EP0929696A2 - Verfahren und reagentiensatz zur erkennung und/oder quantifizierung von nukleinsäuren durch sandwich-hybridisierung an der festphase - Google Patents

Verfahren und reagentiensatz zur erkennung und/oder quantifizierung von nukleinsäuren durch sandwich-hybridisierung an der festphase

Info

Publication number
EP0929696A2
EP0929696A2 EP97939883A EP97939883A EP0929696A2 EP 0929696 A2 EP0929696 A2 EP 0929696A2 EP 97939883 A EP97939883 A EP 97939883A EP 97939883 A EP97939883 A EP 97939883A EP 0929696 A2 EP0929696 A2 EP 0929696A2
Authority
EP
European Patent Office
Prior art keywords
nucleotide sequence
target
sequence
standard
bases
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Withdrawn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
EP97939883A
Other languages
English (en)
French (fr)
Inventor
José REMACLE
Isabelle Alexandre
Nathalie Zammatteo
Isabelle Ernest
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.)
Eppendorf Array Technologies SA
Original Assignee
Eppendorf Array Technologies 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
Priority claimed from BE9600755A external-priority patent/BE1010608A3/fr
Priority claimed from BE9700244A external-priority patent/BE1011052A3/fr
Application filed by Eppendorf Array Technologies SA filed Critical Eppendorf Array Technologies SA
Publication of EP0929696A2 publication Critical patent/EP0929696A2/de
Withdrawn legal-status Critical Current

Links

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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the labeled nucleotide sequence will be immobilized if the target or standard nucleotide sequence is present and immobilized on a trapping nucleotide sequence.
  • the target or standard nucleotide sequence is then sandwiched between the nucleotide sequence trapper and the nucleotide sequence marked, by a double recognition, which increases the specificity, reduces the background noise, and allows a quantification of the nucleotide sequences. This is only possible if the hybridization is carried out quantitatively and reproducibly. This is all the more true as the yield from this hybridization is high.
  • the target and standard nucleotide sequences to be detected and / or quantified consist of any type of nucleic acid, DNA or RNA.
  • the trapped, "helper”, labeled and standard nucleotide sequences used according to the present invention consist of DNA so as to avoid any destruction of these sequences by RNase possibly present in the biological sample. .
  • the term “content in close GC / AT bases” is understood to mean that the ratio of GC / AT bases of the standard is less than 20% of the ratio of GC / AT bases of the target nucleotide sequence.
  • the nucleotide sequence labeled has a length sufficient to specifically recognize the target or standard nucleotide sequence to be detected and / or quantified, this specificity depending on the type of target or standard nucleotide sequence to be detected and / or quantified, and can be characterized by recognition by hybridization a specific complementary portion of at least 10 bases, preferably more than 20 bases, of the target or standard nucleotide sequence.
  • FIG. 7 is a schematic description of the principle of the quantification of a target DNA by using a competitive standard according to the invention and their respective measurement by specific probes after capture on a common immobilized sequence.
  • Another property of the method and of the kit of the invention is the advantageous use of trapping nucleotide sequences having a minimum size of 50 bases if possible 100 and at best 150 or more bases. This observation is unexpected based on the following considerations.
  • the stability of the hybrids in the case where the ionic strength is constant and the size sufficient (beyond 50 cells), only depends on the composition (% G + C i and not on the size of the hybrids
  • the importance of the size to favor or not the hybridization depends on the speed of the hybridization and not on their stability. Indeed, in solution, the size of the nucleic acid strands influences the rate of re-pairing. This is proportional to the square root of the length (Wetmur, JG and Davidson, N. 1968, J. Mol. Biol.
  • the target nucleotide sequence even after sandwich hybridization keeps a free sequence, this can always serve as a pairing site for the other strand of the complementary target nucleotide sequence, which will destabilize the hybrid and may even dissociate according to the experimental conditions (temperature, salts, etc.), because once the pairing has started, the propagation of the formation of the double strand is very rapid and thermodynamically favorable.
  • this sandwich hybridization also allows quantification of these two amplicons with the same efficiency by operating as follows: the preparation containing the 2 amplicons is diluted adequately and is added to 2 or a double series of wells
  • Two types of trappers were used, corresponding to 180 and 360 bases complementary to the amplicon sequence.
  • the hybridizations were carried out in the presence of increasing concentrations of target amplicons at 45 ° C. for 2 hours in a solution consisting of SSC 2x concentrated, ⁇ e Denhart 5x concentrated, Denatured salmon sperm DNA at 0.1 mg / ml.
  • the kinase activity is revealed in 20 mM Tris buffer pH 7.75 containing 60 ⁇ M DTT, 100 ⁇ M EDTA, 5 mM MgCl 2 , 8 ⁇ M Luciferine, 6 mU luciferase per 20 mM KCl well, 1 mM Phosphoenolpyruvate and ADP 3.2 ⁇ M.
  • the light emission is followed for one hour with a luminometer (Lum oskan, Labsystem, Finland) and the results are expressed in RLU. mm.
  • Example 6 Differential hybridization of a small and a
  • tubes are prepared containing increasing concentrations of external standard as defined in FIG. 5. All the tubes have undergone a PCR of 40 cycles using primers corresponding to the sequence
  • 0.1 ml included 100 pmol of each of the 2 primers, 200 mM of the various dNTPs and 2.5 U of Taq DNA polymerase in a 10 mM TRIS-HCl buffer pH 8.4 with 1.5 mM MgCl 2 and KCl
  • Each point represents the average of 3 measurements. There is a correlation between the number of starting copies and the signal obtained. In addition, the curves obtained for the target and the standard are very close, which makes it possible to use the curve of the standard as a reference for determining the number of copies of the target DNA CMV in the starting sample.
  • the composition of the internal standard is given in FIG. 5.
  • Each tube was subjected to amplification by 40 cycles of PCR under the conditions presented in example 15 using the primers corresponding to the sequence 3191-3217 and 3504-3481 of CMV gene ( Figure 5). After amplification, 314 base pair amplicons corresponding to the CMV target and to the standard are therefore obtained. From each of the PCR solutions, 0.04 ml are taken and incubated in 2 wells containing an identical trapper for the common sequences " of the 2 amplicons. In one of the wells, is added the biotinylated 40 base probe specific for the CMV target and in the other, the 40-base biotinylated probe specific for the standard.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (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)
EP97939883A 1996-09-09 1997-09-09 Verfahren und reagentiensatz zur erkennung und/oder quantifizierung von nukleinsäuren durch sandwich-hybridisierung an der festphase Withdrawn EP0929696A2 (de)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
BE9600755 1996-09-09
BE9600755A BE1010608A3 (fr) 1996-09-09 1996-09-09 Procede de quantification d'une sequence d'acides nucleiques.
BE9700244 1997-03-20
BE9700244A BE1011052A3 (fr) 1997-03-20 1997-03-20 Procede et trousse de diagnostic et/ou de quantification par hybridation de type sandwich de sequences d'acides nucleiques sur support solide.
PCT/BE1997/000102 WO1998011253A2 (fr) 1996-09-09 1997-09-09 Procede et trousse de diagnostic et/ou de quantification par hybridation de type sandwich de sequences d'acides nucleiques sur support solide

