Title:
AMPLIFICATION AND DETECTION OF OBJECTIVE NUCLEIC ACID SEQUENCE USING HEAT-RESISTANT ENZYME
Document Type and Number:
Japanese Patent JP3241555
Kind Code:
B2
Abstract:
PURPOSE: To amplify an objective nucleic acid without using the successive addition of enzymes while suppressing the deactivation of enzymes by using heat-resistant enzymes as DNA polymerase, RNA polymerase and ribonuclease necessary in a replicative RNA base amplification system.
CONSTITUTION: A single strand 1st template RNA produced by modifying a target nucleic acid is reacted with a 1st primer having a promoter sequence at the 5' terminal to effect the extension of the chain by a heat-resistant RNA- dependent DNA polymerase to obtain a 2nd template DNA. The DNA is reacted with a heat-resistant ribonuclease H to form a single strand 2nd template DNA, which is extended with a 2nd primer to obtain an extended 2nd primer. A single strand 3rd template RNA complementary to the target nucleic acid is produced by using the extended 2nd primer and the RNA is extended by reacting with the 2nd primer. The obtained 4th template complementary to the 3rd template is decomposed with ribonuclease H. The obtained single strand 4th template DNA is extended by reacting with the 1st primer and the produced double strand DNA is used for increasing the copy of the sequence complementary to the target nucleic acid.
Inventors:
Hiroshi Takarada
Hiroaki Inoue
Shuji Shibata
Yoshihisa Kawamura
Hiroaki Inoue
Shuji Shibata
Yoshihisa Kawamura
Application Number:
JP29498094A
Publication Date:
December 25, 2001
Filing Date:
November 29, 1994
Export Citation:
Assignee:
TOYO BOSEKI KABUSHIKI KAISHA
International Classes:
C12N15/09; C12N15/00; C12Q1/68; (IPC1-7): C12Q1/68; C12N15/09
Domestic Patent References:
| JP4507197A |
Other References:
【文献】国際公開92/8800(WO,A1)
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