Analog probe complexes

• US 20060014191A1
• Filed: 06/29/2005
• Published: 01/19/2006
• Est. Priority Date: 06/30/2004
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Straus, Neil A., Lao, Kai Qin, Geiser, Timothy G.
• Application Number:
  US11/172,280
• Publication Number:
  US 20060014191A1
• 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 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine

Abstract

The present invention relates to the detection of target sequences. The present description discloses compositions and methods involving analog nucleic acids, such as PNA and L-DNA, for the detection of nucleic acids. Additionally, hybrid detectable markers are provided.

Claims

1. A L-DNA/PNA analog probe complex comprising:

a first segment comprising a protein nucleic acid (PNA) segment, wherein said PNA segment comprises a PNA probe sequence that is capable of binding to a target sequence;

a fluorescent marker attached to the first segment; and

a second segment comprising a left-handed DNA (L-DNA) sequence that is capable of dissociably binding to said PNA probe sequence.

2. The L-DNA/PNA analog probe complex of claim 1, wherein the PNA segment further comprises a second PNA sequence that is configured to not hybridize to the L-DNA sequence, wherein said second PNA sequence binds to the target sequence.

3. The L-DNA/PNA analog probe complex of claim 2, wherein the first segment and the second segment have the same number of nucleotides.

4. The L-DNA/PNA analog probe complex of claim 1, wherein the first and the second segments are bound together via the hybridization of the first PNA sequence and the L-DNA sequence.

5. The L-DNA/PNA analog probe complex of claim 1, wherein the PNA probe sequence and the L-DNA sequence are complementary sequences.

6. The L-DNA/PNA analog probe complex of claim 1 further comprising a first fluorescent moiety as the fluorescent marker on the first segment and a second fluorescent moiety on the second segment, wherein hybridization of the first and second segments results in a first fluorescent signature from the first and second moieties, and a second fluorescent signature is obtained from the first and second moieties in the absence of hybridization.

7. The L-DNA/PNA analog probe complex of claim 6, wherein the first PNA sequence binds to a target sequence, wherein said target sequence is a RNA sequence.

8. The L-DNA/PNA analog probe complex of claim 7, wherein the first segment is longer in length than the second segment on an end of the first segment distal to the fluorescent moiety.

9. The L-DNA/PNA analog probe complex of claim 8, wherein the nucleotides in the L-DNA segment have a conformation of 1′

S, 3′

R, and 4′

S.

10. A method of in-situ hybridization comprising:

contacting an analog probe complex with a sample comprising a target sequence, said analog probe complex comprising a) a first segment comprising a first PNA sequence, wherein said first PNA sequence will hybridize to a target sequence, b) a second segment, said second segment comprising a first L-DNA sequence, wherein said first L-DNA sequence can dissociably bind to said first PNA sequence, c) a fluorophore attached to the first segment, and d) a quencher attached to the second segment;

allowing the PNA sequence to bind to the target sequence;

fixing the sample; and

observing a fluorescence from the fluorophore.

11. The method of claim 10, wherein the PNA sequence is longer than the L-DNA sequence.

12. The method of claim 10, wherein the target sequence is mRNA.

13. The method of claim 10, wherein no wash step is performed after contacting an analog probe and after observing a fluorescence.

14. The method of claim 10, wherein a temperature of a mixture in which the contacting step occurs is lower than a temperature of a mixture that would be required for the PNA segment from an otherwise identical PNA/D-DNA analog probe complex to dissociate in the presence of a target.

15. The method of claim 10, wherein the fixing of the sample occurs before the analog probe complex is contacted with the sample.

16. A kit for wash free in situ hybridization comprising:

an analog probe complex comprising

     1) a first section comprising a fluorescent marker and a PNA sequence, wherein said PNA sequence will hybridize to a target sequence and can dissociably bind to a L-DNA sequence, and

     2) a second section comprising an L-DNA sequence and a quencher, wherein said L-DNA sequence can dissociably bind to said PNA sequence; and

a fixation reagent.

17. The kit of claim 16, further comprising a low ionic strength solution, wherein the low ionic strength is sufficient for dissociation of a L-DNA/PNA hybridized probe, but insufficient for effective dissociation of a D-DNA/PNA probe.

18. The kit of claim 16, wherein the fluorescent marker is a quantum dot.

19. An analog probe complex for the detection of nucleic acids, said analog probe complex comprising:

a first nucleic acid analog segment comprising a means for detecting a target sequence; and

a second nucleic acid analog segment comprising a means for detecting the second nucleic acid analog segment and a means for hybridizing said first segment with said second segment.