Method of identifying gene transcription patterns

• US 6,461,814 B1
• Filed: 03/05/1999
• Issued: 10/08/2002
• 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: Spinella, Dominic G.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US09/263,196
• Time in Patent Office: 1,313 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/287.2, 435/320.1, 436/694, 536/23.5
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

This invention provides an rapid, artifact free, improved method of obtaining short DNA “tag” or arrays thereof, allowing for determination of the relative abundance of a gene transcript within a given mRNA population and is useful to identify patterns of gene transcription, as well as identify new genes.

Claims

1. A method of obtaining a set of tags from a source of interest comprising the steps of:

(a) providing a double-stranded cDNA, having a poly A or a poly T tail,

(b) cleaving said double-stranded cDNA with a punctuating restriction endonuclease providing a cDNA fragment,

(c) ligating to said cDNA fragment a 5′

adapter comprising a recognition sequence for a type IIs restriction endonuclease which allows DNA cleavage at a site in the cDNA fragment distant from the recognition sequence for a type IIs restriction endonuclease providing a first cDNA construct,

(d) cleaving said first cDNA construct with said type IIs restriction endonuclease providing a second cDNA construct,

(e) ligating to said second cDNA construct a 3′

adapter, thereby producing a recognition sequence for a punctuating endonuclease located 3′

adjacent to said second cDNA construct, providing a third cDNA construct,

(f) digesting said third cDNA construct with said punctuating restriction endonuclease providing a set of tags comprising cDNA sequences, and

(g) determining the nucleotide sequences of said set of tags.

2. The method of claim 1, wherein the punctuating restriction endonuclease is MspI and the type IIs restriction endonuclease is BsgI.

3. A method of claim 1 further comprising the step of ligating said set of tags providing an array of tags.

4. The method of claim 3 further comprising the step of introducing said array of tags into a vector.

5. A method of obtaining an array of tags, comprising the steps of:

(a) providing double-stranded cDNA from an mRNA using a biotinylated oligo dT primer,

(b) cleaving within said double-stranded cDNA with a punctuating restriction endonuclease providing a population of a cDNA fragments,

(c) ligating to said cDNA fragment a 5′

adapter comprising,

i) a single-stranded end compatible with ends produced by cleavage with said punctuating restriction endonuclease;

ii) a recognition sequence for a type IIs restriction endonuclease located 5′

to said single-stranded end;

iii) a recognition sequence for a 5′

-cloning restriction endonuclease located 5′

to a recognition sequence for said type IIs restriction endonuclease, providing a first cDNA construct,

(d) isolating said first cDNA construct by affinity capture on solid phase streptavidin,

(e) cleaving said first cDNA construct with said type IIs restriction endonuclease providing a second cDNA construct,

(f) ligating to said second cDNA construct a 3′

adapter comprising,

i) a degenerate single-stranded end compatible with ends produced by said type IIs restriction endonuclease;

ii) a recognition sequence for said punctuating restriction endonuclease located 3′

to said degenerate end;

iii) a recognition sequence for a 3′

-cloning restriction endonuclease located 3′

to a recognition sequence for said punctuating restriction endonuclease, providing a third cDNA construct,

(g) digesting said third cDNA construct with said 5′ and

3′

-cloning restriction endonucleases providing a fourth cDNA construct;

(h) inserting said fourth cDNA construct into a cloning vector digested with said 5′ and

3′

-cloning restriction endonucleases,

(i) replicating said vector DNA in a suitable host strain,

(j) isolating said vector DNA,

(k) digesting said vector DNA with said punctuating restriction endonuclease providing a set of tags comprising cDNA sequences and a GC clamp,

(l) ligating said set of tags providing arrays of tags comprising at least 10 tags and GC rich clamps,

(m) optionally inserting said arrays of tags into a sequence vector, and

(n) optionally determining the nucleotide sequences of the arrays of tags.

6. The method of claim 5, wherein the punctuating restriction endonuclease is MspI and the type IIs restriction endonuclease is BsgI.

7. The method of claim 5, wherein the 5′

cloning restriction endonuclease is EcoRI and the 3′

-cloning restriction endonuclease is NotI.

8. A method of obtaining a set of tags from a source of interest comprising the steps of:

(a) providing a double-stranded cDNA, prepared from mRNA,

(b) cleaving said double-stranded cDNA with a punctuating restriction endonuclease providing a cDNA fragment,

(c) ligating to said cDNA fragment a 5′

adapter comprising a recognition sequence for a type IIs restriction endonuclease which allows DNA cleavage at a site in the cDNA fragment distant from the recognition sequence for a type IIs restriction endonuclease providing a first cDNA construct,

(d) cleaving said first cDNA construct with said type IIs restriction endonuclease providing a second cDNA construct,

(e) ligating to said second cDNA construct a 3′

adapter, thereby producing a recognition sequence for a punctuating endonuclease located 3′

adjacent to said second cDNA construct, providing a third cDNA construct,

(f) digesting said third cDNA construct with said punctuating restriction endonuclease providing a set of tags comprising cDNA sequences, and

(g) determining the nucleotide sequences of said set of tags.