US5643761A
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Method for generating a subtracted cDNA library and uses of the generated library
Assignee: Columbia University
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Filing: 1993-10-27
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Granted: 1997-07-01
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Expires: 2013-10-27
Abstract
Abstract This invention provides a method of generating a subtracted cDNA library of a cell comprising: a) generating a cDNA library of the cell; b) isolating double-stranded DNAs from the cDNA library; c) releasing the double-stranded cDNA inserts from the double-stranded DNAs; d) denaturing the isolated double-stranded cDNA inserts; e) hybridizing the denatured double-stranded cDNA inserts with a labelled single-stranded nucleic acid molecules which are to be subtracted from the cDNA library; and f) separating the hybridized labeled single-stranded nucleic acid molecule from the double-stranded cDNA inserts, thereby generating a subtracted cDNA library of a cell.
Inventors
- 👤 Paul B. Fisher
- 👤 Hongping Jiang
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 00:00
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/54; C07K14/46; C07K14/47; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1096; C12Q; C12Q1/00; C12Q1/68; C12Q1/6809; C12Q1/6876; C12Q1/6883; C12Q1/6886; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/575; G01N33/57505; G01N33/5751; G01N33/68; G01N33/6863; G01N33/6866; C07K2319/00; C12Q2600/00; C12Q2600/106; C12Q2600/112; C12Q2600/136; C12Q2600/158; G01N2333/00; G01N2333/435; G01N2333/705; Y; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
127 words
A method of generating a subtracted cDNA library of a population of cells comprising: a) generating a λ ZAP cDNA library of the cells; b) converting the λ ZAP cDNA library into a phagemid cDNA library; c) preparing double-stranded phagemid DNAs; d) isolating double-stranded DNAs from the prepared phagemid DNAs; e) releasing the double-stranded cDNA inserts from the double-stranded DNAs by digestion of the nucleic acids with appropriate restriction enzyme; f) denaturing the isolated double-stranded cDNA inserts; g) hybridizing the denatured double-stranded cDNA inserts with labelled single stranded nucleic acid molecules, which are to be subtracted from the cDNA library; and h) separating the hybridized labelled single-stranded nucleic acid molecules from the double stranded cDNA inserts, thereby generating a subtracted cDNA library of a population of cells.
Claim 2
Independent Claim
127 words
A method of generating a subtracted cDNA library of a population of cells comprising: a) generating a λ ZAP cDNA library of the cells; b) converting the λ ZAP cDNA library into a phagemid cDNA library; c) preparing double-stranded phagemid DNAs; d) isolating double-stranded DNAs from the prepared phagemid DNAs; e) releasing the double-stranded cDNA inserts from the double-stranded DNAs by digestion of the nucleic acids with appropriate restriction enzyme; f) denaturing the isolated double-stranded cDNA inserts; g) hybridizing the denatured double-stranded cDNA inserts with labelled single stranded nucleic acid molecules, which are to be subtracted from the cDNA library; and h) separating the hybridized labelled single-stranded nucleic acid molecules from the double stranded cDNA inserts, thereby generating a subtracted cDNA library of a population of cells.
Claim 3
Dependent Claim
13 words
A method of claim 1, wherein the single-stranded nucleic acid molecules are DNAs.
Claim 4
Dependent Claim
16 words
A method of claim 2, wherein the single-stranded nucleic acid molecules are from another cDNA library.
Claim 5
Dependent Claim
16 words
A method of claim 2, wherein said another cDNA library allows propagation in single-stranded circle forms.
Claim 6
Dependent Claim
16 words
A method of claim 1, wherein said another cDNA library is a λ ZAP cDNA library.
Claim 7
Dependent Claim
15 words
A method of claim 1, wherein the single-stranded nucleic acis molecules are labelled with biotin.
Claim 8
Dependent Claim
17 words
A method of claim 6, wherein the separating of step h) is performed by extraction with streptavidin-phenol:chloroform.
Claim 9
Dependent Claim
17 words
A method of claim 5, wherein the cells are H0-1 melanoma cells treated with IFN-β and MEZ.
Claim 10
Dependent Claim
21 words
A method of claim 8, wherein the single-stranded nucleic acid molecules are from said another cDNA library of H0-1 melanoma cells.
Claim 11
Dependent Claim
25 words
A method of claim 4, wherein the cells are terminally differentiated and the single-stranded nucleic acid molecules are from another cDNA library of undifferentiated cells.
Claim 12
Dependent Claim
25 words
A method of claim 4, wherein the cells are undifferentiated and the single-stranded nucleic acid molecules are from another cDNA library of terminally differentiated cells.
Claim 13
Dependent Claim
42 words
A method of claim 11, wherein the cells are selected from a group consisting of neuroblastoma cells, glioblastoma multiform cells, myeloid leukemic cells, breast carcinoma cells, colon carcinoma cells, endometrial carcinoma cells, lung carcinoma cells, ovarian carcinoma cells and prostate carcinoma cells.
Claim 14
Dependent Claim
31 words
A method of claim 4, wherein the cells are induced to undergo reversible growth arrest or damage and the single-stranded nucleic acid molecules are from another cDNA library of uninduced cells.
Claim 15
Dependent Claim
28 words
A method of claim 4, wherein the cells are uninduced cells and the single-stranded nucleic acid molecules are from cells induced to undergo reversible growth arrest or damage.
Claim 16
Dependent Claim
32 words
A method of claim 4, wherein the cells are at one developmental stage and the single-stranded nucleic acid molecules are from another cDNA library of the cells at a different developmental stage.
Claim 17
Dependent Claim
24 words
A method of claim 4, wherein the cells are cancerous and the single-stranded nucleic acid molecules are from another cDNA library from normal cells.
Claim 18
Dependent Claim
26 words
A method of claim 4, wherein the cells are selected from the group consisting of breast, brain, meninges, spinal cord, colon, endometrium, lung, prostate and ovary.
Claim 19
Dependent Claim
14 words
A method of claim 1, further comprising introducing the subtracted library into host cells.
Claim 20
Dependent Claim
15 words
A method of claim 1, further comprising ligating the subtracted inserts into λ Uni-ZAP arms.
Claim 21
Dependent Claim
10 words
A subtracted library generated by the method of claim 1.
Claim 22
Dependent Claim
10 words
A subtracted library generated by the method of claim 1.
Claim 23
Dependent Claim
96 words
A method of identifying a melanoma differentiation associated gene comprising: a) generating probes from clones of the subtracted library of claim 1; and b) hybridizing the probe with the total RNAs or mRNAs from H0-1 cells treated with IFN-β and MEZ and the total RNAs or mRNAs from untreated H0-1 cells, hybridization of the probe with the total RNAs or mRNAs from the treated H0-1 cells but no or reduced hybridization with the total RNAs or mRNA from untreated cells indicating that the clone from which the probe is generated carries a melanoma differentiation associated gene.
References Cited (Backward Citations)
No citation records indexed.
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| 3.5 Years (Year 4) | 2000-06-30 | 2000-12-30 | 2001-07-01 | $2000.00 | Window Closed / Lapsed |
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