Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-23
2002-11-26
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06485917
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel method for synthesizing a cDNA and a kit used for the method, which are useful in a field of genetic engineering.
BACKGROUND ART
Analysis of mRNA molecules derived from various genes is very important in order to elucidate biological phenomena. Discovery of an RNA-dependent DNA polymerase, so-called a reverse transcriptase, from a retrovirus has enabled a reverse transcription reaction in which a cDNA is synthesized using an RNA as a template. As a result, methods for analyzing mRNA molecules have made rapid progress. Then, the methods for analyzing mRNA molecules using a reverse transcriptase have now become indispensable experimental methods for studying genes. Furthermore, these methods, which have been applied to cloning techniques and PCR techniques, have become indispensable techniques not only for studying genes but also in wide variety of fields including biology, medicine and agriculture.
However, the conventional reverse transcription methods had many problems such as interruption of a cDNA synthesis reaction, inability to synthesize a long cDNA, low fidelity, and damage of a template RNA in the course of reaction due to long time required for the reaction.
It is considered that the interruption of the cDNA synthesis reaction is due to the secondary structure formed by the RNA as a template. Optimal reaction temperature for a reverse transcriptase from a retrovirus is low. RNAs form complicated secondary structures during the reaction at the temperature. Then, the cDNA synthesis reaction is interrupted at the sites of such secondary structures. A method in which a heat-resistant reverse transcriptase is used has been proposed in order to solve the above-mentioned problem. However, the reactivity of this method is not satisfactory. In addition, reverse transcriptases have not been known to exhibit a proofreading activity during a reverse transcription reaction, and a method for synthesizing a cDNA with high fidelity has not been known.
As described above, it was difficult to synthesize a cDNA with high efficiency and with high fidelity according to the conventional methods. Thus, a more efficient method for synthesizing a cDNA has been desired.
OBJECTS OF INVENTION
The present invention has been made in view of the prior art as described above. The main object of the present invention is to solve the problems associated with the prior art and to provide a method for synthesizing a cDNA with high reaction efficiency and accuracy.
SUMMARY OF INVENTION
The present invention is outlined as follows. The first aspect of the present invention relates to a method for synthesizing a cDNA, characterized in that the method comprises conducting a reverse transcription reaction in the presence of an enzyme having a reverse transcription activity and another enzyme having a 3′-5′ exonuclease activity.
The second aspect of the present invention relates to a method for amplifying a cDNA, characterized in that the method comprises conducting a gene amplification reaction using a cDNA synthesized according to the method of the first aspect as a template.
The third aspect of the present invention relates to a kit for cDNA synthesis, which contains an enzyme having a reverse transcription activity and another enzyme having a 3′-5′ exonuclease activity.
The fourth aspect of the present invention relates to a kit for amplifying a cDNA by conducting a gene amplification reaction using a cDNA synthesized according to the method of the first aspect as a template, which contains an enzyme having a reverse transcription activity and another enzyme having a 3′-5′ exonuclease activity as well as a reagent for the gene amplification reaction.
The present inventors have found the efficiency of cDNA synthesis and the fidelity are increased by conducting a reverse transcription reaction in the presence of an enzyme having a 3′-5′ exonuclease activity in a cDNA synthesis reaction. Thus, the present invention has been completed.
DETAILED DESCRIPTION OF THE INVENTION
One of the main features of the method for synthesizing a cDNA of the present invention is that a cDNA is synthesized using an RNA as a template in a reverse transcription reaction system containing an enzyme having a 3′-5′ exonuclease activity.
Examples of the samples containing RNAs which can be used in the method of the present invention include, but are not limited to, samples from organisms such as a cell, a tissue and a blood, and samples that may contain an organism such as a food, a soil and a waste water. The sample may be a preparation containing a nucleic acid obtained by processing the above-mentioned sample according to a known method. Examples of the preparations that can be used in the present invention include a cell destruction product or a sample obtained by fractionating the product, the total RNA in the sample, or a sample in which specific RNA molecules such as mRNAs are enriched.
The RNAs to which the method of the present invention can be applied include, but are not limited to, RNA molecules such as total RNA, mRNA, tRNA and rRNA in a sample, as well as specific RNA molecules (e.g., RNA molecules each having a common base sequence motif, transcripts obtained using an RNA polymerase and RNA molecules concentrated by a subtraction method). Any RNAs for which a primer used for a reverse transcription reaction can be prepared may be used.
The primer used for synthesizing a cDNA from an RNA as a template in the present invention is not limited to specific one as long as it is an oligonucleotide that has a nucleotide sequence complementary to that of the template RNA and that can anneal to the template RNA under reaction conditions used. The primer may be an oligonucleotide such as an oligo(dT) or an oligonucleotide having a random sequence (a random primer).
In view of specific annealing, the length of the primer is preferably 6 nucleotides or more, more preferably 10 nucleotides or more. In view of oligonucleotide synthesis, the length is preferably 100 nucleotides or less, more preferably 30 nucleotides or less. The oligonucleotide can be synthesized using, for example, the DNA synthesizer type 394 from Applied Biosystems Inc. (ABI) according to a phosphoramidite method. Alternatively, any methods including a phosphate triester method, an H-phosphonate method and a thiophosphonate method may be used to synthesize the oligonucleotide. The oligonucleotide may be derived from a biological sample. For example, it may be isolated and prepared from a DNA prepared from a natural sample digested with a restriction endonuclease. In view of synthesis of a cDNA from a template RNA, the concentration of the primer in the reverse transcription reaction mixture is preferably 0.1 &mgr;M or more, more preferably 0.5 &mgr;M or more. In view of inhibition of the reaction, the concentration is preferably 10 &mgr;M or less, more preferably 5 &mgr;M or less.
Any enzymes having reverse transcription activities can be used in the present invention as long as they have activities of synthesizing cDNAs using RNAs as templates. However, enzymes having reverse transcription activities at a high temperature (i.e., heat-resistant reverse transcriptases) are preferable for the purpose of the present invention. Examples of such enzymes which can be used include a DNA polymerase from a bacterium of genus Thermus (e.g., Tth DNA polymerase) and a DNA polymerase from a thermophilic bacterium of genus Bacillus. The presence of a manganese ion in a reaction mixture is indispensable for the exertion of the reverse transcription activity of Tth DNA polymerase. The manganese ion is known to reduce the fidelity of a PCR. Thus, it is required to eliminate the manganese ion when a reverse transcription reaction mixture in which Tth DNA polymerase is used is used for a PCR. DNA polymerases from thermophilic bacteria of genus Bacillus do not require the addition of a manganese ion for the exertion of its reverse transcription activity and the remov
Hino Fumitsugu
Kato Ikunoshin
Miyake Kazue
Mukai Hiroyuki
Yamamoto Junko
Browdy and Neimark
Chunduru Suryaprabha
Takara Shuzo Co. Ltd.
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