In situ cloning from pathological tissue specimens

• US 20090023593A1
• Filed: 04/09/2007
• Published: 01/22/2009
• Est. Priority Date: 04/07/2006
• Status: Active Grant

• Current Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Kelz, Max B., Eberwine, James H.
• Granted Patent: US 8,530,196 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2539/103 Serial analysis of gene expression [SAGE]

Abstract

The present invention pertains to methods related to cloning nucleic acids from biological samples, particularly pathological tissue samples. This method includes hybridizing a population of oligonucleotide sequence probes comprising degenerate sequence tags to a fixed tissue, isolating the hybridized oligonucleotide sequence probes and amplifying the sequence tags in the hybridized oligonucleotide sequence probes. This method can be utilized to identify genes associated with disease and to quantitate the expression of disease-related transcripts. The method can also be used to identify truncated mRNAs.

Claims

1. A method of cloning a nucleic acid from a biological sample comprising:

(a) providing a population of oligonucleotide sequence probes, wherein each of said oligonucleotide sequence probe comprises a sequence tag flanked by a 5′

-end extension sequence and a 3′

-end extension sequence, wherein said sequence tag is a degenerate sequence and wherein at least one of said 5′

-end extension sequence and said 3′

-end extension sequence comprises a detection sequence;

(b) hybridizing said population of oligonucleotide sequence probes with said nucleic acid in said biological specimen, thereby forming a population of hybridized oligonucleotide sequences probes and a population of unhybridized oligonucleotide sequence probes;

(c) washing away said population of unhybridized oligonucleotide sequence probes; and

(d) isolating said population of hybridized oligonucleotide sequence probes thereby forming an isolated population of hybridized oligonucleotide sequence probes, thereby cloning said nucleic acid from said biological sample.

2. The method of claim 1, further comprising the step of:

(e) amplifying said isolated population of hybridized oligonucleotide sequence probes to produce a population of amplified fragments comprising sequence tags.

4. The method of claim 2, further comprising sequencing said sequence tags in said population of amplified fragments comprising sequence tags.

5. The method of claim 1, wherein said biological sample is a pathological sample.

6. The method of claim 1, wherein said biological sample is fixed.

7. The method of claim 6, wherein said fixed biological sample is fixed by a method of fixation selected from the group consisting of formaldehyde, Bouin'"'"'s, xylene and an ethanol precipitating fixative.

8. The method of claim 7, wherein said fixed biological sample is treated with a permeabilization agent prior to the hybridizing step.

9. The method of claim 8, wherein said permeabilization agent is selected from the group consisting of proteinase K, pronase and triethanolamine.

10. The method of claim 2, wherein said 5′

-end extension sequence and said 3′

-end extension sequence each comprise a restriction site for a class II restriction endonuclease or a homing endonuclease.

11. The method of claim 10, further comprising:

(f) cleaving said population of amplified fragments comprising sequence tags with said class II restriction endonuclease or homing endonuclease to produce cleaved fragments, and

(g) subcloning said cleaved fragments into a vector.

12. The method of claim 11, further comprising ligating said cleaved fragments comprising sequence tags together prior to said subcloning step.

13. The method of claim 1, wherein at least one of said 5′

-end extension sequence and said 3′

-end extension sequence comprises a detection sequence.

15. The method of claim 2, wherein said amplifying step comprises the polymerase chain reaction.

16. The method of claim 4, further comprising comparing sequences of said sequence tags to a database of gene sequences, thereby identifying one or more genes comprising said sequence tags.

17. The method of claim 1, wherein said biological sample is from a human.

18. The method of claim 2, further comprising hybridizing said population of amplified fragments comprising sequence tags to a microarray, thereby determining the sequences of the sequence tags.

19. The method of claim 18, further comprising comparing sequences of said sequence tags to a database of gene sequences, thereby identifying genes comprising said sequence tags.

20. A method for identifying differentially expressed genes in a test biological sample, said method comprising:

(a) providing a test biological sample and a control biological sample;

(b) identifying one or more genes expressed in said test biological sample and in said control sample using the method of claim 16, and

(c) comparing the one or more genes expressed in said test sample to the one or more genes expressed in said control sample to identify differentially expressed genes.

