 Genes, primers and specific PCR products
  • Primer sequences Numb Primer FWD: gag gtt cct aca ggc ace tgc cca g
  • the three bases of these primers can hybridize together and give at low temperature primer-dimers.
  • NCBI reference The target is identified as NT _ 026437.11 in the NCBI database (sequence location:
  • the target is identified as NT_07592.14 in the NCBI database.
  • Primer sequences t-PAl Primer FWD: aga cag tac age cag cct ca Primer REVl : gac ttc aaa ttt ctg etc etc During PCR, in some conditions, these primers can generate the formation of primer- dimers.
  • NCBI reference The target is identified as NT 007995.14 in the NCBI database. Amplicon length: 374bp
  • HLA-C but also formation of primer-dimers and of non-specific product.
  • Taq-cyclodextrin preparation increases the amplification of specific
  • a non-specific fragment is always seen on the gel.
  • SAl 250 mM dilute 2x with H2O the SAl stock 50OmM and stored at -20 0 C. This SAl stock 50OmM was resuspended in 2x storage buffer.
  • SAR 250 mM Weigh 0,0408 g SAR, add 56 ⁇ l 2x storage buffer and vortex to obtain a final concentration of 500 mM -> the SAR isn't dissolved in the solution.
  • Aim Perform PCR reaction without initial heating step at 95°C with TAQ polymerase
  • PCR begins directly with cycles (no activation step): 35 cycles: 10 sec at 95°C 10 sec at 60 0 C
  • HotGoldStar Taq polymerase requires an activation step at 95°C before PCR cycling. Without this activation step, there are not amplification of specific genes with HotGoldSar. We observe an amplification of the specific genes with the non-modified TAQ polymerase and with TAQ-cyclodextrin preparation even if the initial heating step is removed. The addition of cyclodextrin to TAQ polymerase, improves the efficiency and sensitivity of the PCR reaction, the specific fragment is amplified and there is much less primer-dimers that them obtained without cyclodextrin.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés de synthèse d'ADN in vitro catalysée par une ADN polymérase et utilisant des cyclodextrines. L’invention concerne également des procédés, des compositions et des kits comprenant des cyclodextrines destinés à l'amplification d'un acide nucléique. L'utilisation des cyclodextrines améliore la spécificité, la sensibilité et/ou le rendement de la réaction d'amplification. L'invention concerne plus particulièrement des kits, des compositions et des procédés destinés à mettre en œuvre des réactions PCR comprenant une cyclodextrine.
EP09796681A 2008-12-12 2009-12-14 Utilisation de cyclodextrines pour améliorer la spécificité, la sensibilité et le rendement de réactions d'amplification d'acide nucléique Withdrawn EP2373811A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP09796681A EP2373811A1 (fr) 2008-12-12 2009-12-14 Utilisation de cyclodextrines pour améliorer la spécificité, la sensibilité et le rendement de réactions d'amplification d'acide nucléique

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP2008067435 2008-12-12
PCT/EP2009/067110 WO2010066908A1 (fr) 2008-12-12 2009-12-14 Utilisation de cyclodextrines pour améliorer la spécificité, la sensibilité et le rendement de réactions d'amplification d'acide nucléique
EP09796681A EP2373811A1 (fr) 2008-12-12 2009-12-14 Utilisation de cyclodextrines pour améliorer la spécificité, la sensibilité et le rendement de réactions d'amplification d'acide nucléique

Publications (1)

Publication Number Publication Date
EP2373811A1 true EP2373811A1 (fr) 2011-10-12

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP09796681A Withdrawn EP2373811A1 (fr) 2008-12-12 2009-12-14 Utilisation de cyclodextrines pour améliorer la spécificité, la sensibilité et le rendement de réactions d'amplification d'acide nucléique

Country Status (1)

Country Link
EP (1) EP2373811A1 (fr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2010066908A1 *

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