Real-time monitoring of nucleic acid target-identifying signals

• US 20070048762A1
• Filed: 05/22/2006
• Published: 03/01/2007
• Est. Priority Date: 09/09/2002
• Status: Active Grant

• Current Assignee: Ghazala Hashmi, Joachim Messing, Michael Seul
• Original Assignee: Ghazala Hashmi, Joachim Messing, Michael Seul
• Inventors: Seul, Michael, Messing, Joachim, Hashmi, Ghazala
• Granted Patent: US 7,344,841 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 20/00 Nanooptics, e.g. quantum optics or photonic crystals
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• 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 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 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels

Abstract

This invention provides compositions and methods for genetic testing of an organism and for correlating the results of the genetic testing with a unique marker that unambiguously identifies the organism. The markers may be internal markers, such as for example single nucleotide polymorphisms (SNPs), short tandem repeats (STRs), or other sites within a genomic locus. Alternatively, the markers may be external, such that they are separately added to the genetic sample before testing.

Claims

1-38. -38. (canceled)

39. A method of sequence-specific amplification of assay signals produced in the analysis of a target nucleic acid sequence, the method permitting real-time monitoring of amplified signal and comprising the following steps:

(a) providing a temperature-controlled sample containment device that permits real-time recording of optical assay signals produced within said device and a temperature control means for controlling the temperature of said device;

(b) providing within said sample containment device a set of oligonucleotide probes, said probes being capable of forming a hybridization complex with a target nucleic acid and being attached to beads, wherein said beads are associated with a chemically or physically distinguishable characteristic that identifies the probes attached thereto;

(c) contacting said oligonucleotide probes with the target sequence to form a hybridization complex between the probes and the target sequence;

(d) contacting said hybridization complex with a second oligonucleotide probe, said second probe comprising a label and capable of being ligated to the interrogation probes contained within the hybridization complex;

(e) providing conditions suitable for ligating said second labeled oligonucleotide probe to the interrogation probe;

(f) detecting the optical signals from the second labeled probes ; and

(g) performing one or more annealing-ligating-detecting-denaturing cycles, each cycle increasing the number of extended probes in arithmetic progression and involving the following steps;

(i) providing a first temperature for the formation of the hybridization complex;

(ii) providing a second temperature for ligase-catalyzed ligation of oligonucleotide probe and the second labeled probe to occur, wherein ligation is associated with a change in optical signature of beads associated with the ligated probe;

(iii) imaging and/or recording optical signals from the probes; and

(iv) providing a third temperature for denaturing all hybridization complexes.

40. The method of claim 39, wherein steps (c) to (g) are repeated one or more times.

41. The method of claim 39, wherein the target nucleic acid sequences are derived from the patient genomic sample by amplification of one or more nucleic acid subsequences within the patient genomic sample.

42. The method of claim 41, wherein the amplification is performed using the polymerase chain reaction.

43. The method of claim 39, wherein the target nucleic acid sequence includes a mutation site or a polymorphic site and said set oligonucleotide probes includes members designed to detect the different nucleotides at the mutation or polymorphic site.

44. The method of claim 39, wherein the target sequence includes a mutation site or a polymorphic site and the first set of oligonucleotides includes members capable of annealing to subsequences in the target sequence including the mutation site or the polymorphic site, within a range of proximity to the mutation site or the polymorphic site.

45. The method of claim 43 or 44, wherein the polymorphic site is a single nucleotide polymorphism.

46. The method of claim 43 or 44, wherein the polymorphic site is a short-term tandem repeat site.

47. The method of claim 39, wherein the label is an inorganic nanoparticle label, including semiconductor Quantum Dot particles and Resonant Light Scattering metal nanoparticles.