Method of analyzing expression of gene

• US 20090325185A1
• Filed: 08/18/2009
• Published: 12/31/2009
• Est. Priority Date: 12/12/2000
• Status: Abandoned Application

• Current Assignee: ABE: 5%, MASUMI, MASUMI ABE: 7%, Maze, Inc., Messengerscape Co., Ltd., National Institutes for Quantum and Radiological Science and Technology, TOSHIYUKI SAITO: 7%
• Original Assignee: Aisin Seiki Kabushiki Kaisha National Institute of Radiological Sciences
• Inventors: Sato, Shinji, Saito, Toshiyuki, Hattori, Atsushi, Kasama, Yasuji, Abe, Masumi
• Application Number:
  US12/583,348
• Publication Number:
  US 20090325185A1
• Time in Patent Office: Days
• 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 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

A cDNA is prepared from mRNA. The prepared cDNA is subjected to incision with two different types of restriction enzymes. The sequence of a portion of each of the fragments obtained as a result of treatment is determined, and the fragments are classified on the basis of the determined sequence by using a primer set. At the same time, the fragments are amplified in a manner that the relative expression magnitude thereof continues to be reflected therein. When the fragments have been classified into a predetermined number of fractions, the respective fractions are subjected to electrophoresis, and a gene expression profile is produced from the results.

Claims

1. A method of producing a gene expression profile, comprising:

(a) a step of synthesizing cDNA to mRNA extracted from a cell, such that a tag substance is added to the 3′

terminal of the cDNA;

(b) a step of cutting the product obtained as a result of the reaction in step (a) with a first restriction enzyme X;

(c) a step of connecting, to a fragment obtained in step (b), an “

X”

adaptor having a sequence complementary to a sequence of a site of the fragment at which site incision with the first restriction enzyme X has been effected;

(d) a step of connecting the fragment obtained in step (c) to a substance having high affinity with respect to the tag substance, thereby collecting the fragment;

(e) a step of cutting the fragment collected in step (d) with a second restriction enzyme Y and removing a fragment connected to the tag substance, thereby obtaining a fragment including the 5′

side-portion of the cut cDNA;

(f) a step of adding, to a fragment obtained in step (e), a “

Y”

adaptor having a sequence complementary to a sequence of a site of the fragment at which site incision with the second restriction enzyme Y has been effected;

(g) a step of carrying out a PCR reaction, for the fragment obtained in step (f), by using an “

X”

primer which has a sequence complementary to the sequence of the “

X”

adaptor and an optional two-nucleotide sequence (NN) at the 3′

terminal thereof, and a “

Y”

primer which has a sequence complementary to the sequence of the “

Y”

adaptor and an optional two-nucleotide sequence (NN) at the 3′

terminal thereof; and

(h) a step of subjecting the obtained PCR product to electrophoresis and detecting a migration distance and a peak, thereby producing a gene expression profile.

2. A method of producing a gene expression profile according to claim 1, wherein the “

X”

primer further includes a fluorescent substance added to the 5′

terminal thereof, and thereby the result of the electrophoresis of the PCR product is analyzed by detecting a magnitude of fluorescence of the fluorescence substance.

3. A method of producing a gene expression profile according to claim 1, wherein the restriction enzyme X and the restriction enzyme Y are selected, respectively, from the group consisting of the following enzymes:

AccII, AfaI, AluI, AspLEI, BfaI, BscFI, Bsh1236I, BshI, BsiSI, Bsp143I, BstUI, BsuRI, CfoI, Csp6I, DpnII, FnuDII, HaeIII, HapII, HhaI, Hin2I, Hin6I, HinPlI, HpaII, Hsp92II, HspAI, Kzo9I, MaeI, MboI, MseI, MspI, MvnI, NdeII, NlaIII, PalI, RsaI, Sau3AI, Sse9I, TaqI, ThaI, TrulI, Tru9I, Tsp509I, TspEI and TthHB8I.

4. A method of producing a gene expression profile according to claim 1, wherein the restriction enzyme X is MspI and the restriction enzyme Y is MseI.

5. A method of producing a gene expression profile according to claim 1, wherein the NN included in the “

X”

primer and the NN included in the “

Y”

adaptor are designed as a combination of adenine, thymine, guanine and cytosine, and thus totally 256 types of the “

X” and

“

Y”

primer are used.

