Method for generating a subtracted cDNA library and uses of the generated library

• US 5,643,761 A
• Filed: 10/27/1993
• Issued: 07/01/1997
• Est. Priority Date: 10/27/1993
• Status: Expired due to Fees

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Fisher, Paul B., Jiang, Hongping
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/143,576
• Time in Patent Office: 1,343 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/69.1, 435/810, 435/172.3, 436/501, 536/22.1, 536/23.5, 536/24.5, 536/24.3-24.33, 935/9, 935/77, 935/78
• US Class Current: 506/9
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/54 Interleukins [IL]
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 33/57426 leukemia
• CPC Class Codes: G01N 33/5743 of skin, e.g. melanoma
• CPC Class Codes: G01N 33/6866 Interferon
• CPC Class Codes: Y10S 435/81 Packaged device or kit

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.

Claims

1. 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.

2. A method of claim 1, wherein the single-stranded nucleic acid molecules are DNAs.

3. A method of claim 2, wherein the single-stranded nucleic acid molecules are from another cDNA library.

4. A method of claim 2, wherein said another cDNA library allows propagation in single-stranded circle forms.

5. A method of claim 1, wherein said another cDNA library is a λ

ZAP cDNA library.

6. A method of claim 1, wherein the single-stranded nucleic acis molecules are labelled with biotin.

7. A method of claim 6, wherein the separating of step h) is performed by extraction with streptavidin-phenol:

chloroform.

8. A method of claim 5, wherein the cells are H0-1 melanoma cells treated with IFN-β

and MEZ.

9. A method of claim 8, wherein the single-stranded nucleic acid molecules are from said another cDNA library of H0-1 melanoma cells.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. A method of claim 1, further comprising introducing the subtracted library into host cells.

19. A method of claim 1, further comprising ligating the subtracted inserts into λ

Uni-ZAP arms.

20. A subtracted library generated by the method of claim 1.

21. A subtracted library generated by the method of claim 1.

22. 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.