Title:
METHOD OF AMPLIFYING TARGET GENE CONTAINED IN BIOLOGICAL SAMPLE
Document Type and Number:
WIPO Patent Application WO/2006/054492
Kind Code:
A1
Abstract:
It is intended to provide a method of efficiently isolating and amplifying a specific gene even from a single cell without resorting to any complicated procedure. Namely, a method of amplifying a target gene contained in a biological sample which comprises: (1) subjecting the biological sample to reverse transcription to thereby prepare an RNA-DNA complex; (2) forming Poly-N at the 3’-end of the DNA strand of the RNA-DNA complex obtained above by a tailing reaction; (3) synthesizing a double-stranded DNA with the use of the DNA strand of the RNA-DNA complex carrying Poly-N as a template and an oligo-dN adaptor as one of primers; and (4) further carrying out PCR with the use of a single-stranded DNA containing the oligo-dN adaptor in the obtained double-stranded DNA a template and a sequence contained in the adaptor as the other primer. The RNA-DNA complex prepared in (1) is subjected to the tailing reaction in (2) as such without purification (including separation from the substrate of the reverse transcription). In the tailing reaction in (2), the concentration of dNTP forming a poly strand is adjusted to twice or more higher than other dNTP concentrations.
Inventors:
OZAWA TATSUHIKO (JP)
Application Number:
PCT/JP2005/020718
Publication Date:
May 26, 2006
Filing Date:
November 11, 2005
Export Citation:
Assignee:
TOYAMA NEW INDUSTRY ORGANIZATI (JP)
OZAWA TATSUHIKO (JP)
OZAWA TATSUHIKO (JP)
International Classes:
C12N15/10
Other References:
JENA P.K. ET AL.: "Amplification of genes, single transcripts and cDNA libraries from one cell and direct sequence analysis of amplified products derived from one molecule", J. IMMUNOL. METHODS, vol. 190, no. 2, 1996, pages 199 - 213, XP004020876
HIROAKI MATSUNAMI: "Tan'itsu Saibo kara no cDNA Library Sakusei. (Construction of single-cell cDNA libraries)", EXPERIMENTAL MEDICINE, vol. 16, no. 13, 1998, pages 1673 - 1677, XP002998064
BERTIOLI D.: "Rapid Amplification of cDNA Ends", METHODS IN MOLECULAR BIOLOGY, vol. 67, 1997, pages 233 - 238, XP002998065
FROHMAN M.A. ET AL.: "Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer", PROC. NATI. ACAD. SCI. USA, vol. 85, 1988, pages 8998 - 9002, XP000604678
WANG X. AND STOLLAR B.D.: "Human immunoglobulin variable region gene analysis by single cell RT-PCR", J. IMMUNOL. METHODS, vol. 244, 2000, pages 217 - 225, XP004218461
BAHRING S. ET AL.: "Mapping of transcriptional start and capping points by a modified 5'RACE technique", BIOTECHNIQUES, vol. 16, no. 5, 1994, pages 807 - 808, XP002998066
SHI X. AND KAMINSKYJ G.W.: "5'RACE by tailing a general template-switching oligonucleotide", BIOTECHNIQUES, vol. 29, no. 6, 2000, pages 1192, 1194 - 1195, XP001121212
HIROAKI MATSUNAMI: "Tan'itsu Saibo kara no cDNA Library Sakusei. (Construction of single-cell cDNA libraries)", EXPERIMENTAL MEDICINE, vol. 16, no. 13, 1998, pages 1673 - 1677, XP002998064
BERTIOLI D.: "Rapid Amplification of cDNA Ends", METHODS IN MOLECULAR BIOLOGY, vol. 67, 1997, pages 233 - 238, XP002998065
FROHMAN M.A. ET AL.: "Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer", PROC. NATI. ACAD. SCI. USA, vol. 85, 1988, pages 8998 - 9002, XP000604678
WANG X. AND STOLLAR B.D.: "Human immunoglobulin variable region gene analysis by single cell RT-PCR", J. IMMUNOL. METHODS, vol. 244, 2000, pages 217 - 225, XP004218461
BAHRING S. ET AL.: "Mapping of transcriptional start and capping points by a modified 5'RACE technique", BIOTECHNIQUES, vol. 16, no. 5, 1994, pages 807 - 808, XP002998066
SHI X. AND KAMINSKYJ G.W.: "5'RACE by tailing a general template-switching oligonucleotide", BIOTECHNIQUES, vol. 29, no. 6, 2000, pages 1192, 1194 - 1195, XP001121212
Attorney, Agent or Firm:
SIKs & Co. (Kyobashi-Nisshoku Bldg. 8-7, Kyobashi 1-chome, Chuo-ku Tokyo 31, JP)
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