Fluorescent in situ rt-pcr

• US 20030059801A1
• Filed: 09/18/2002
• Published: 03/27/2003
• Est. Priority Date: 12/09/1999
• Status: Active Grant

• Current Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Original Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Inventors: Kher, Rajesh, Bacallao, Robert
• Granted Patent: US 7,186,507 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention describes an in situ reverse transcriptase PCR method in which the background fluorescence is greatly reduced as compared to traditional in situ PCR.

Claims

1. A method for in situ amplification of a target nucleic acid in a selected cell comprising:

a) contacting a fixed, permeabilized cell with a restriction endonuclease composition comprising at least one restriction endonuclease to produce a restriction digest;

b) contacting said cell with a DNase to produce a DNase digested cell;

c) incubating said cell with a reverse transcriptase (RT) cocktail comprising an RT enzyme and a RT primer specific for said target nucleic acid to produce a cDNA; and

d) amplifying said cDNA using a PCR reaction in the presence of forward and reverse PCR primers specific for said target nucleic acid wherein at least one of said PCR primers is labeled.

7. The method of claim 1, wherein said PCR primer is labeled with biotin and digoxigenin.

8. The method of claim 1, wherein said restriction endonuclease composition comprises one or more tetra-cutter restriction endonucleases.

9. The method of claim 1, wherein said restriction endonuclease composition comprises one or more restriction endonucleases selected from the group consisting of Hae III, Hpa II, Mbo I, Cfo I Hha I, and Bst 98 I.

10. The method of claim 1, wherein said DNase is DNase I.

11. The method of claim 1, wherein said cell has been permeabilized by contacting said cell with proteinase selected from the group consisting of trypsinase, pepsinogen, and proteinase K.

12. The method of claim 1, wherein said reverse transcriptase is selected from the group consisting of Superscript™

;

AMV Reverse Transcriptase, M-MLV Reverse Transcriptase, Retrotherm™

;

Thermoscript™ and

Tth reverse transcriptase.

13. The method of claim 1, wherein said fixed, permeabilized cell is in a tissue sample selected from the group consisting of kidney, heart, lung, liver, blood, pancreas, cervix, breast and muscle.

14. The method of claim 13, wherein said tissue sample has been obtained from a subject.

15. The method of claim 14, wherein said subject is suffering from a disease selected from the group consisting of cancer, cystic fibrosis, cardiac hypertrophy, and autoimmune diseases.

16. The method of claim 1, wherein said fixed, permeabilized cell is a tumor cell.

17. The method of claim 1, wherein said target nucleic acid encodes a marker for an infectious particle.

18. The method of claim 17, wherein said infectious particle is a viral particle selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, HIV 1, HIV 2, and Epstein-Barr virus particles.

19. The method of claim 17, wherein said infectious particle is M. tuberculin, or M avian.

20. The method of claim 17, wherein said target nucleic acid is a disease specific nucleic acid.

21. The method of claim 20, wherein said target nucleic acid is selected from the group consisting of Muc 1, CCAM, RB, APC, DCC, MEN-I, MEN-II, zacl, MMAC1, FCC, MCC p16, p21, p27, p53, p73, zacl, MMAC1, Rb, WT-1, DCC, NF-1, NF-2, BRCA-1, BRCA-2, MTS, CA125, prostate specific antigen, melanoma antigen gp75, CD9, CD63, CD53, CD37, CD63, R2, CD81, CO029, TI-1, L6 and SAS.

22. The method of claim 1, wherein said cells have been fixed in a fixative selected from the group consisting of formaldehyde, formalin, paraformaldehyde and glutaraldehyde.

23. The method of claim 1, wherein said fixed, permeabilized cells are located in a histochemical section affixed to a microscope slide.

24. The method of claim 1, further comprising the step of detecting said cDNA.

25. The method of claim 1, wherein said PCR reaction employs a DNA polymerase selected from the group consisting of DNA Polymerase I, T4 DNA Polymerase, DNA Polymerase I Klenow fragment, PLATINUM taq™

, Tfl DNA Polymerase, Taq DNA Polymerase, Tli DNA Polymerase, Tth DNA Polymerase, Vent™

, Deepvent™ and

pfu.

