In situ cloning from pathological tissue specimens

• US 8,530,196 B2
• Filed: 04/09/2007
• Issued: 09/10/2013
• 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: Eberwine, James H., Kelz, Max B.
• Primary Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US11/784,677
• Publication Number:
  US 20090023593A1
• Time in Patent Office: 2,346 Days
• Field of Search: 435/91.2
• US Class Current: 435/91.2
• 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, wherein said biological sample is fixed, said method comprising:

(a) providing a population of oligonucleotide sequence probes to said fixed biological sample, wherein each of said oligonucleotide sequence probes comprises a sequence tag flanked by a 5′

-end extension sequence and a 3′

-end extension sequence, wherein said 5′

-end and 3′

-end extension sequences are not found in the mammalian genome, further 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 sample, 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,

wherein said fixed biological sample comprises fixed cells.

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.

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

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

5. The method of claim 1, 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.

6. The method of claim 5, wherein said fixed biological sample is treated with a permeabilization agent prior to the hybridizing step.

7. The method of claim 6, wherein said permeabilization agent is selected from the group consisting of proteinase K, pronase and triethanolamine.

8. 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.

9. The method of claim 8, 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.

10. The method of claim 9, further comprising ligating said cleaved fragments comprising sequence tags together prior to said subcloning step.

11. 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.

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

13. The method of claim 3, further comprising comparing sequences of said sequence tags to a database of gene sequences, thereby identifying one or more genes comprising said sequence tags.

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

15. 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.

16. The method of claim 15, further comprising comparing sequences of said sequence tags to a database of gene sequences, thereby identifying genes comprising said sequence tags.

17. A method of identifying a truncated mRNA sequence from a biological sample, wherein said biological sample is fixed, said method comprising:

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

-end extension sequence and a 3′

-end extension sequence, wherein said 5′

-end and 3′

-end extension sequences are not found in the mammalian genome, further 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 sample, 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,

wherein the fixed biological sample comprises fixed cells.