PROBE DENSITY SELF-CONSIDERATIONS AND ELONGATION OF COMPLEMENTARY LOOPED PROBES WHERE PROBES ARE ATTACHED TO A SOLID PHASE

• US 20100267578A1
• Filed: 02/18/2010
• Published: 10/21/2010
• Est. Priority Date: 04/12/2006
• Status: Active Grant

• Current Assignee: BioArray Solutions Limited (Werfen SA)
• Original Assignee: BioArray Solutions Limited (Werfen SA)
• Inventors: SEUL, MICHAEL, Banerjee, Sukanta, Zhang, Yi, Yang, Jiacheng, Chau, Chiu
• Granted Patent: US 8,795,966 B2
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

In a multiplexed assay method carried out in solution, wherein the solution contains nucleic acid targets and, wherein several different types of oligonucleotide probes, each type having a different sequence in a region designated as a target binding domain, are used to detect the nucleic acid targets, said assay method including a method for increasing the effective concentration of the nucleic acid targets at the surface of a bead to which the oligonucleotide probes are bound, by one or more of the following steps:

adjusting assay conditions so as to increase the effective concentration of the targets available for binding to the probes, by one or more of the following: (i) selecting a particular probe density on the surface of the bead; (ii) selecting a solution having an ionic strength greater than a threshold; (ii) selecting a target domain of a size less than a threshold; or (iii) selecting target domains within a specified proximity to a terminal end of the targets.

Claims

1-3. -3. (canceled)

4. In a multiplexed assay method carried out in solution, wherein the solution contains nucleic acid targets and, wherein several different types of oligonucleotide probes, each type having a different sequence in a target binding domain, are bound to a substrate and used to detect the nucleic acid targets, and wherein said probes have a target binding domain and a complementary closing domain capable of forming a duplex with the target binding domain wherein when the duplex is formed, no signal is emitted by the probe, and a joining region between the target binding domain and the closing domain, and wherein the same signal is generated by a probe in a non-duplex as by a probe bound to the target or by an elongated probe bound to the target, the method comprising:

placing the probes in contact with the targets under conditions suitable for capture of the target and formation of a probe-target duplex;

generating conditions suitable for enzyme-mediated probe elongation wherein the 3′

terminal end of the probe is elongated if a nucleotide in the target sequence which is aligned with the 3′

terminal end of the target binding domain is complementary; and

detecting the increase in cumulative signal associated with each type of probe, resulting from probe elongation.

5. The method of claim 4 wherein the detection of elongation is by detecting a signal associated with labeled nucleotides incorporated into the elongated probe.

6. The method of claim 5 wherein labeled dNTPs or ddNTPs are incorporated into the elongated probe.

7. The method of claim 4 wherein detection of elongation is performed by conducting a thermal stability analysis, by cycling to a temperature above the de-annealing temperature of non-elongated duplexes and then monitoring probe fluorescence to determine probe-target-associated fluorescence.

13. The method of any of claim 4, 8, or 9, wherein the substrate is a microparticle.

14. The method of any of claim 4, 8, or 9, wherein the ionic strength of the solution is increased by adding salt.

15. The method of any of claim 4, 8, or 9, wherein there are several different types of oligonucleotide probes on each microparticle.

16. The method of any of claim 4, 8, or 9, wherein the target binding domain is fully complementary to the target domain.

17. The method of any of claim 4, 8, or 9, wherein the oligonucleotide probes and the nucleic acid targets can either be DNA or RNA.

8. A method of expanding the operating range of stringencies of a multiplexed format of nucleic acid analysis, wherein a solution contains nucleic acid targets and, wherein several different types of oligonucleotide probes, each type having a different sequence in a target binding domain, are bound to a substrate and are used to detect the nucleic acid targets, and wherein said probes have a target binding domain and a complementary closing domain capable of forming a duplex with the target binding domain, wherein when the duplex is formed, no signal is emitted by the probe, and a joining region between the target binding domain and the closing domain, and wherein the same signal is generated by a probe in a non-duplex as by a probe bound to the target or by a probe bound to the target and elongated, the method comprising:

stabilizing the duplex by elongating the 3′

terminal ends of certain probes which have a nucleotide in the target sequence aligned with a complementary nucleotide in the target binding domain to thereby generating a stable duplex capable of withstanding a wider range of reaction conditions without causing changes in the assay results.

11. The method of claim 8 or 9 wherein the reaction time is selected to reflect the stringency of the imposed conditions, the stringency determining the probability of random formation of a probe-target-enzyme-substrate intermediate state in the formation of elongation product, such that a sufficient number of stable elongated duplex states are capable of being formed.

9. A method of conducting a multiplexed format of nucleic acid analysis, wherein a solution containing nucleic acid targets is placed in contact with several different types of oligonucleotide probes, each different type having a different sequence in a region designated as a target binding domain, wherein when the duplex is formed, no signal is emitted by the probe), and a joining region between the target binding domain and the closing domain, and wherein the same signal is generated by a probe in a non-duplex as by a probe bound to the target, the method comprising:

said target binding domain joined to a complementary closing domain through a joining region, the method comprising;

adjusting assay conditions so as to permit stabilization of probe-target complexes by target-mediated enzymatic elongation; and

detecting capture by monitoring probe fluorescence from the target-associated state of the probe and comparing it to the pre-assay signal.

10. The method of claim 9 further including increasing the effective concentration of the targets available for binding to the probes by one or more of the following:

(i) adjusting the solution'"'"'s ionic strength to greater than a threshold;

(ii) selecting a target domain of a size less than a threshold;

or (iii) selecting target domains within a specified proximity to a terminal end of the targets.

18. The method of claim 10, wherein the ionic strength threshold corresponds to a buffer concentration of 50 mM salt.

12. (canceled)