Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-15
2001-10-16
Fredman, Jeffrey (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06303306
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for amplifying a specific nucleotide sequence, a method for detecting the target nucleotide sequence from RNA copy or DNA copy of the specific nucleotide sequence obtained by said method for amplification, and a kit used for said methods.
PRIOR ART
Methods for diagnosing diseases by detecting genes such as DNA and RNA have been developed to detect bacteria, viruses and the like. In certain samples, there exists sufficient amount of nucleic acids to be directly detected, however it is difficult to detect the target gene directly when the amount of the target gene is very small or when abundance ratio thereof is very small. Conventionally, the target gene is increased by culturing cells or bacteria. However, there has been a defect that these methods require a long time.
Polymerase chain reaction (PCR; JP-B 4-67957) is also known as another method for amplifying the target gene. In this method, the degree of amplification of the target gene is adjusted by a number of the cycle. Theoretically, the amplification factor is calculated to be 2
n
(n is a number of the cycle). Twenty-five to thirty cycles have been required to amplify the target gene to a level where it can be detected effectively.
In addition, amplification systems based on replicated RNA are known as a further method for amplifying nucleic acids (JP-A 2-5864, JP-A 2-500565 and JP-A 2-501532). In these methods, using a primer containing a promoter sequence of DNA dependent RNA polymerase, a double stranded DNA is synthesized from the target nucleic acid, and the RNA corresponding to the target nucleic acid is synthesized by DNA dependent RNA polymerase using the double stranded DNA synthesized as a template. Then, a DNA/RNA chain is synthesized from the RNA which is synthesized by RNA dependent DNA polymerase, and the DNA/RNA chain is separated to obtain a single stranded DNA. As methods for separation to DNA, a heat-denaturation (JP-A 2-500565 and JP-A 2-501532) and a method using ribonuclease H (JP-A 2-5864) are known. Using the single stranded DNA thus obtained and a primer, a double stranded DNA containing a promoter sequence of DNA dependent RNA polymerase is synthesized, and transcription to RNA is carried out. According to this method, dozens to thousands of RNA molecules can be transcribed and amplified from a molecule of the double stranded nucleic acid by DNA dependent RNA polymerase, so that the amplification efficiency per cycle is higher than PCR method. When ribonuclease H is used, a thermal cycling which has been required in PCR method is not required, so that amplification can be carried out more easily.
PROBLEMS TO BE SOLVED BY THE INVENTION
Amplification efficiency is high in the amplification system based on replicated RNA. However, because of a poor heat stability of conventional enzymes, namely RNA dependent DNA polymerase, DNA dependent RNA polymerase and DNA dependent DNA polymerase, the reaction temperature does not have to be high, and non-specific hybridization between the nucleic acid as a template and the primer cannot be avoidable, so that decrease of the specificity is a problem. In addition, the instability of the enzymes creates a severe problem in supplying and storing enzymes, and storage in a frozen state or in a refrigerator is required.
Moreover, heat is used to denature a double stranded nucleic acid in a method for amplification without using ribonuclease H. In this method, it is necessary to add the enzymes sequentially every time heating is carried out because of instability of the enzymes.
The object of the invention is to solve the problems that the specificity is decreased by non-specific hybridization, and that the enzymes are unstable in supply and storage.
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Inoue Hiroaki
Kawamura Yoshihisa
Shibata Shuji
Takarada Yutaka
Fredman Jeffrey
Leydig , Voit & Mayer, Ltd.
Toyo Boseki Kabushiki Kaisha
Tung Joyce
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