In situ cloning from pathological tissue specimens
First Claim
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.
1 Assignment
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Accused Products
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.
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Citations
45 Claims
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1. A method of cloning a nucleic acid from a biological sample comprising:
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(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. - View Dependent Claims (2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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3. A method of obtaining a cell or tissue of interest from a biological specimen, said method comprising:
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(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). - View Dependent Claims (14)
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25. A kit for identifying a nucleic acid in a biological specimen of an organism of interest, said kit comprising:
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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. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33)
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34. A kit for identifying a nucleic acid in a human biological specimen, said kit comprising:
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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. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42)
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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.
- -end extension sequence and a 3′
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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.
- -end extension sequence and a 3′
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45. A method of identifying a truncated mRNA sequence comprising:
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(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.
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Specification