Methods for identifying differentially expressed genes and differences between genomic nucleic acid sequences

• US 5,804,382 A
• Filed: 05/10/1996
• Issued: 09/08/1998
• Est. Priority Date: 05/10/1996
• Status: Expired due to Fees

• Current Assignee: New England Deaconess Hospital Corporation
• Original Assignee: Beth Israel Deaconess Medical Center, Inc. (Beth Israel Lahey Health, Inc.)
• Inventors: Sytkowski, Arthur J., Yang, Meiheng
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/644,326
• Time in Patent Office: 851 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.14
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• 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 2600/158 Expression markers

Abstract

Methods for identifying differentially expresses genes and differences between genomic nucleic acid sequences are described. These methods typically include: (a) providing a tester DNA molecule with an amplification tag at the 5'"'"' and 3'"'"' ends of the molecule and a driver DNA or RNA molecule lacking said amplification tag; (b) hybridizing said tester and said driver molecules to form a reaction mixture, wherein said reaction mixture comprises a tester--tester homoduplex, a tester-driver heteroduplex, a driver--driver homoduplex, a single stranded driver DNA molecule and a single stranded tester DNA molecule; (c) treating said reaction mixture to reduce the number of single stranded molecules in said mixture; (d) treating said reaction mixture to remove said amplification tag from said tester-driver heteroduplex; and (e) amplifying said tester--tester homoduplex from said reaction mixture to form an amplification product, wherein steps (c) and (d) occur before step (e).

Claims

1. A method for identifying a differentially expressed gene, comprising:

(a) providing a tester DNA molecule with an amplification tag at the 5'"'"' and 3'"'"' ends of the molecule and a driver DNA or RNA molecule lacking said amplification tag;

(b) hybridizing said tester and said driver molecules to form a reaction mixture, wherein said reaction mixture comprises a tester--tester homoduplex, a tester-driver heteroduplex, a driver--driver homoduplex, a single stranded driver DNA molecule and a single stranded tester DNA molecule;

(c) treating said reaction mixture to reduce the number of single stranded molecules in said mixture;

(d) treating said reaction mixture to remove said amplification tag from said tester-driver heteroduplex; and

(e) amplifying said tester--tester homoduplex from said reaction mixture to form an amplification product, wherein steps (c) and (d) occur before step (e).

2. The method of claim 1 wherein said tester DNA molecule is isolated from a cancer cell.

3. The method of claim 1 wherein said driver DNA molecule is isolated from a normal cell.

4. The method of claim 1 wherein said tester DNA molecule is a double stranded cDNA molecule.

5. The method of claim 1 wherein said driver DNA molecule is a double stranded cDNA molecule.

6. The method of claim 1 wherein said amplification tag comprises a double stranded oligodeoxynucleotide sequence.

7. The method of claim 6 wherein said double stranded oligodeoxynucleotide sequence is the AluI/SacI linker which comprises the nucleic acid sequences shown in SEQ ID NO:

6 and SEQ ID NO;

7.

8. The method of claim 1 wherein said steps (c) and (d) occur simultaneously.

9. The method of claim 1 wherein said hybridization step (b) occurs in the presence of an excess of said driver DNA molecule.

10. The method of claim 1 wherein said reaction mixture is treated with a DNA nuclease which digests single stranded DNA molecules.

11. The method of claim 1 wherein said DNA nuclease is selected from the group consisting of mung bean nuclease, exonuclease VII, S1 nuclease, nuclease BAL-31 and nuclease P1.

12. The method of claim 1 wherein said method further comprises repeating steps (b) through (e) at least one additional time.

13. The method of claim 1 wherein said method further comprises the steps of inserting said amplification product into a vector and constructing a subtraction library.

14. A method for identifying a differentially expressed gene, comprising:

(a) providing a tester DNA molecule with an amplification tag at the 5'"'"' and 3'"'"' ends of the molecule and a driver DNA or RNA molecule lacking said amplification tag;

(b) hybridizing said tester and said driver molecules to form a reaction mixture, wherein said reaction mixture comprises a tester--tester homoduplex, a tester-driver heteroduplex, a driver--driver homoduplex, a single stranded driver DNA molecule and a single stranded tester DNA molecule;

(c) treating said reaction mixture with a DNA nuclease which digests single stranded DNA molecules to reduce the number of said single stranded molecules in said mixture and to remove said amplification tag from said tester-driver heteroduplexes; and

(d) amplifying said tester--tester homoduplex from said reaction mixture to form an amplification product, wherein step (c) occurs before step (d).

