Method for analyzing gene expression frequency

• US 6,806,049 B1
• Filed: 08/16/2001
• Issued: 10/19/2004
• Est. Priority Date: 02/17/1999
• Status: Expired due to Fees

• Current Assignee: Ajinomoto Co., Inc.
• Original Assignee: Ajinomoto Co., Inc.
• Inventors: Mitsui, Akira, Date, Masayo, Takahara, Yoshiyuki, Fukuda, Hisao, Maekawa, Takami
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): SAKELARIS, SALLY A
• Application Number:
  US09/926,028
• Time in Patent Office: 1,160 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/23.5, 536/24.31, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

In gene expression frequency analysis, a vector primer to which each cDNA is ligated is formed by synthesizing the cDNA from each mRNA using by a vector primer having a poly(T) sequence; each cDNA sequence is converted to a tag on the vector primer; a concatemer is formed by ligating the obtained tags via a sequence that enables recognition of ends of the tags; and the nucleotide sequence of the concatemer is analyzed.

Claims

1. A method for analyzing expression frequencies of genes, which comprises the following steps:

(a) forming a vector primer to which each cDNA is ligated, by annealing the vector primer with each mRNA derived from a cell of which expression frequencies of genes is to be analyzed, and synthesizing the cDNA, said vector primer comprising a linear plasmid vector having a single-stranded poly(T) sequence at one 3′

end of one strand of the linear plasmid vector, said linear plasmid vector comprising a recognition sequence for a first restriction enzyme, a recognition sequence for a type IIS restriction enzyme, and a recognition sequence for a second restriction enzyme in order from the poly(T) sequence wherein (1) the first restriction enzyme and the second restriction enzyme each digest the vector primer at one position, (2) the cleavage site of the type IIS restriction enzyme is positioned beyond the recognition sequence of the second restriction enzyme, and (3) the vector primer digested with the first restriction enzyme and the type IIS restriction enzyme can be cyclized,

(b) digesting the vector primer to which the cDNA is ligated, with the second restriction enzyme and a third restriction enzyme that does not digest the vector primer and forms a digested end of the same shape as a digested end obtained with the second restriction enzyme, to excise an upstream region of the cDNA, and cyclizing the vector primer,

(c) digesting the cyclized vector primer with the first restriction enzyme and the type IIS restriction enzyme to excise a downstream region of the cDNA so that a tag consisting of a part of the cDNA is left, and cyclizing the vector primer again,

(d) performing polymerase chain reaction (PCR) by using the vector primer as a template and primers to amplify the tag, wherein said primers are oligonucleotides having nucleotide sequences corresponding to known nucleotide regions on each side of the tag that are maintained in the vector primer following digestion in step (c),

(e) ligating the amplification products to form a concatemer of the tags, wherein the tags are separated by known nucleotide sequences introduced by the primers for tag amplification so that no ditags are present in the concatemer, and

(f) determining the nucleotide sequence of the concatemer and investigating types and frequencies of tags occurring in the nucleotide sequence.

2. The method according to claim 1, wherein the ligation reaction in the step (e) is performed in the presence of an adaptor having one end of the same shape as an end of the tag to arrange the adaptor at each end of the concatemer, and the concatemer is amplified by performing PCR using an oligonucleotide having a sequence corresponding to the sequence of the adaptor as a primer.

3. The method according to claim 1, wherein, after the step (e), the concatemer is cloned in a cloning vector for nucleotide sequencing, and then the nucleotide sequence of the concatemer is determined.

4. The method according to claim 1, wherein the recognition sequence for the third restriction enzyme consists of four nucleotides.

5. The method according to claim 4, wherein the vector primer has a recognition sequence for a fourth restriction enzyme of which digestion point is in the same position as or a position downstream from the digestion point of the recognition sequence for the second restriction enzyme, which is not excised from the vector primer by the digestion with the type IIS restriction enzyme;

the primer for the downstream side of the tag among the primers used in the step (d) has a recognition sequence for a fifth restriction enzyme that forms an end of the same shape as the end digested with the fourth restriction enzyme; and

the concatemer is formed after the amplified primers are digested with the fourth restriction enzyme and the fifth restriction enzyme.

6. The method according to claim 5, wherein the vector primer has a nucleotide sequence different from the recognition sequence for the fifth restriction enzyme by one nucleotide in at a position upstream from the recognition sequence for the first restriction enzyme, and the nucleotide sequence different by one nucleotide is converted to the recognition sequence for the fifth restriction enzyme by PCR using the primer for the downstream side of the tag.

7. The method according to claim 6, wherein the third, fourth and fifth restriction enzymes are identical to one another.

8. The method according to claim 1, wherein the vector primer is formed by ligating a linear plasmid obtained by digesting a plasmid having a multicloning site at two sites in the multicloning site, and a partially double-stranded DNA having an end of the same shape as one end of the linear plasmid and a single-stranded poly(T) sequence.

9. The method according to claim 2, wherein, after the step (e), the concatemer is cloned in a cloning vector for nucleotide sequencing, and then the nucleotide sequence of the concatemer is determined.

10. The method according to claim 1, wherein said vector primer is prepared from a vector selected from the group consisting of pUC 18, pUC19, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, and pMW218.

11. The method according to claim 1, wherein said vector primer is prepared from pUC19.

12. The method according to claim 1, wherein said vector primer is prepared by cloning SEQ ID NO:

1 between EcoRI and HindIII within the multiple cloning site of a vector selected from the group consisting of pUC18, pUC19, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, and pMW218.

13. The method according to claim 1, wherein said first restriction enzyme is PmeI.

14. The method according to claim 1, wherein said second restriction enzyme is BglII.

15. The method according to claim 1, wherein said type IIS restriction enzyme is BsgI or BsmFI.

16. The method according to claim 1, wherein said single-stranded poly(T) sequence comprises 10-50 consecutive thymine nucleotides.

17. The method of claim 1, wherein said concatemer comprises 2 to 50 ligated tags.

18. The method according to claim 1, wherein at least one of said primers to amplify the tag is selected from the group consisting of SEQ ID NO:

2 and SEQ ID NO;

3.

19. The method according to claim 2, wherein the molar ratio of the tags to the adaptor ranges from 1:

1 to 1;

0.01.

20. The method according to claim 6, wherein at least one of the third, fourth and fifth restriction enzymes is MboI.