Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-21
2002-11-26
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S091100, C435S091200, C435S183000, C435S252300, C536S025400, C536S025300
Reexamination Certificate
active
06485916
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a preparation method of nucleic acid sample, in particular to a preparation method of nucleic acid sample suitable for analysis of rare expressed genes in the sample, to nucleic acid samples obtained by the preparation method and to an analyzing method using the prepared nucleic acid samples.
2. Description of the Related Art
Constitutive cells of a human body can be classified into 200 or more species, and each one cell type has subtly different subtypes. Each of the cells has a common genome coding for about 10×10
4
genes and several ten thousands genes are expressed therein according to its cell type. Investigations on such gene expression have become more important not only for obtaining findings on functions of individual genes but also for clarifying biological phenomena. Detailed analyses on comparatively limited few genes have revealed that plural genes operate in coordination in majority of the biological phenomena. In the course of the human genome project, investigations on networks of genes for macroscopic comprehension of relations between genes and biological phenomena are to start. Under these circumstances, demands have been made to provide techniques for further detailed analyses of a multitude of genes.
Recently, there have been developed analyzing methods of gene expression by the comparison between RNA fingerprintings (e.g., differential display method (Liang, P., and Pardee, A. B. (1992)
Science
257,967-971) and molecular index method (Kikuya Kato (1995)
Nucleic Acids Res.,
23,3685-3690)) and methods using DNA chips, suggesting the possibility of complete analysis of expressed genes.
Expressed Genes can be roughly classified according to its expressing amount into three classes, i.e., abundant class where a gene is expressed 12,000 copies or more per cell, intermediate class where a gene is expressed about 300 copies per cell and rare class where a gene is expressed about 15 copies. In mammals, species of expressed genes amount to several ten thousands per cell and most genes belong to the rare class. In other words, expressed genes in a cell comprise enormous species of rare expressed genes coresident with extremely few species of abundant expressed genes yet expressed on three or four orders of magnitude greater than the rare expressed genes (e.g., Alberts, B., et al. (1989)
Molecular Biology of the Cell,
2nd edition, Garland Publishing Inc., NY, USA).
As analyzing methods of rare expressed gene are known an equalized library (Minoru S. H. Ko (1990)
Nucleic Acids Res.,
18,5705-5711), methods based upon multiplex PCR (polymerase chain reaction) including the differential display method and the molecular index method, and methods using DNA chips. It is known, however, that the equalized library technique would lose information on expression amounts because the amounts of genes are made uniform among gene species; and that in an analysis using multiplex PCR such as in the differential display method a comparative PCR has a strong bias towards abundant expressed genes to result in less sensitivity than a usual PCR and thereby to fail to detect rare expressed genes (David J. Bertioli, et al. (1995)
Nucleic Acids Res.,
23,4520-4523).
SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to provide a preparation method of nucleic acid sample suitable for analyses of rare expressed genes, and nucleic acid samples prepared thereby, and to provide an analyzing method of rare expressed genes using the prepared nucleic acid samples.
The present inventors focused that abundant expressed gene (abundant class) in a nucleic acid sample can relatively easily be analyzed and readily removed in a selective manner, as there is a strong bias towards abundant expressed genes in the process of gene expression analysis using the differential display method, and hence the expression analysis of rare expressed genes using a nucleic acid obtained by removing abundant expressed genes in advance from a nucleic acid sample to be analyzed is expected to achieve higher precision. The present invention has been accomplished on the basis of the above findings.
REFERENCES:
patent: 5888792 (1999-03-01), Bandman et al.
patent: 6180778 (2001-01-01), Bastian et al.
Analytical Biochemistry, vol. 162, 1987, P. Chomczynski et al, pp. 156-159.
Nucleic Acids Research, 1995, vol. 23, No. 21, D. J. Bertioli et al, pp. 4520-4523.
FEBS Letters, 1994, T. Ito et al, pp. 231-236.
Nucleic Acids Research, vol. 18, No. 19, M. Ko et al, pp. 5705-5711.
Science, vol. 257, Aug. 14, 1992, P. Liang et al, pp. 967-971.
Nucleic Acids Research, 1995, vol. 23, No. 18, K. Kato, pp. 3685-3690.
Science, vol. 270, Oct. 20, 1995, V. Velculescu et al, pp. 484-487.
Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989, J. Sambrook et al, “Synthesis of the First Strand of cDNA & Amplification of cDNA Generated by Reverse Transcription of mRNA”.
Molecular Biology of the Cell, Second Edition, Garland Publishing, Inc., B. Alberts et al, “RNA Synthesis and RNA Processing”.
Fujita Takeshi
Irie Takashi
Kiyama Masaharu
Muramatsu Takamichi
Okano Kazunori
Jones W. Gary
Mattingly Stanger & Malur, P.C.
Taylor Janell E.
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