26. The method of claim 1, wherein said cell comprises between about 0.1 picograms and 10 micrograms of poly (A)+RNA.

27. The method of claim 1, wherein said cell comprises between 1 and 10⁸ copies of said poly(A)+RNA.

28. The method of claim 1, further comprising the step of counterstaining said cell with a non-fluorescent dye.

29. The method of claim 1, wherein one of said forward and reverse PCR primers is the same as said RT primer.

2. The method of claim 2, wherein at least one of said PCR primers is labeled with a radioactive label, an immunocytochemical label or a fluorescent label to facilitate detection of said amplified cDNA.

3. The method of claim 2, wherein at least one of said PCR primers is labeled with a fluorescent label that fluoresces in the far red range of fluorescence range.

4. The method of claim 3 wherein said fluorescent label fluoresces at about 545 nm or higher.

5. The method of claim 3 wherein said fluorescent label fluoresces at about 645 nm or higher.

6. The method of claim 3, wherein said label is selected from the group consisting of Cy-5, Cy-3, rhodamine and Texas Red.

30. A method for in situ amplification of a target nucleic acid in a selected cell comprising:

a) contacting a fixed, permeabilized cell with a restriction endonuclease composition comprising Sau 96I to produce a restriction digest;

d) contacting said restriction digest with a DNase to produce a DNase digested cell;

e) incubating said DNase digested cell with a reverse transcriptase (RT) cocktail contacting an RT enzyme and a RT primer specific for said target nucleic acid to produce a cDNA; and

f) amplifying said cDNA using a polymerase chain reaction (PCR) reaction in the presence of forward and reverse PCR primers specific for said target nucleic acid wherein at least one of said PCR primers is labeled to facilitate detection

wherein said contacting of said cell with a said restriction endonuclease composition produces an increased signal for detection as compared to an in situ RT PCR reaction conducted in the absence of said restriction endonuclease composition.

31. The method of claim 30, wherein said restriction endonuclease composition comprises a further restriction endonuclease selected from the group consisting of Hae III, Hpa II, Mbo I, Cfo I Hha I, and Bst 98 I.

32. A method for improving the signal from in situ RT PCR reaction comprising subjecting the target cell to a restriction endonuclease reaction and DNase digestion.

33. A method for improving the signal from in situ RT PCR reaction comprising conducting PCR reaction in the presence of at least one PCR primer labeled with a fluorescent label that fluoresces in the far red range of fluorescence range.

34. A kit for use in in situ RT PCR, said kit comprising discrete containers of a first restriction endonuclease, a second restriction endonuclease, and an RNase free DNase.

35. The kit of claim 34, further comprising a reverse transcriptase.

36. The kit of claim 34, further comprising a DNA polymerase.

37. The kit of claim 34, wherein said first restriction endonuclease is selected from the group consisting of Sau 96I, Hae III, Hpa II, Mbo I, Cfo I Hha I, and Bst 98 I.

38. The kit of claim 37, wherein said second restriction endonuclease is selected from the group consisting of Sau 96I, Hae III, Hpa II, Mbo I, Cfo I Hha I, and Bst 98 I.

39. The kit of claim 35, wherein said reverse transcriptase is selected from the group consisting of Superscript™

;

AMV Reverse Transcriptase, M-MLV Reverse Transcriptase, Retrotherm™

;

Thermoscript™ and

Tth reverse transcriptase.

40. The kit of claim 36, wherein said DNA polymerase is selected from the group consisting of DNA Polymerase I, T4 DNA Polymerase, DNA Polymerase I Klenow fragment, PLATINUM taq™

, Tfl DNA Polymerase, Taq DNA Polymerase, Tli DNA Polymerase, Tth DNA Polymerase, Vent™

, Deepvent™ and

pfu.

41. In a method for carrying out an in situ RT PCR reaction on a fixed cell, the improvement comprising the steps of:

(a) contacting the fixed cell with a restriction endonuclease composition to produce a restriction digest, and

(b) contacting the fixed cell with a DNase composition to produce a DNase digest.

42. In a method for carrying out an in situ RT PCR reaction on a fixed cell, the improvement comprising the step of conducting PCR reaction in the presence of at least one PCR primer labeled with a fluorescent label that fluoresces in the far red range of fluorescence range.