DEVICE AND METHOD FOR PARALLEL QUANTITATIVE ANALYSIS OF MULTIPLE NUCLEIC ACIDS

• US 20110039720A1
• Filed: 12/16/2008
• Published: 02/17/2011
• Est. Priority Date: 12/19/2007
• Status: Abandoned Application

• Current Assignee: Biocartis SA (Biocartis Group NV)
• Original Assignee: Biocartis SA (Biocartis Group NV)
• Inventors: Klunder, Derk J. W., Van Houten, Henk, Van Dijk, Erik M. H. P., Van Gemen, Bob, Stapert, Hendrik R., Vossenaar, Erik R.
• Application Number:
  US12/809,412
• Publication Number:
  US 20110039720A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/549 characterised by the capture oligonucleotide being a reporter labelled capture oligonucleotide

Abstract

The present invention relates to a process for conducting real-time PCR, and to a device for conducting the method of the present invention. The invention is especially suited for the simultaneous identification and quantification of nucleic acids present in a sample, e.g. a biological sample. Further, this invention describes a method for simultaneous quantitative analysis of multiple nucleic acid sequences in a single compartment by using an integrated nucleic acid microarray combined with a highly surface-specific readout device. The invention relates to a device wherein a surface which is either part of the chamber surface or a surface that is created in the reaction chamber, such as bead surface, is coated with capture probes and in the same chamber, a PCR reaction takes place.

Claims

1-16. -16. (canceled)

17. A method for simultaneous real-time quantitative detection of multiple target nucleic acid sequences during amplification using capture probes,

wherein the number of simultaneously and quantitatively detected target nucleic acids that are amplified and detected in a single compartment is greater than six, preferably greater than seven, more preferably greater than ten and, wherein the capture probes are probes with a fluorescent label and a quencher in close proximity due to the structure of the probes, and a signal may be detected in case an amplicon hybridizes to said capture probes.

18. The method according to claim 17,

wherein the capture probes are probes with a fluorescent label and a quencher, and a signal may be detected in case an amplicon hybridized to said capture probes causing enzymatic hydrolysis of the capture probes thereby liberating said quencher from said fluorophore.

19. The method according to claim 17,

wherein the capture probes are folded probes with a fluorescent label and a quencher, and a signal may be detected in case an amplicon hybridizes to said capture probes thereby separating said quencher from said fluorophore.

20. The method according to claim 17,

wherein surface-immobilized oligonucleotide probes complementary to said multiple nucleic acid sequences act as capture probes for said multiple amplified nucleic acid sequences,

and wherein detection of said multiple amplified nucleic acid sequences captured to the capture probes is performed with a surface-specific detection method detecting a signal in proximity to the surface.

21. The method according to claim 20, wherein the detection of a signal in proximity to the surface is done by a surface specific detection system and/or by surface-specific generation of the signal to be detected.

22. The method according to claim 20,

wherein the capture probes are immobilized on individually identifiable beads.

23. The method according to claim 22, wherein different beads have different capture probes.

24. The method according to claim 22, wherein said beads are brought to the surface of the compartment and captured amplicons are measured by a surface specific detection method detecting a signal in proximity to said surface of the compartment.

25. The method according to claim 17 comprising the following steps:

providing a device having a reaction compartment, wherein the reaction compartment comprises a surface that is coated with multiple capture probes for said multiple nucleic acid sequences,

adding multiple target nucleic acids and an amplification mixture comprising amplification primer for the amplification of said multiple target nucleic acids to the compartment,

starting amplification thermocycling during which specific segments of said target nucleic acids are amplified thereby creating amplicons,

wherein during the annealing phase of the amplification reaction a reproducible portion of the amplicons hybridize to the capture probes, and

wherein amplicons which are hybridized to said capture probes are specifically and quantitatively detected, wherein the respective signal which is detected is indicative of the initial respective target nucleic acid concentration and thereby the multiple target nucleic acid sequences are quantitatively detected.

26. The method according to claim 17 wherein the capture probes are arranged on the hybridization surface in a patterned array.

27. The method according to claim 17, wherein the same label is used for detection of each of said multiple target nucleic acid sequences.

28. A device for conducting a method according to claim 17 comprising a compartment of which an inner surface is coated with capture probes for multiple target nucleic acid sequences and wherein the capture probes are labeled and a signal may be detected in case an amplicon hybridizes to said capture probes.

29. A device for conducting a method according to claim 22, comprising a compartment with individually identifiable beads wherein capture probes for multiple target nucleic acid sequences are immobilized on said individually identifiable beads.

30. The device according to claim 29, wherein the device further comprises means for bringing the beads to the surface of the compartment and a surface-specific detector that detects those labels which are bound to the surface but does essentially not detect labels which are in solution.

31. The device according to any one of claim 28, wherein the capture probes are probes with a fluorescent label and a quencher, and a signal may be detected in case an amplicon hybridized to said capture probes causing enzymatic hydrolysis of the capture probes thereby liberating said quencher from said fluorophore.

32. The device according to claim 28, wherein the capture probes are folded probes with a fluorescent label and a quencher, and a signal may be detected in case an amplicon hybridized to said capture probes thereby separating said quencher from said fluorophore.

33. Use of a device according to claim 28 for simultaneous quantitative analysis of multiple target nucleic acid sequences.