Oligonucleotides and assemblies thereof useful in the detection of the presence or absence of target nucleic acid sequences in a sample

• US 6,887,662 B1
• Filed: 12/04/2000
• Issued: 05/03/2005
• Est. Priority Date: 11/29/1999
• Status: Expired due to Term

• Current Assignee: DiaSorin S.p.A.
• Original Assignee: DiaSorin S.p.A.
• Inventors: Alajem, Sara, Reinhartz, Avraham, Waksman, Michal
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/727,480
• Time in Patent Office: 1,611 Days
• Field of Search: 435/6, 435/91.2, 435/91.21, 435/91.5, 536/231, 536/24.3, 536/24.31, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

Oligonucleotides useful in the detection of a nucleic acid target sequence in a sample.

Claims

1. A method of detecting a presence or an absence of a target nucleic acid sequence in a sample, the method comprising the steps of:

(a) contacting the sample with an oligonucleotide system under hybridization conditions so as to form a reaction mixture, said oligonucleotide system including an anchor oligonucleotide and an amplifier oligonucleotide, each of said anchor and said amplifier oligonucleotides includes a first region complementary with the target nucleic acid sequence, each of said anchor and said amplifier oligonucleotides further including a second region, said second regions of said anchor and said amplifier oligonucleotides being at least partially complementary and thus capable of forming a duplex structure including a nucleic acid cleaving agent recognition sequence following hybridization of said first regions of said anchor and said amplifier oligonucleotides with the target nucleic acid sequence, said anchor and said amplifier oligonucleotides are selected such that when hybridized with the target nucleic acid sequence in a presence of a nucleic acid cleaving agent recognizing said nucleic acid cleaving agent recognition sequence, only said amplifier oligonucleotide is cleavable by said nucleic acid cleaving agent, wherein cleavage of said amplifier oligonucleotide leads to dissociation of said amplifier oligonucleotide from the target nucleic acid sequence while said anchor oligonucleotide remains hybridized to, and does not dissociate from, the target nucleic acid sequence to form a stabilized anchor oligonucleotide-target nucleic acid sequence hybrid, thereby allowing a second and uncleaved amplifier oligonucleotide to hybridize with said anchor oligonucleotide-target nucleic acid sequence hybrid, thus enabling recycling of said anchor oligonucleotide-target nucleic acid sequence hybrid with respect to said amplifier oligonucleotide;

(b) adding said nucleic acid cleaving agent to said reaction mixture under predetermined reaction conditions, such that, if the target nucleic acid sequence is present in the sample, said nucleic acid cleaving agent recognition sequence is cleaved by said nucleic acid cleaving agent; and

(c) monitoring cleavage of said nucleic acid cleaving agent recognition sequence by said nucleic acid cleaving agent;

wherein cleavage of said nucleic acid cleaving agent recognition sequence by said nucleic acid cleaving agent indicates hybridization of the oligonucleotide system to the target nucleic acid sequence and therefore the presence of the target nucleic acid in the sample.

2. The method of claim 1, wherein under said hybridization conditions said first region said amplifier oligonucleotide is stably hybridizable with said target nucleic acid sequence only if said first region of said anchor oligonucleotide is stably hybridizable with said nucleic acid target sequence.

3. The method of claim 1, wherein at least one nucleotide or internucleotidic bond of said anchor oligonucleotide which forms a part of said nucleic acid cleaving agent recognition sequence includes a modification selected so as to prevent cleavage of said anchor oligonucleotide by said nucleic acid cleaving agent.

4. The method of claim 1, wherein said duplex structure is formed in part by self annealing of a portion of said second region of said amplifier oligonucleotide.

5. The method of claim 1, wherein a sequence of said first region of said anchor oligonucleotide is selected such that said anchor oligonucleotide remains annealed with said target nucleic acid sequence under said predetermined reaction conditions, whereas said sequence of said first region of said amplifier oligonucleotide is selected such that said amplifier oligonucleotide dissociates from said target nucleic acid sequence under said predetermined reaction conditions, following cleavage of said nucleic acid cleaving agent recognition sequence by said nucleic acid cleaving agent.

6. The method of claim 5, wherein a Tm of said first region of said anchor oligonucleotide is at least 10°

C. higher than said Tm of said first region of said amplifier oligonucleotide.