Methods for identifying nucleic acid sequences

• US 9,556,473 B2
• Filed: 08/22/2013
• Issued: 01/31/2017
• Est. Priority Date: 02/15/2011
• Status: Active Grant

• Current Assignee: Leica Biosystems Newcastle Ltd. (Danaher Corporation)
• Original Assignee: Leica Biosystems Newcastle Ltd. (Danaher Corporation)
• Inventors: Bernitz, Mats Nilsson, Larsson, Chatarina, Grundberg, Ida
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/973,814
• Publication Number:
  US 20140120534A1
• Time in Patent Office: 1,258 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/327 RNAse, e.g. RNAseH
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• 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 2543/10 the purpose being "in situ" analysis
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Embodiments relate to the detection of RNA in a sample of cells. More particularly, methods concern the localized detection of RNA in situ. The method relies on the conversion of RNA to complementary DNA prior to the targeting of the cDNA with a padlock probe(s). The hybridization of the padlock probe(s) relies on the nucleotide sequence of the cDNA which is derived from the corresponding nucleotide sequence of the target RNA. Rolling circle amplification of the subsequently circularized padlock probe produces a rolling circle product which may be detected. Advantageously, this allows the RNA to be detected in situ. In additional methods, rolling circle amplification products are sequenced.

Claims

1. A method for evaluating a nucleic acid sequence in a tissue section comprising:

incubating an RNA containing the nucleic acid sequence in the tissue section with a reverse transcriptase and a reverse transcription primer that is complementary to the RNA to generate cDNA, wherein the reverse transcription primer is modified so as to be capable of immobilization in the cells of the tissue section;

digesting all or part of the RNA;

hybridizing one or more padlock probes to the cDNA, wherein the padlock probe(s) comprise one or two terminal regions having the nucleic acid sequence;

incubating the hybridized padlock probe(s) and cDNA with ligase under conditions to ligate the ends of the padlock probe(s);

incubating the padlock probe(s) with a polymerase to create an amplified rolling circle amplification product; and

,

sequencing the amplified rolling circle amplification product.

2. The method of claim 1, wherein the sample is on a solid support.

3. The method of claim 2, wherein the solid support is a slide.

4. The method of claim 3, wherein the slide has a cover.

5. The method of claim 2, wherein the tissue sample is removed from the solid support prior to sequencing.

6. The method of claim 1, wherein the cells are stained.

7. The method of claim 6, wherein the cells are stained with hematoxylin and eosin.

8. The method of claim 1, wherein the reverse transcription primer has a functional moiety capable of binding to or reacting with a cell or cellular component or an affinity binding group capable of binding to a cell or cellular component.

9. The method of claim 1, wherein the reverse transcription primer comprises at least one nucleotide modified with biotin, an amine group, a lower alkylamine group, an acetyl group, DMTO, fluoroscein, a thiol group, or acridine.

10. The method of claim 9, wherein the reverse transcription primer comprises one or more locked nucleic acid residues.

11. The method of claim 10, wherein the reverse transcription primer comprises 2 or more locked nucleic acids separated by 1 or more natural or synthetic nucleotides in the primer sequence.

12. The method of claim 1, further comprising adding a ribonuclease to digest RNA hybridized to the cDNA.

13. The method of claim 1, wherein the rolling circle amplification uses a DNA polymerase having 3′

-5′

exonuclease activity wherein if necessary the exonuclease activity digests the cDNA to generate a free 3′

end which acts as a primer for the RCA.

14. The method of claim 13, wherein the DNA polymerase is a Φ

29 polymerase.

15. The method of claim 1, further comprising contacting the sample with an exonuclease to digest the cDNA to generate a free 3′

end which acts as a primer for the RCA.

16. The method of claim 1, wherein sequencing involves one or more chain terminating nucleotides.

17. The method of claim 1, wherein sequencing comprises mass spectroscopy.

18. The method of claim 1, wherein in the contacting step, the sample is contacted with at least a first and a second padlock probe, wherein the first padlock probe comprises terminal regions complementary to immediately adjacent regions on the cDNA, and wherein the second padlock probe comprises terminal regions that differ from the terminal regions of the first padlock probe only by a single nucleotide at the 5′

or 3′

terminus of the second padlock probe.

19. The method of claim 1, wherein multiple different RNAs are detected using multiple different padlock probes.

20. The method of claim 1, wherein subjecting the circularized padlock probe(s) to rolling circle amplification comprises adding labeled nucleotides to generate labeled, amplified padlock(s).

21. The method claim of 1, wherein RNA is detected in a single cell.

22. The method of claim 1, wherein the sample comprises a fixed tissue section, touch imprint samples, a formalin-fixed paraffin-embedded tissue section or a cytological preparation comprising one or more cells.

23. The method of claim 22, wherein the tissue section is a formalin-fixed paraffin-embedded tissue section.

24. The method of claim 1, wherein the tissue section is a colon, lung, pancreas, prostate, skin, thyroid, liver, ovary, endometrium, kidney, brain, testis, lymphatic fluid, blood, plasma, urinary bladder, or breast sample.

25. A method for determining in situ a nucleic acid sequence in a formalin-fixed paraffin-embedded tissue section comprising:

generating a cDNA complementary to an RNA containing the nucleic acid sequence in the tissue section;

incubating the cDNA with a ribonuclease in the sample to digest the RNA;

hybridizing one or more padlock probes to the cDNA, wherein the padlock probe(s) comprise one or two terminal regions having the nucleic acid sequence;

incubating the hybridized padlock probes and cDNA with ligase under conditions to ligate the ends of the padlock probe(s);

replicating the padlock probe(s) using a polymerase to create an amplified product;

sequencing the complement of the nucleic acid sequence in the amplified product to determine the nucleic acid sequence and/or its complement.

26. The method of claim 25, wherein the sequencing comprises performing mass spectroscopy.