21. The method of claim 20, wherein said test biological sample and said control biological sample are fixed tissue samples.

22. The method of claim 20, wherein said biological sample is from a human.

23. A method for in vitro detection of a pathological condition or a susceptibility to a pathological condition in a subject, said method comprising:

(a) providing a sample comprising RNA from said subject;

(b) identifying a gene expressed in said sample using the method of claim 16; and

(c) detecting a pathological condition or a susceptibility to a pathological condition by assessing a change of expression of said gene in said sample compared to expression in a subject without said pathological condition or susceptibility to said pathological condition.

24. The method of claim 23, wherein said subject is human.

3. A method of obtaining a cell or tissue of interest from a biological specimen, said method comprising:

(a) providing a population of oligonucleotide sequence probes, wherein each of said oligonucleotide sequence probe comprises a sequence tag flanked by a 5′

-end extension sequence and a 3′

-end extension sequence, wherein said sequence tag is a degenerate sequence and wherein at least one of said 5′

-end extension sequence and said 3′

-end extension sequence comprises a detection sequence;

(b) hybridizing said population of oligonucleotide sequence probes with said nucleic acid in said biological specimen, thereby forming a population of hybridized oligonucleotide sequences probes and a population of unhybridized oligonucleotide sequence probes;

(c) washing away said population of unhybridized oligonucleotide sequence probes;

(d) annealing a detection oligonucleotide comprising a detectable label to at least one of said 5′

-end extension sequence and said 3′

-end extension sequence of each of said oligonucleotide sequence probe, wherein said detection oligonucleotide is complementary to a detection sequence;

(e) detecting said detectable label to identify a cell or tissue comprising a hybridized oligonucleotide sequence probe; and

(f) separating said cell or tissue that comprises said detectable label from cells or tissue that does not comprise said detectable label, wherein step (d) is performed before or after step (b).

14. The method of claim 3, wherein said detectable label is a fluorescent label.

25. A kit for identifying a nucleic acid in a biological specimen of an organism of interest, said kit comprising:

an oligonucleotide sequence probe, wherein said oligonucleotide sequence probe comprises, in 5′

to 3′

order, a first sequence element comprising a sequence of about 10 to about 22 nucleotides that is not found in the genome of the organism of interest, a second sequence element that is a degenerate sequence and is about 18 to about 28 nucleotides long, and a third sequence element that comprises a sequence of about 10 to about 22 nucleotides that is not found in the genome of the organism of interest;

wherein said first sequence element and said third sequence element are not identical and further wherein said oligonucleotide sequence probe is about 36 to about 200 nucleotides long;

a first primer and a second primer, wherein said first primer comprises a sequence that is identical to at least about 8 consecutive nucleotides of said first sequence element and wherein said second primer comprises a sequence that is 100% complementary to at least about 8 consecutive nucleotides of said third sequence element; and

an instructional material.

26. The kit of claim 25, wherein said oligonucleotide sequence probe further comprises:

a fourth sequence element of about 10 to about 30 nucleotides that comprises a first restriction site, wherein said fourth sequence element is between said first and second sequence elements; and

a fifth sequence element of about 10 to about 30 nucleotides that comprises a second restriction site, wherein said fifth sequence element is between said second and third sequence elements.

27. The kit of claim 25, wherein said first restriction site and said second restriction site are Fok I sites.

28. The kit of claim 25, wherein said first restriction site and said second restriction site are I-CeuI sites.

29. The kit of claim 25 wherein at least one of said first and second primers comprises biotin.

30. The kit of claim 25, wherein at least one of said first and second primers comprises a detectable label.

31. The kit of claim 25, wherein one of said first and second primers comprises biotin and the other of said first and second primers comprises a detectable label.

32. The kit of claim 25, wherein at least one of said first and second primers comprises one or more aminoallyl-substituted nucleotides.

33. The kit of claim 30, wherein said detectable label is selected from the group consisting of a chromogenic moiety, a radioactive moiety and a fluorescent moiety.

