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Gene-specific unbiased amplification method

Foreign code F200010134
Posted date May 22, 2020
Country EPO
Application number 16755460
Gazette No. 3263716
Gazette No. 3263716
Date of filing Feb 23, 2016
Gazette Date Jan 3, 2018
Gazette Date Apr 8, 2020
International application number JP2016055192
International publication number WO2016136716
Date of international filing Feb 23, 2016
Date of international publication Sep 1, 2016
Priority data
  • P2015-033236 (Feb 23, 2015) JP
  • 2016JP55192 (Feb 23, 2016) WO
Title Gene-specific unbiased amplification method
Abstract 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.
Outline of related art and contending technology 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.
Citation List
Non-Patent Literature
Non-Patent Literature 1: Kobayashi H. et al., PLoS One 8: e76385 doi: 10. 137 1/journal.pone.0076385.
Scope of claims [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 such that each uracil can be removed by treating an adapter portion with uracil DNA glycosylase (UNG) and the antisense strand can be completely degraded via heat treatment;
(d) at least one end of the adapter DNA is in the form of a blunt end;
(e) the blunt end of the adapter DNA binds to a target gene to be amplified;
(f) a part or all of the sense strand corresponds to a forward primer sequence used for gene amplification;
(g) a restriction enzyme site is not inserted to the adapter DNA; and
(h) an amino group is bound to the 3' end of the antisense strand of the double-stranded adapter DNA.

[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 or 2, wherein a phosphate group is bound to the 5' end of the antisense strand.

[claim4]
4. A pair of the double-stranded adapter DNAs, comprising two double stranded adapter DNA according to any one of claims 1 to 3.

[claim5]
5. An unbiased gene amplification kit, which includes the adapter DNA according to any one of claims 1 to 3, a forward primer comprising a part or all of a sequence of the sense strand of the adapter DNA and a reverse primer which is annealed in the vicinity of the 3' end of the sense strand of a target gene.

[claim6]
6. 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 any one of claims 1 to 3 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 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 in the course of elongating the reverse primer, 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.

[claim7]
7. 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 5, and conducting sequencing using a sequencer.
  • Applicant
  • TOHOKU UNIVERSITY
  • Inventor
  • OGASAWARA KOETSU
IPC(International Patent Classification)
Specified countries Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
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