Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-13
2002-04-16
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024330, C536S025300
Reexamination Certificate
active
06372437
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for reverse transcription which can produce a full length cDNA from a mRNA. In addition, the present invention relates to a method for improving heat stability of RNA.
2. Related Art
It is known that cDNAs can be obtained from mRNAs in vitro using a reverse transcriptase (RNA-dependent DNA polymerase). A project elucidating whole human gene sequences is moving on and, in that project, mRNA strands are produced by using genes as templates and full length cDNA strands are produced in turn by using the mRNA strands as templates. That is, synthesis of first chains of cDNA from mRNA strands is used as a first step of production of cDNA libraries, RT-PCR and the like.
Reverse transcription is utilized in order to obtain full length cDNA strands from the mRNAs as described above. However, conventional reverse transcription can not afford full length cDNAs from mRNAs because the conventional reverse transcription method could not complete reverse transcription to the most end cap site of mRNAs.
According to the present inventor's examination, it was found that the failure of complete reverse transcription is caused as follows. That is, a long chain mRNA may form a secondary structure like secondary structure of protein and the elongation by reverse transcriptase is sterically hindered at the site forming the secondary structure. As a result, reverse transcription was not completed to the end of mRNA.
That is, current techniques for reverse transcription have a technical limitation that the reaction is ended prematurely because of a stable secondary structure of mRNA and thus the probability of complete transcription over the whole transcription unit including its 5′ end is extremely low. This technical limitation affects the quality of libraries. That is, most of cloned cDNAs synthesized from the poly A at the 3′ end using an oligo dT as a primer have only the 3′ end and do not have the full length because of the premature termination of the synthesis. Several attempts have been made to overcome this problem. For example, it was proposed that the mRNAs are pre-treated at 70° C. to unfold the secondary structure before the synthesis of the first chains. It is also possible to treat the mRNAs with methylmercury hydroxide instead of the heat treatment. Though these techniques are effective for increasing efficiency of the synthesis of the first chain to some extent, they are not yet sufficient to efficiently obtain full length cDNAs. In particular, they show particularly low efficiency for the reverse transcription of long mRNAs of several kbp or more.
Therefore, the first object of the present invention is to provide a method capable of reverse transcription of mRNA over the full length and hence capable of providing a full length cDNA even if a long chain mRNA is used as a template.
In this respect, the present inventor has found that the above first object of the present invention can be achieved by performing reverse transcription at a temperature at which mRNA does not form a secondary structure. Though the temperature range where mRNAs do not form a secondary structure may change depending on buffer composition and the like, it is for example a range of 45° C. or more, especially, 60° C. or more.
In such a temperature range, mRNAs can be maintained in a condition that it does not take the secondary structure and the synthesis of the first chain can be effected efficiently. However, it was also found that, in such a temperature range as mentioned above, (1) the reverse transcriptase may be disadvantageously inactivated depending on the kind of the enzyme, and (2) stability of mRNA may be disadvantageously deteriorated (mRNA is fragmented) when metal ions necessary for activation of reverse transcriptase such as magnesium ions and a buffer agent such as Tris [Tris(hydroxymethyl)aminomethane] are present simultaneously.
Therefore, the second object of the present invention is to provide a method which is capable of reverse transcription of mRNA over the full length of the mRNA even if a long chain mRNA is used as a template by performing the reverse transcription of mRNA at a temperature at which the mRNA does not form the secondary structure and, in addition, which can prevent inactivation of the enzyme by heat, i.e., activate it at an elevated temperature even when a heat-labile reverse transcriptase is used and, as a result, provide a full length cDNA with high reliability.
The third object of the present invention is to provide a method which is capable of reverse transcription of mRNA over the full length of mRNA even if a long chain mRNA is used as a template by performing the reverse transcription of mRNA at a temperature at which the mRNA does not form the secondary structure and, in addition, which can provide a full length cDNA with high reliability by using a heat-resistant reverse transcriptase.
The fourth object of the present invention is to provide a method which is capable of reverse transcription of mRNA over the full length of mRNA even if a long chain mRNA is used as a template by performing the reverse transcription of mRNA at a temperature at which the mRNA does not form the secondary structure and, in addition, which can maintain stability of mRNA and hence provide a full length cDNA with high reliability even when metal ions necessary for activation of reverse transcriptase is present, in particular, when a buffer agent such as Tris is further present simultaneously.
The fifth object of the present invention is to provide a method improve heat stability of mRNA even when metal ions necessary for activation of reverse transcriptase is present, in particular, when a buffer agent such as Tris is further present simultaneously.
SUMMARY OF THE INVENTION
As the first embodiment of the present invention, which can achieve the above first object of the present invention, there is provided a method for preparing a cDNA from a mRNA using a reverse transcriptase wherein reverse transcription is performed at a temperature at which temperature the mRNA does not take a secondary structure.
As the second embodiment of the present invention, which can achieve the above second object of the present invention, there is provided a method for preparing a cDNA from a mRNA using a reverse transcriptase wherein reverse transcription is performed at a temperature at which the mRNA does not take a secondary structure using a heat-labile reverse transcriptase in the presence of a substance exhibiting chaperone function.
As the third embodiment of the present invention, which can achieve the above third object of the present invention, there is provided a method for preparing a cDNA from a mRNA using a reverse transcriptase wherein reverse transcription is performed at a temperature at which the mRNA does not take a secondary structure using a heat-resistant reverse transcriptase.
As the fourth embodiment of the present invention, which can achieve the above fourth object of the present invention, there is provided a method for preparing a cDNA from a mRNA using a reverse transcriptase wherein reverse transcription is performed at a temperature at which the mRNA does not take a secondary structure in the presence of metal ions necessary for activation of reverse transcriptase, a Tris buffer and a chelating agent for the metal ions.
As the fifth embodiment of the present invention, which can achieve the above fifth object of the present invention, there is provided a method for improving heat stability of RNAs in a solution containing metal ions wherein the solution further contains a chelating agent for the metal ions.
One of the preferred embodiments of the invention is a method for preparing a cDNA from a mRNA using a reverse transcriptase wherein:
(1) the reverse transcription is performed at a temperature at which the mRNA does not take a secondary structure,
(2) the reverse transcription is performed using a heat-labile reverse transcriptase in the presenc
Burns Doane , Swecker, Mathis LLP
McGarry Sean
Shibuya Mark L.
The Institute of Physical & Chemical Research
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