Publications (1)

Publication Number Publication Date
EP0929696A2 true EP0929696A2 (de) 1999-07-21

Family

ID=25663054

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97939883A Withdrawn EP0929696A2 (de) 1996-09-09 1997-09-09 Verfahren und reagentiensatz zur erkennung und/oder quantifizierung von nukleinsäuren durch sandwich-hybridisierung an der festphase

Country Status (2)

Country Link
EP (1) EP0929696A2 (de)
WO (1) WO1998011253A2 (de)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1096024A1 (de) * 1999-10-28 2001-05-02 Remacle, José Verfahren und Reagentiensatz zum Auswählen und/oder Quantifizieren von multiplen homologen Nukleinsäuresequenzen unter Verwendung von Sondenanordnungen
US7205104B2 (en) * 2000-03-24 2007-04-17 Eppendorf Array Technologies Sa (Eat) Identification of biological (micro) organisms by detection of their homologous nucleotide sequences on arrays
US7202026B2 (en) 2000-03-24 2007-04-10 Eppendorf Array Technologies Sa (Eat) Identification of a large number of biological (micro)organisms groups at different levels by their detection on a same array
US7829313B2 (en) 2000-03-24 2010-11-09 Eppendorf Array Technologies Identification and quantification of a plurality of biological (micro)organisms or their components
US7875442B2 (en) 2000-03-24 2011-01-25 Eppendorf Array Technologies Identification and quantification of a plurality of biological (micro)organisms or their components
EP1164201A1 (de) * 2000-06-14 2001-12-19 Facultés Universitaires Notre-Dame de la Paix Umgekehrter nachweis zur Identifizierung und/oder Quantifizierung von Nukleotid-Zielsequenzen mittels Biochips
EP2441520A1 (de) 2010-10-12 2012-04-18 Eppendorf AG Echtzeitverstärkungs- und Mikroarray-basierte Erkennung von Nukleinsäuretargets in einem Fluss-Chip-Assay

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5213961A (en) * 1989-08-31 1993-05-25 Brigham And Women's Hospital Accurate quantitation of RNA and DNA by competetitive polymerase chain reaction
GB9016163D0 (en) * 1990-07-24 1990-09-05 Cemu Bioteknik Chemical method
FR2683827B1 (fr) * 1991-11-15 1994-03-04 Institut Nal Sante Recherc Medic Procede de determination de la quantite d'un fragment d'adn d'interet par une methode d'amplification enzymatique.
ZA936015B (en) * 1992-08-24 1994-03-10 Akzo Nv Elimination of false negatives in nuleic acid detection.
AU5298393A (en) * 1992-10-08 1994-05-09 Regents Of The University Of California, The Pcr assays to determine the presence and concentration of a target
WO1996009407A1 (en) * 1994-09-21 1996-03-28 Pharmacia Biosensor Ab Process for quantification of nucleic acids

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9811253A3 *

Also Published As

Publication number Publication date
WO1998011253A2 (fr) 1998-03-19
WO1998011253A3 (fr) 1998-05-07

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