Method for analyzing quantitative expression of genes

• US 5,968,784 A
• Filed: 01/15/1997
• Issued: 10/19/1999
• Est. Priority Date: 01/15/1997
• Status: Expired due to Fees

• Current Assignee: Chugai Pharmaceutical Co., Ltd. (Roche Holding AG)
• Original Assignee: Chugai Pharmaceutical Co., Ltd. (Roche Holding AG)
• Inventors: Sajjadi, Fereydoun G., Spinella, Dominic G.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/784,208
• Time in Patent Office: 1,007 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/320.1, 435/270, 435/252.3, 435/91.4, 435/91.51, 435/810, 436/94, 935/76, 935/77, 536/23.1
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The present invention provides novel methods for identifying gene expression patterns in mRNA populations. The methods are useful for determining differential gene expression among various cells or tissues, including cells or tissues of a target organism. The invention also provides methods of determining the frequency of gene expression in mRNA populations, thus providing a method of comparing gene expression frequency among various cells or tissues. The present invention also provides methods for isolating genes corresponding to tag sequences identified according to the methods of the present invention. Furthermore, sequences that are identified according to the present invention may be used to diagnose the presence of disease.

Claims

1. A method of identifying gene transcription patterns in an mRNA population, comprising the steps of:

(a) preparing double-stranded cDNAs from an mRNA population using a primer, wherein said primer contains a recognition sequence of six bases or greater for a priming restriction endonuclease;

(b) cleaving said double-stranded cDNAs with;

a first restriction endonuclease which cleaves within said cDNA sequence and not within said primer, and

the priming restriction endonuclease which cleaves within said primer to obtain a population of cDNA inserts;

(c) inserting said cDNA inserts into insertion sites of cloning vectors to obtain a population of DNA constructs, wherein said cloning vectors comprise

a second restriction endonuclease recognition sequence located 5'"'"' to said insertion sites, and

a third restriction endonuclease recognition sequence located 5'"'"' to or overlapping with said second restriction endonuclease recognition sequence,

and wherein said cDNA inserts are inserted into said cloning vectors in an orientation in which an end cleaved by the first restriction endonuclease is proximal to the second restriction enzyme recognition site and an end cleaved by the primer restriction endonuclease is distal to the second restriction enzyme recognition site;

(d) replicating said DNA constructs;

(e) isolating said DNA constructs;

(f) digesting said DNA constructs with a second restriction endonuclease that is a Type IIs restriction endonuclease, thereby cleaving within the cDNA inserts;

(g) digesting said DNA constructs with a third restriction endonuclease, thereby cleaving the DNA constructs 5'"'"' to cleavage sites of the second restriction endonuclease to obtain tags comprising cDNA sequences;

(h) causing said tags to have blunt 5'"'"' and 3'"'"' ends, if one or more ends has an overhang resulting from restriction endonuclease digestion;

(i) ligating said tags to obtain ligated tandem arrays of tags comprising at least 10 tags;

(j) inserting said ligated tandem arrays of tags into a sequencing vector; and

(k) determining the nucleotide residue sequence of said tags to identify gene transcription patterns in said mRNA population.

2. The method of claim 1 wherein said DNA constructs are amplified in a host cell capable of replicating said DNA constructs.

3. The method of claim 2 wherein said host cell is E. coli.

4. The method of claim 1 wherein said first restriction endonuclease recognizes a sequence consisting of less than six bases.

5. The method of claim 1 wherein said first restriction endonuclease recognizes a sequence consisting of four bases.

6. The method of claim 5 wherein said first restriction endonuclease is MboI.

7. The method of claim 1 wherein said third restriction endonuclease is PmlI or FokI.

8. The method of claim 1, wherein said second restriction endonuclease is BsgI.

9. The method of claim 1 wherein said third restriction endonuclease recognition sequence is located within 20 to 40 nucleotides 5'"'"' of a cleavage site of said second restriction endonuclease.

10. The method of claim 1 wherein said third restriction endonuclease recognition sequence is located within 10 to 15 nucleotides 5'"'"' of a cleavage site of said second restriction endonuclease.