34. A kit for identifying a nucleic acid in a human biological specimen, said kit comprising:

an oligonucleotide sequence probe, wherein said oligonucleotide sequence probe comprises, in 5′

to 3′

order, a first sequence element comprising a sequence of about 18 to about 22 nucleotides that is not found in the human genome, a second sequence element that is a degenerate sequence and is about 18 to about 28 nucleotides long, and a third sequence element that comprises a sequence of about 18 to about 22 nucleotides that is not found in the human genome;

wherein said first sequence element and said third sequence element are not identical and further wherein said oligonucleotide sequence probe is about 54 to about 200 nucleotides long;

a first primer and a second primer, wherein said first primer comprises a sequence that is identical to at least about 15 consecutive nucleotides of said first sequence element and wherein said second primer comprises a sequence that is 100% complementary to at least about 15 consecutive nucleotides of said third sequence element; and

an instructional material.

35. The kit of claim 34, wherein said oligonucleotide sequence probe further comprises:

a fourth sequence element of about 10 to about 30 nucleotides that comprises a first restriction site, wherein said fourth sequence element is between said first and second sequence elements; and

a fifth sequence element of about 10 to about 30 nucleotides that comprises a second restriction site, wherein said fifth sequence element is between said second and third sequence elements.

36. The kit of claim 34, wherein said first restriction site and said second restriction site are Fok I sites.

37. The kit of claim 34, wherein said first restriction site and said second restriction site are I-CeuI sites.

38. The kit of claim 34 wherein at least one of said first and second primers comprises biotin.

39. The kit of claim 34, wherein at least one of said first and second primers comprises a detectable label.

40. The kit of claim 34, wherein one of said first and second primers comprises biotin and the other of said first and second primers comprises a detectable label.

41. The kit of claim 34, wherein at least one of said first and second primers comprises one or more aminoallyl-substituted nucleotides.

42. The kit of claim 39, wherein said detectable label is selected from the group consisting of a chromogenic moiety, a radioactive moiety and a fluorescent moiety.

43. A composition comprising an oligonucleotide sequence probe, wherein said oligonucleotide sequence probe comprises a sequence tag flanked by a 5′

-end extension sequence and a 3′

-end extension sequence, wherein said sequence tag is a degenerate sequence and wherein at least one of said 5′

-end extension sequence and said 3′

-end extension sequence comprises a detection sequence.

44. A composition comprising a population of oligonucleotide sequence probes, wherein each of said oligonucleotide sequence probe comprises a sequence tag flanked by a 5′

-end extension sequence and a 3′

-end extension sequence, wherein said sequence tag is a degenerate sequence and wherein at least one of said 5′

-end extension sequence and said 3′

-end extension sequence comprises a detection sequence.

45. A method of identifying a truncated mRNA sequence comprising:

(a) providing a population of oligonucleotide sequence probes, wherein each of said oligonucleotide sequence probe comprises a sequence tag flanked by a 5′

-end extension sequence and a 3′

-end extension sequence, wherein said sequence tag is a degenerate sequence and wherein at least one of said 5′

-end extension sequence and said 3′

-end extension sequence comprises a detection sequence;

(b) hybridizing said population of oligonucleotide sequence probes with said nucleic acid in said biological specimen, thereby forming a population of hybridized oligonucleotide sequences probes and a population of unhybridized oligonucleotide sequence probes;

(c) washing away said population of unhybridized oligonucleotide sequence probes;

(d) isolating said population of hybridized oligonucleotide sequence probes thereby forming an isolated population of hybridized oligonucleotide sequence probes,

(e) amplifying said isolated population of hybridized oligonucleotide sequence probes to produce a population of amplified fragments comprising sequence tags;

(f) annealing a detection oligonucleotide comprising a detectable label to at least one of said 5′

-end extension sequence and said 3′

-end extension sequence of each of said oligonucleotide sequence probe to form a population of detectably labeled amplified fragments, wherein said detection oligonucleotide is complementary to a detection sequence;

(g) hybridizing said population of detectably labeled amplified fragments to a microarray, wherein said microarray is capable of detecting sequences for at least two different regions of at least one mRNA;

(h) detecting the detectable label to obtain a signal for each of said at least two different regions of said at least one mRNA;

(i) comparing said signals from said at least two different regions of said at least one mRNA, whereby a difference in signal strength or an absence of a signal from one region and not another is indicative of a truncation in said mRNA.