Title:
ONE-STEP REVERSE TRANSCRIPTION TEMPLATE-SWITCHING PCR
Document Type and Number:
WIPO Patent Application WO/2017/222057
Kind Code:
A1
Abstract:
The present invention provides technology for carrying out one-step reverse transcription template-switching PCR more quickly, more easily, and with high specificity. The present invention provides a nucleic acid amplification method for amplifying at least a partial region of RNA using a modified oligonucleotide primer, said nucleic acid amplification method being characterized by the following: a nucleic acid amplification reaction comprises a reverse transcription step a) in which RNA is used as a template, a template switching step b) in which a template-switching oligonucleotide is added to cDNA synthesized in step a), and a DNA amplification step c) in which DNA amplification is carried out by PCR in which the template-switch cDNA synthesized in step b) is used as a template; the steps a) to c) are performed in a single stage in the same reaction system; as a result of being modified, some or all of the primer function of the modified oligonucleotide primer is blocked in the reverse transcription step a); and blocking of the primer function is cancelled in the DNA amplification step c).
Inventors:
SHIROGUCHI KATSUYUKI (JP)
Application Number:
PCT/JP2017/023254
Publication Date:
December 28, 2017
Filing Date:
June 23, 2017
Export Citation:
Assignee:
RIKEN (JP)
International Classes:
C12Q1/68; C12N15/09
Foreign References:
| JP2009538621A | 2009-11-12 | |||
| JP2000502905A | 2000-03-14 | |||
| JP2011521646A | 2011-07-28 | |||
| US8133669B2 | 2012-03-13 | |||
| US8361753B2 | 2013-01-29 |
Other References:
JEREMY KAPTEYN ET AL.: "Incorporation of non- natural nucleotides into template-switching oligonulceotides reduces background and improves cDNA synthesis from very small RNA samples", BMC GENOMICS, vol. 11, no. 413, 2010, pages 1 - 9, XP021072710
"Curr Protoc Nucleic Acid Chem", September 2009, pages: 1 - 17
NUCLEIC ACIDS RES., vol. 36, no. 20, November 2008 (2008-11-01), pages e131
NUCLEIC ACIDS RES., vol. 35, 2007, pages 4238
J. AM. CHEM. SOC., vol. 121, 1999, pages 11585
J. AM. CHEM. SOC., vol. 131, 2009, pages 14620
PROC. NATL. ACAD. SCI. USA, vol. 98, 2001, pages 4922
J. AM. CHEM. SOC., vol. 127, 2005, pages 17286
NUCLEIC ACIDS RES., vol. 33, 2005, pages el29
BIOTECHNIQUES, vol. 40, 2006, pages 711
J. AM. CHEM. SOC., vol. 126, 2004, pages 13298
NAT. METHODS, vol. 3, 2006, pages 729
J. AM. CHEM. SOC., vol. 129, 2007, pages 15549
NUCLEIC ACIDS RES., vol. 37, 2009, pages el4
J. AM. CHEM. SOC., vol. 126, 2004, pages 11826
J. AM. CHEM. SOC., vol. 131, 2009, pages 1644
ANGEW. CHEM. INT. ED., vol. 44, 2005, pages 596
See also references of EP 3476944A4
"Curr Protoc Nucleic Acid Chem", September 2009, pages: 1 - 17
NUCLEIC ACIDS RES., vol. 36, no. 20, November 2008 (2008-11-01), pages e131
NUCLEIC ACIDS RES., vol. 35, 2007, pages 4238
J. AM. CHEM. SOC., vol. 121, 1999, pages 11585
J. AM. CHEM. SOC., vol. 131, 2009, pages 14620
PROC. NATL. ACAD. SCI. USA, vol. 98, 2001, pages 4922
J. AM. CHEM. SOC., vol. 127, 2005, pages 17286
NUCLEIC ACIDS RES., vol. 33, 2005, pages el29
BIOTECHNIQUES, vol. 40, 2006, pages 711
J. AM. CHEM. SOC., vol. 126, 2004, pages 13298
NAT. METHODS, vol. 3, 2006, pages 729
J. AM. CHEM. SOC., vol. 129, 2007, pages 15549
NUCLEIC ACIDS RES., vol. 37, 2009, pages el4
J. AM. CHEM. SOC., vol. 126, 2004, pages 11826
J. AM. CHEM. SOC., vol. 131, 2009, pages 1644
ANGEW. CHEM. INT. ED., vol. 44, 2005, pages 596
See also references of EP 3476944A4
Attorney, Agent or Firm:
YAMAMOTO Shusaku et al. (JP)
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