11. The method of claim 10 wherein said second restriction endonuclease is BsgI and said third restriction endonuclease is FokI.

12. The method of claim 1 wherein said third restriction endonuclease recognition sequence is located within said second restriction endonuclease recognition sequence.

13. The method of claim 12 wherein said second restriction endonuclease is BsgI and said third restriction endonuclease is PmlI.

14. The method of claim 1 wherein said primer comprises an oligo dT sequence.

15. The method of claim 14 wherein said oligo dT sequence consists of 7 to 40 T residues.

16. The method of claim 14 wherein said recognition sequence for a priming restriction endonuclease is linked to the 5'"'"' end of said oligo dT sequence.

17. The method of claim 16 wherein said priming restriction endonuclease recognizes a sequence consisting of more than six bases.

18. The method of claim 17 wherein said priming restriction endonuclease recognizes a sequence consisting of at least 8 bases.

19. The method of claim 18 wherein said priming restriction endonuclease recognizes a sequence comprising at least one CG dinucleotide.

20. The method of claim 1, wherein said priming restriction endonuclease is Not I.

21. The method of claim 1 wherein insertion sites of said cloning vectors have a first end compatible with a cleavage site of said priming restriction endonuclease and a second end compatible with a cleavage site of said first restriction endonuclease.

22. The method of claim 1 wherein said tags are treated with T4 DNA polymerase to cause tags to have blunt 5'"'"' and 3'"'"' ends.

23. The method of claim 1 wherein said ligating step uses DNA ligase.

24. The method of claim 1 wherein said ligated tandem arrays of tags comprise at least 50 tags.

25. The method of claim 1 wherein said ligated tandem arrays of tags comprise at least 100 tags.

26. The method of claim 1 wherein said ligated tandem arrays of tags comprise at least 200 tags.

27. A method for determining the frequency of gene transcription in an mRNA population comprising the steps of:

(a) preparing a cDNA library comprising DNA constructs according to steps (a) through (d) of claim 1;

(b) preparing an oligonucleotide probe comprising a nucleotide sequence of a tag identified according to the method of claim 1; and

(c) probing said cDNA library with said oligonucleotide probe to determine the frequency of transcription of a gene which comprises said nucleotide sequence.

28. The method of claim 27, further comprising the step of isolating from said cDNA library a gene sequence which comprises said nucleotide sequence for which the frequency of gene transcription was determined in step (c).

29. A method for detecting a difference in gene transcription between two or more mRNA populations, comprising the steps of:

(a) identifying a gene transcription pattern from a first mRNA population according to claim 1;

(b) identifying a gene transcription pattern from at least one additional mRNA population according to the method of claim 1; and

(c) comparing the gene transcription pattern of said first mRNA population with the gene transcription pattern of at least one additional mRNA population, thereby detecting differences in gene transcription between said first and additional mRNA populations.

30. The method of claim 29 wherein said first mRNA population is obtained from a normal cell or tissue and said additional mRNA population is obtained from a cell or tissue from a target organism having a disease or disorder.

31. The method of claim 30, further comprising the step of isolating a tag nucleotide sequence that is differentially expressed in said first mRNA population compared to said additional mRNA population to produce a probe containing said tag nucleotide sequence.

32. The method of claim 31, further comprising the steps of:

probing a second additional mRNA population with said probe containing said tag nucleotide sequence to determine gene transcription of said tag nucleotide sequence in said second additional mRNA population; and

determining whether gene transcription of said tag nucleotide sequence in said second additional mRNA population is similar to the gene transcription pattern on said first mRNA population obtained from a normal cell or tissue or similar to the gene transcription pattern of the additional mRNA population obtained from a cell or tissue from a target organism having a disease or disorder.

33. The method of claim 26 wherein said first and said additional mRNA populations are obtained from cells or tissues at different developmental stages.

34. The method of claim 29 wherein said first and additional mRNA populations are obtained from cells derived from different tissues or organs of a single target organism.

35. The method of claim 29 wherein said first and said additional mRNA populations are obtained from different target organisms.

36. The method of claim 29, further comprising the step of isolating a tag nucleotide sequence that is differentially expressed in said first mRNA population compared to said additional mRNA population to produce a probe containing said tag nucleotide sequence.

