In situ cloning from pathological tissue specimens

• US 8,945,883 B2
• Filed: 03/21/2013
• Issued: 02/03/2015
• 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:
  US13/848,416
• Publication Number:
  US 20130189698A1
• Time in Patent Office: 684 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 obtaining a cell or tissue of interest from a fixed biological specimen, 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 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 a nucleic acid in said fixed 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).

2. The method of claim 1, wherein said detectable label is a fluorescent label.

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

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

wherein said test fixed biological sample comprises a cell, and wherein said control biological sample comprises a cell;

(b) identifying one or more genes expressed in said test fixed biological sample and in said control fixed sample by;

(i) providing a population of oligonucleotide sequence probes to said fixed 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 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;

(ii) hybridizing said population of oligonucleotide sequence probes with a nucleic acid in said fixed sample, thereby forming a population of hybridized oligonucleotide sequences probes and a population of unhybridized oligonucleotide sequence probes;

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

(iv) isolating said population of hybridized oligonucleotide sequence probes thereby forming an isolated population of hybridized oligonucleotide sequence probes; and

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

4. The method of claim 3, wherein said biological sample is from a human.

7. The method of claim 3, wherein identifying one or more genes expressed in said test fixed biological sample and in said control fixed sample further comprises the step of:

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

8. The method of claim 7, wherein identifying one or more genes expressed in said test fixed biological sample and in said control fixed sample further comprises the step of:

(vi) sequencing said sequence tags in said population of amplified fragments comprising sequence tags.

9. The method of claim 8, wherein identifying one or more genes expressed in said test fixed biological sample and in said control fixed sample further comprises the step of:

(vii) comparing sequences of said sequence tags to a database of gene sequences thereby identifying one or more genes.

5. 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 fixed sample comprising a cell and RNA from said subject;

(b) identifying one or more genes expressed in said fixed sample by;

(i) providing a population of oligonucleotide sequence probes to said fixed 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 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;

(ii) hybridizing said population of oligonucleotide sequence probes with a nucleic acid in said fixed sample, thereby forming a population of hybridized oligonucleotide sequences probes and a population of unhybridized oligonucleotide sequence probes;

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

(iv) isolating said population of hybridized oligonucleotide sequence probes thereby forming an isolated population of hybridized oligonucleotide sequence probes; and

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

6. The method of claim 5, wherein said subject is human.

10. The method of claim 5, wherein identifying one or more genes expressed in said fixed sample further comprises the step of:

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

11. The method of claim 10, wherein identifying one or more genes expressed in said fixed sample further comprises the step of:

(vi) sequencing said sequence tags in said population of amplified fragments comprising sequence tags.

12. The method of claim 11, wherein identifying one or more genes expressed in fixed sample further comprises the step of:

(vii) comparing sequences of said sequence tags to a database of gene sequences thereby identifying one or more genes.