Method of analyzing expression of gene
First Claim
Patent Images
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.
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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.
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Citations
18 Claims
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1. A method of producing a gene expression profile, comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method of producing a gene expression profile, comprising:
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(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 thereof, and a “
Y”
primer which has a sequence complementary to the sequence of the “
Y”
adaptor and a 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. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16)
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17. A method of analyzing frequency of gene expression, comprising:
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(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 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.
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18. A method of analyzing frequency of gene expression, comprising:
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(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 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 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 a primer which has a sequence complementary to the sequence of the “
X”
adaptor and a two-nucleotide sequence (NN) at the 3′
terminal thereof, and a primer which has a sequence complementary to the sequence of the “
Y”
adaptor and a 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;
(1) 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 set including 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 a set including 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.
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Specification