Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-08-13
2001-08-28
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06280931
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for selectively amplifying a cDNA. More specifically, the invention is concerned with a method for selectively amplifying a cDNA synthesized from an mRNA expressed only in an extremely small quantity.
2. Prior Art
Inherited diseases, cancers and some types of progeria and dementia are caused by occurrence of variation in specific genes. Thus, these diseases are often called generically “genetic diseases”. In these genetic diseases, abnormal characters appear only in such an organ or tissue where a specific gene involved in the disease is expressed. However, the variation in the gene itself is present in any tissue in the body. Therefore, as a technique for diagnosing a genetic disease, a method may be considered which comprises extracting genomic DNA from leukocytes in peripheral blood that is most easy to sample and then amplifying the genomic DNA.
As a method for amplifying genomic DNA, firstly, a method by PCR (polymerase chain reaction) may be given. Then, a genetic disease is diagnosed by searching the amplified DNA for variation in the base sequence.
However, it is difficult in general to amplify an entire genomic DNA by performing PCR just one time because the size of genomic DNA is too big.
As a means to amplify genomic DNA, secondly, a method of amplifying exons alone may be given because variation in bases involved in a genetic disease only occurs in the region of “exons” encoding proteins. In order to amplify exons alone, information on genetic structure, i.e. information as to which regions of the genomic DNA are exons is necessary.
However, it is seldom that such information has been previously known.
Due to these reasons, in many cases it is necessary to use as a sample for genetic diagnosis not peripheral blood but a sample obtained by biopsy from a tissue manifesting symptoms. Generally, mRNA (precursors of proteins) is extracted from the tissue sample obtained and cDNA is synthesized therefrom. Subsequently, a PCR is performed (this method is called “RT-PCR”).
However, biopsy gives a great pain to a patient and, moreover, some tissues such as brain are extremely difficult to perform biopsy.
For the reasons described above, it is the present situation that genetic diagnosis using easy-to-sample blood has many restrictions, and that many restrictions are also present in the case of examining a tissue obtained by biopsy to find out at which part of the coding region of a candidate causative gene of an inherited disease or the like variation in specific base sequences has occurred.
On the other hand, recently, it has been made clear by RT-PCR or the like that such a gene that is expressed only tissue-specifically (e.g., in brain or heart) is actually expressed in an extremely small quantity in leukocytes in peripheral blood, fibroblasts in skin and the like (Chelly, J. et al., Proc. Natl. Acad. Sci. USA 86, 2617-2621 (1989)). It is appropriate to interpret this phrase “expressed in an extremely small quantity” that inhibition of gene expression in the body is not 100% complete but there is some “leakage”, rather than to interpret this as physiological gene expression. In other words, the “expressed in an extremely small quantity” does not mean that a specific gene is expressed naturally and properly though in an extremely small quantity. It is appropriate to interpret that an mRNA being expressed in a specific tissue such as brain may leak out of the tissue though it should be expressed only in that specific tissue, and that since the body cannot inhibit the leakage of such mRNA completely, the mRNA which has leaked out in an extremely small quantity exists in tissues other than the specific tissue as well (e.g., peripheral blood leukocyte).
Such RNA which has thus leaked out is called “leaky RNA” or “illegitimate transcription”. If a target cDNA can be amplified selectively from such “leaky” mRNA by RT-PCR, it is believed that, in principle, diagnosis of any genetic disease can become possible by examining peripheral blood.
In conventional RT-PCR, total RNA is extracted from a tissue and then mRNAs are isolated and purified. Thereafter, using oligo(dT) primers or random primers, a pool of cDNAs from mRNAs being expressed in the tissue is prepared (Michael A. Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press Inc. (1990)). Then, a PCR is performed using an appropriate set of oligo DNA primers specific to a target cDNA.
Since the template for the above PCR is a mixture of cDNAs from the entire tissue, contamination with (mixing of) a large number of non-specific amplified products may occur when the amount of cDNAs or the number of PCR cycles is increased. Accordingly, it has been difficult to apply this method to the “illegitimate transcription” described above.
OBJECTS AND SUMMARY OF THE INVENTION
It is the object of the present invention to provide a method for specifically amplifying a cDNA, in particular, a cDNA synthesized from an mRNA expressed only in an extremely small quantity.
As a result of extensive and intensive researches toward the solution of the above assignment, the present inventors have found that it is possible to selectively amplify a cDNA from an mRNA expressed only in an extremely small quantity by locating a 3′ primer for cDNA amplification at 5′ upstream to that of a primer for cDNA synthesis from the mRNA. Thus, the present invention has been achieved.
The present invention relates to a method for selectively amplifying a cDNA of an extremely small quantity, comprising synthesis of a cDNA from a target mRNA using an oligonucleotide primer complementary to at least a part of the 3′ untranslated region of the mRNA; and amplification of the resultant cDNA using a 5′ primer and a 3′ primer which is located upstream of the oligonucleotide primer for synthesis of a cDNA.
As examples of the cDNA, a cDNA encoding dystroglycan, &agr;-sarcoglycan or endothelin B receptor may be given.
The distance between the primer for cDNA synthesis and the 3′ primer for cDNA amplification is preferably 1 kb or less, more preferably in the range from 1 to 300 bp. As the 5′ primer and the 3′ primer for cDNA amplification, primers located adjacent to the coding region of the target mRNA in the upstream and the downstream thereof, respectively, may be used.
REFERENCES:
patent: 5057410 (1991-10-01), Kawasaki et al.
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patent: 2 260 811 (1993-04-01), None
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Cooper et al., Ann. Med. 26, 9-14 (1994).*
Naylor et al., Lancet 337, 635-639 (1991).*
Chelly et al., Proc. Natl. Acad. Sci. USA 86, 2617-2621 (1989).*
Chelly et al., Biochem. Biophys. Res. Comm. 178(2), 553-557 (1991).*
Bertling W.M. et al., “Determination of 5′Ends of Specific MRNAS by DNA Ligase-Dependent Amplification”PCR Methods Applications, vol. 3, No. 2, Oct. 1, 1993, pp. 95-99.
Leister, et al. Trends in Genetics (1996) vol. (12)(1) p. 11.*
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Duggan, et al (1996) Journal of Neurological Sciences vol. 140 p. 30-39.
Hanaoka Fumio
Sakamoto Aiji
Fish & Richardson P.C.
Horlick Kenneth R.
The Institute of Physical and Chemical Research
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