6. A method of producing a gene expression profile according to claim 1, wherein examples of combination of the tag substance and the substance having high affinity with respect to the tag substance include:

biotin and streptoavidin;

biotin and avidin;

FITC and FITC antibody;

DIG and anti-DIG;

protein A and mouse IgG;

latex particles; and

the like.

7. A method of producing a gene expression profile according to claim 1, further comprising, after step (h) of subjecting the obtained PCR product to electrophoresis and detecting a migration distance and a peak, thereby producing a gene expression profile, the step of:

collecting a molecule separated by electrophoresis and corresponding to the detected peak, and determining by sequencing the sequence of the PCR product contained therein, thereby identifying the expressed gene.

8. A method of producing a gene expression profile according to claim 1, further comprising the step of:

identifying the expressed gene, by comparing the identification sequence of the restriction enzyme X and the restriction enzyme Y, the length of the fragments produced as a result of the incision of the reaction product obtained in step (a) with the restriction enzyme X and the restriction enzyme Y, and data from any suitable data banks, with each other.

9. A method of producing a gene expression profile, comprising:

(a) a step of synthesizing cDNA from mRNA extracted from a cell, such that a tag substance is added to the 3′

terminal of the cDNA, and dividing the product obtained as a result of the synthesis into two fractions;

(b) a step of cutting the first fraction of the synthesis product obtained in step (a) with a first restriction enzyme X;

(c) a step of connecting, to a fragment obtained in step (b), an “

X”

adaptor having a sequence complementary to a sequence of a site of the fragment at which site incision with the first restriction enzyme X has been effected;

(d) a step of connecting the fragment obtained at the step (c) to a substance having high affinity with respect to the tag substance, thereby collecting the fragment;

(e) a step of cutting the fragment collected at the step (d) with a second restriction enzyme Y and removing a fragment connected to the tag substance, thereby obtaining a fragment including the 5′

side-portion of the cut cDNA;

(f) a step of adding, to a fragment obtained in step (e), a “

Y”

adaptor having a sequence complementary to a sequence of a site of the fragment at which site incision with the second restriction enzyme Y has been effected;

(g) a step of carrying out a PCR reaction, for the fragment obtained in step (f), by using an “

X”

primer which has a sequence complementary to the sequence of the “

X”

adaptor and a two-nucleotide sequence (NN) at the 3′

terminal, and a “

Y”

primer which has a sequence complementary to the sequence of the “

Y”

adaptor and an optional two-nucleotide sequence (NN) at the 3′

terminal thereof;

(h) a step of cutting the second fraction of the synthesis product obtained in step (a) with the restriction enzyme Y;

(i) a step of connecting, to a fragment obtained in step (h), a “

Y′

”

adaptor having a sequence complementary to a sequence of a site of the fragment at which site incision with the restriction enzyme Y has been effected;

(j) a step of connecting the fragment obtained in step (i) to a substance having high affinity with respect to the tag substance, thereby collecting the fragment;

(k) a step of cutting the fragment collected in step (j) with the restriction enzyme X and removing a fragment connected to the tag substance, thereby obtaining a fragment including the 5′

side-portion of the cut cDNA;

(l) a step of adding, to a fragment obtained in step (k), an “

X′

”

adaptor having a sequence complementary to a sequence of a site of the fragment at which site incision with the restriction enzyme X has been effected;

(m) a step of carrying out a PCR reaction, for the fragment obtained in step (1), by using a “

Y′

”

primer which has a sequence complementary to the sequence of the “

Y′

”

adaptor and an optional two-nucleotide sequence (NN) at the 3′

terminal thereof, and an X primer which has a sequence complementary to the sequence of the “

X′

”

adaptor and an optional two-nucleotide sequence (NN) at the 3′

terminal thereof; and

(n) a step of subjecting the PCR product obtained in steps (g) and (m) to electrophoresis and detecting a migration distance and a peak, thereby producing a gene expression profile.

10. A method of producing a gene expression profile according to claim 9, wherein the primer which has a sequence complementary to the sequence of the “

X”

primer and an optional two-nucleotide sequence (NN) at the 3′

terminal thereof, used in step (g), further includes a fluorescent substance added to the 5′

terminal thereof, and

the primer which has a sequence complementary to the sequence of the “

Y′

”

adaptor and an optional two-nucleotide sequence (NN) at the 3′

terminal thereof, used in step (m), further includes a fluorescent substance added to the 5′

terminal thereof, and the result of the electrophoresis of the PCR product is analyzed by detecting a magnitude of fluorescence of the fluorescence substance.

