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Gene-specific unbiased amplification method UPDATE

外国特許コード F200010131
掲載日 2020年5月22日
出願国 アメリカ合衆国
出願番号 201615552971
公報番号 20180245130
出願日 平成28年2月23日(2016.2.23)
公報発行日 平成30年8月30日(2018.8.30)
国際出願番号 JP2016055192
国際公開番号 WO2016136716
国際出願日 平成28年2月23日(2016.2.23)
国際公開日 平成28年9月1日(2016.9.1)
優先権データ
  • 特願2015-033236 (2015.2.23) JP
  • 2016JP55192 (2016.2.23) WO
発明の名称 (英語) Gene-specific unbiased amplification method UPDATE
発明の概要(英語) The present invention is intended to provide a method of amplifying a target gene without bias and an adapter DNA use therefor. The adapter DNA of the present invention is double-stranded adapter DNA, which is used for unbiased gene amplification and has the following features: (a) the double-stranded adapter DNA has a sense strand and an antisense strand that are annealed with each other, the base length of the sense strand being equal to or longer than the base length of the antisense strand; (b) the base length of the sense strand is 15 to 40 bp; (c) the antisense strand includes a plurality of uracil bases, each uracil is removed by treating an adapter portion with uracil DNA glycosylase (UNG), and then, heat treatment is performed to degrade the antisense strand; (d) at least one end of the adapter DNA is in the form of a blunt end; (e) the other end of the adapter DNA binds to a target gene to be amplified; and (f) a part or all of the sense strand corresponds to a forward primer sequence used for gene amplification.
従来技術、競合技術の概要(英語) BACKGROUND ART
Hitherto, PCR methods have been used as methods of amplifying particular genes. In addition, in the case of unbiased amplification for uniform amplification, an adapter is provided, and gene amplification has been conducted using primers that are a partial adapter sequence and a gene-specific sequence, respectively. However, since the provided adapter is added to both ends of DNA, the adapter portion alone induces PCR amplification, which has been problematic. Although it has been considered possible to solve such problem via digestion with a restriction enzyme, it has been technically problematic.
Conventional techniques are intended to carry out amplification of a specific gene without bias by inserting a restriction enzyme site into an adapter portion or a cDNA end (3′ end side) and carrying out restriction enzyme treatment. However, restriction enzyme treatment cannot be achieved with 100% certainty, and PCR amplification takes place only at the adapter portion due to untreated DNA or the remaining adapter, which has been problematic.
特許請求の範囲(英語) [claim1]
1. Double-stranded adapter DNA, which is used for unbiased gene amplification and has the following features:
(a) the double-stranded adapter DNA has a sense strand and an antisense strand that are annealed with each other, the base length of the sense strand being equal to or longer than the base length of the antisense strand;
(b) the base length of the sense strand is 15 to 40 bp;
(c) the antisense strand includes a plurality of uracil bases, each uracil is removed by treating an adapter portion with uracil DNA glycosylase (UNG), and then, heat treatment is performed to degrade the antisense strand;
(d) at least one end of the adapter DNA is in the form of a blunt end;
(e) the other end of the adapter DNA binds to a target gene to be amplified; and
(f) a part or all of the sense strand corresponds to a forward primer sequence used for gene amplification.

[claim2]
2. The double-stranded adapter DNA according to claim 1, wherein the number of uracil bases included in the antisense strand accounts for 10% to 25% of the number of bases of the antisense strand, and uracil is present every 5 to 10 bases.

[claim3]
3. The double-stranded adapter DNA according to claim 1, wherein a phosphate group is bound to the 5′ end of the antisense strand and an amino group is bound to the 3′ end thereof.

[claim4]
4. An unbiased gene amplification kit, which includes the adapter DNA according to claim 1 and a primer comprising a part or all of a sequence of the sense strand of the adapter DNA.