37. The method of claim 36, further comprising the step of probing a second additional mRNA population with said probe containing said tag nucleotide sequence to determine a gene transcription pattern of said tag nucleotide sequence in said second additional mRNA population.

38. The method of claim 36, further comprising the step of probing a cDNA library with the probe containing said tag nucleotide sequence to isolate a gene sequence that hybridizes to said tag nucleotide sequence.

39. A kit for use in identifying gene transcription patterns in an mRNA population, comprising:

(a) a DNA vector comprising an insertion site that includes a NotI recognition sequence, a first restriction endonuclease recognition sequence, Sequence A, located 5'"'"' to said insertion site, wherein said first recognition sequence is specific for a first restriction endonuclease that cleaves at a site, Sequence B, located 3'"'"' to said first recognition sequence, and a second restriction endonuclease recognition sequence, Sequence C, located 5'"'"' to or overlapping Sequence A;

(b) a primer comprising an oligo dT sequence of 7 to 40 T residues;

(c) a first restriction endonuclease that recognizes said Sequence A and cleaves DNA at a said Sequence B; and

(d) a second restriction endonuclease that recognizes said Sequence C.

40. The kit of claim 39, wherein said primer further comprises a cleavage site of a priming restriction endonuclease linked to the 5'"'"' end of said oligo dT sequence.

41. The kit of claim 40, wherein said priming restriction endonuclease recognizes a sequence of at least 8 bases.

42. The kit of claim 39, wherein said first restriction endonuclease is a Type IIS restriction endonuclease.

43. The kit of claim 39, wherein said second restriction endonuclease is a type IIS restriction endonuclease.

44. The kit of claim 39, wherein said first restriction endonuclease is BsgI, and said second restriction endonuclease is FokI.

45. The kit of claim 39, further comprising

(a) a third restriction endonuclease that recognizes a sequence consisting of at least 8 bases;

and

(b) a fourth restriction endonuclease that recognizes a sequence consisting of four bases,

wherein digestion of said DNA vector with said third and fourth restriction endonucleases cleaves said DNA vector at said insertion site.

46. A method of identifying gene transcription patterns in an mRNA population, comprising the steps of:

(a) preparing double-stranded cDNAs from a mRNA population using a primer, wherein said primer comprises an oligo dT sequence linked at its 5'"'"' end to a NotI cleavage site;

(b) cleaving said double-stranded cDNAs with NotI and with MboI to obtain cDNA inserts;

(c) inserting said cDNA inserts into insertion sites of cloning vectors to obtain DNA constructs, wherein said cloning vectors comprise

a BsgI recognition sequence 5'"'"' to the insertion sites such that digestion of said DNA constructs with BsgI is capable of cleaving said DNA constructs at sites within the cDNA inserts, and

a FokI recognition sequence which is 5'"'"' to said BsgI recognition sequence,

and wherein said cDNA inserts are inserted into said cloning vectors such that an MboI-cleaved end is proximal to the BsgI recognition sequence and a NotI-cleaved end is distal to the BsgI recognition sequence;

(d) replicating said DNA constructs;

(e) isolating said DNA constructs;

(f) digesting said DNA constructs with BsgI;

(g) digesting said DNA constructs with FokI to obtain tags, wherein said tags comprise a portion of said cDNAs;

(h) treating said tags with T4 DNA polymerase to obtain blunt-ended tags;

(i) ligating said blunt-ended tags using DNA ligase to obtain ligated tandem arrays of tags comprising 30 to 60 tags;

(j) inserting said ligated tandem arrays of tags into a sequencing vector;

(k) sequencing said ligated tandem arrays of tags to determine the nucleotide residue sequence of individual tags; and

(h) comparing the sequences of individual tags to known gene sequences to identify gene transcription patterns in said mRNA population.

47. The method of claim 46, wherein sequences of individual tags each consist of a GGATC sequence, containing an MboI recognition sequence, or its reverse complement, depending on a sense or antisense orientation of each tag during ligation, adjacent to ten additional bases of sequence of a transcribed gene.