11. A method of producing a gene expression profile according to claim 9, wherein the restriction enzyme X and the restriction enzyme Y are selected, respectively, from the group consisting of the following enzymes:

AccII, AfaI, AluI, AspLEI, BfaI, BscFI, Bsh1236I, BshI, BsiSI, Bsp143I, BstUI, BsuRI, CfoI, Csp6I, DpnII, FnuDII, HaeIII, HapII, HhaI, Hin2I, Hin6I, HinPlI, HpaII, Hsp921I, HspAI, Kzo9I, MaeI, MboI, MseI, MspI, MvnI, NdeII, NlaIII, PalI, RsaI, Sau3AI, Sse9I, TaqI, ThaI, TrulI, Tru9I, Tsp509I, TspEI and TthHB8I.

12. A method of producing a gene expression profile according to claim 9, wherein the restriction enzyme X is MspI and the restriction enzyme Y is MseI.

13. A method of producing a gene expression profile according to claim 9, wherein the NN included in the “

X”

adaptor and the NN included in the “

Y”

adaptor are designed as a combination of adenine, thymine, guanine and cytosine, and thus totally 256 types of the “

X” and

“

Y”

primer sets are used.

14. A method of producing a gene expression profile according to claim 9, wherein examples of combination of the tag substance and the substance having high affinity with respect to the tag substance include:

biotin and streptoavidin;

biotin and avidin;

FITC and FITC antibody;

DIG and anti-DIG;

protein A and mouse IgG;

latex particles; and

the like.

15. A method of producing a gene expression profile according to claim 9, further comprising, after step (n) of subjecting the obtained PCR product to electrophoresis and detecting a migration distance and a peak, thereby producing a gene expression profile, the step of:

collecting a molecule separated by electrophoresis and corresponding to the detected peak, and determining by sequencing the sequence of the PCR product contained therein, thereby identifying the expressed gene.

16. A method of producing a gene expression profile according to claim 9, further comprising the step of:

identifying the expressed gene, by comparing the identification sequence of the restriction enzyme X and the restriction enzyme Y, the length of the fragments produced as a result of the incision of the reaction product obtained in step (a) with the restriction enzyme X and the restriction enzyme Y, and data from any suitable data banks, with each other.

17. A method of analyzing frequency of gene expression, comprising:

(1) a step of carrying out a method of producing a gene expression profile, for each of a control cell and a subject cell, thereby producing two sets of gene expression profiles; and

(2) a step of analyzing a change in frequency of gene expression at the subject cell, by comparing the two gene expression profiles obtained in step (1),

wherein the method of producing a gene expression profile includes;

(a) a step of synthesizing cDNA to mRNA extracted from a cell, such that a tag substance is added to the 3′

terminal of the cDNA;

(b) a step of cutting the product obtained as a result of the reaction in step (a) with a first restriction enzyme X;

(c) a step of connecting, to a fragment obtained in step (b), an “

X”

adaptor having a sequence complementary to a sequence of a site of the fragment at which site incision with the first restriction enzyme X has been effected;

(d) a step of connecting the fragment obtained in step (c) to a substance having high affinity with respect to the tag substance, thereby collecting the fragment;

(e) a step of cutting the fragment collected in step (d) with a second restriction enzyme Y and removing a fragment connected to the tag substance, thereby obtaining fragments including the 5′

side-portion of the cut cDNA;

(f) a step of adding, to a fragment obtained in step (e), a “

Y”

adaptor having a sequence complementary to a sequence of a site of the fragment at which site incision with the second restriction enzyme Y has been effected;

(g) a step of carrying out a PCR reaction, for the fragment obtained in step (f), by using an “

X”

primer which has a sequence complementary to the sequence of the “

X”

adaptor and an optional two-nucleotide sequence (NN) at the 3′

terminal thereof, and a “

Y”

primer which has a sequence complementary to the sequence of the “

Y”

adaptor and an optional two-nucleotide sequence (NN) at the 3′

terminal thereof; and

(h) a step of subjecting the obtained PCR product to electrophoresis and detecting a migration distance and a peak, thereby producing a gene expression profile.

18. (canceled)