15. The method of claim 14 wherein said tester DNA molecule is isolated from a cancer cell.

16. The method of claim 14 wherein said driver DNA molecule is isolated from a normal cell.

17. The method of claim 14 wherein said tester DNA molecule is a double stranded cDNA molecule.

18. The method of claim 14 wherein said driver DNA molecule is a double stranded cDNA molecule.

19. The method of claim 14 wherein said amplification tag comprises a double stranded oligodeoxynucleotide sequence.

20. The method of claim 19 wherein said double stranded oligodeoxynucleotide sequence is the AluI/SacI linker which comprises the nucleic acid sequences shown in SEQ ID NO:

6 and SEQ ID NO;

7.

21. The method of claim 14 wherein said hybridization step (b) occurs in the presence of an excess of said driver DNA molecule.

22. The method of claim 14 wherein said DNA nuclease is selected from the group consisting of mung bean nuclease, exonuclease VII, S1 nuclease, nuclease BAL-31 and nuclease P1.

23. The method of claim 14 wherein said method further comprises repeating steps (b) through (d) at least one additional time.

24. The method of claim 14 wherein said method further comprises the steps of inserting said amplification product into a vector and constructing a subtraction library.

25. A method for constructing a subtraction library containing a differentially expressed gene, comprising:

(a) providing a tester DNA molecule with an amplification tag at both 5'"'"' and 3'"'"' ends of the molecule and a driver DNA or RNA molecule lacking said amplification tag;

(b) hybridizing said tester and said driver molecules to form a reaction mixture, wherein said reaction mixture comprises a tester--tester homoduplex, a tester-driver heteroduplex, a driver--driver homoduplex, a single stranded driver DNA molecule and a single stranded tester DNA molecule;

(c) treating said reaction mixture with a DNA nuclease which digests single stranded DNA molecules to reduce the number of said single stranded molecules in said mixture and to remove said amplification tag from said tester-driver heteroduplexes;

(d) amplifying said tester--tester homoduplex from said reaction mixture to form an amplification product, wherein step (c) occurs before step (d);

(e) repeating steps (b) through (d) at least one additional time;

(f) inserting an amplification product from step (e) into a vector; and

(g) constructing a subtraction library.

26. The method of claim 25 wherein said method further comprises the steps of isolating a differentially expressed nucleic acid from said subtraction library.

27. A differentially expressed nucleic acid isolated by the method of claim 26.

28. The nucleic acid of claim 27 wherein said nucleic acid is isolated from a cancer cell.

29. A recombinant expression vector comprising the nucleic acid of claim 27.

30. A cell comprising the recombinant expression vector of claim 29.

31. A reaction mixture comprising a tester--tester homoduplex, wherein said homoduplex includes an amplification tag at both 5'"'"' and 3'"'"' ends of the homoduplex molecule, a tester-driver heteroduplex lacking the amplification tags, and a driver--driver homoduplex lacking the amplification tags.

32. The reaction mixture of claim 31 further comprising a DNA nuclease which digests single stranded DNA molecules.

33. A method for identifying a difference between genomic DNA sequences, comprising:

(a) providing a tester DNA molecule with an amplification tag at the 5'"'"' and 3'"'"' ends of the molecule and a driver DNA molecule lacking said amplification tag;

(b) hybridizing said tester and said driver molecules to form a reaction mixture, wherein said reaction mixture comprises a tester--tester homoduplex, a tester-driver heteroduplex, a driver--driver homoduplex, a single stranded driver DNA molecule and a single stranded tester DNA molecule;

(c) treating said reaction mixture to reduce the number of single stranded molecules in the mixture and to remove said amplification tag from the tester-driver heteroduplex; and

(d) amplifying said tester--tester homoduplex from said reaction mixture to form an amplification product, wherein step (c) occurs before step (d).

34. A kit comprising an amplification tag, wherein said tag is a double-stranded oligodeoxynucleotide sequence having two restriction enzyme sites, and a DNA nuclease which digests single-stranded DNA molecules.