[claim5]
5. An inhibitory primer, which is used for unbiased gene amplification by inhibiting gene amplification induced with a forward primer alone in an adapter ligation PCR amplification method in which a double-stranded adapter DNA and a forward primer comprising a partial sequence of a sense strand of the double-stranded adapter DNA are used, wherein
(a) the inhibitory primer has a sequence comprising:
(i) all or a part of a sequence of an anchor sequence portion of an anchored oligo dT primer in which an anchor sequence is ligated to the 5′ end of an oligo dT primer, the anchored oligo dT primer being used when synthesizing single-stranded cDNA from mRNA of a target gene to be amplified; or
(ii) a partial sequence of a sense strand of a double-stranded adapter used in an adapter ligation PCR amplification method, which is a sequence present on the 3′ end side of the sequence of the forward primer, and
(b) the inhibitory primer is modified with a phosphate group, an amino group, or dideoxyl NTP on its 3′ end side.

[claim6]
6. An unbiased gene amplification kit, which includes the adapter DNA according to claim 1, a primer comprising a part or all of a sequence of the sense strand of the adapter DNA, and the an inhibitory primer having a sequence comprising:
(i) all or a part of a sequence of an anchor sequence portion of an anchored oligo dT primer in which an anchor sequence is ligated to the 5′ end of an oligo dT primer, the anchored oligo dT primer being used when synthesizing single-stranded cDNA from mRNA of a target gene to be amplified; or
(ii) a partial sequence of a sense strand of a double-stranded adapter used in an adapter ligation PCR amplification method, which is a sequence present on the 3′ end side of the sequence of the forward primer, and
the inhibitory primer modified with a phosphate group, an amino group, or dideoxyl NTP on its 3′ end side.

[claim7]
7. A method of amplifying a target gene without bias in one direction by PCR, comprising the following steps of:
(i) ligating the double-stranded adapter DNA according to claim 1 to both ends of double-stranded cDNA;
(ii) treating a gene ligated to the double-stranded adapter DNA with uracil DNA glycosylase (UNG) and further conducting heat treatment, thereby degrading the antisense strand of the adapter DNA; and
(iii) conducting PCR amplification using a forward primer comprising a part or all of a sequence of a sense strand of the double-stranded adapter DNA and a reverse primer that is specifically annealed with a target gene.

[claim8]
8. The method of amplifying a target gene without bias in one direction by PCR according to claim 7, wherein the reverse primer induces an elongation reaction while the forward primer alone does not induce an elongation reaction, and after a complementary strand of the sense strand of the adapter is formed, the forward primer is annealed with the complementary strand so that an elongation reaction is induced, indicating that elongation induced by the reverse primer and elongation induced by the forward primer take place in the above order in one direction.

[claim9]
9. The method of amplifying a target gene without bias in one direction by PCR according to claim 7, wherein the inhibitory primer has a sequence comprising:
(i) all or a part of a sequence of an anchor sequence portion of an anchored oligo dT primer in which an anchor sequence is ligated to the 5′ end of an oligo dT primer, the anchored oligo dT primer being used when synthesizing single-stranded cDNA from mRNA of a target gene to be amplified; or
(ii) a partial sequence of a sense strand of a double-stranded adapter used in an adapter ligation PCR amplification method, which is a sequence present on the 3′ end side of the sequence of the forward primer, and
the inhibitory primer modified with a phosphate group, an amino group, or dideoxyl NTP on its 3′ end side, is further used so that an elongation reaction induced by the forward primer alone is inhibited.

[claim10]
10. A method of analyzing a TCR or BCR repertoire, which comprises synthesizing cDNA from total RNA extracted from T cells or B cells, conducting comprehensive amplification of a TCR gene or BCR gene repertoire using, as a reverse primer, a primer that is specifically annealed with a C region sequence of a T cell receptor (TCR) or B cell receptor (BCR) by the method according to claim 7, and conducting sequencing using a sequencer.
  • 発明者/出願人(英語)
  • OGASAWARA KOETSU
  • TOHOKU UNIVERSITY
国際特許分類(IPC)
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