Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1998-03-13
2000-01-25
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 435 71, 435 911, 435 9121, 536 243, 536 2431, 536 2432, 536 2433, C12P 1934, C12Q 168, C07H 2104
Patent
active
06017739&
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method for concentrating a mutant nucleic acid which is capable of selectively separating and removing the normal nucleic acid, or selectively separating and collecting a mutant nucleic acid from a sample including both the nucleic acid wherein the particular segment of the nucleic acid comprises the normal gene and a minute amount of the mutant nucleic acid having a nucleotide sequence slightly different from the normal nucleic acid. The present invention also relates to an assay kit for nucleic acid concentration in order conduct said concentration method.
BACKGROUND TECHNOLOGY
Molecular biology and genetics have recently experienced tremendous progress, and the findings accumulated in these field contributed in the elucidation of biological phenomena in both chemical and physical senses and such findings also gave fundamental impacts on medical researches and practices. DNA medicine working from the level of DNA is rapidly expanding to the clinical fields at a rate far beyond our expectation. Today, DNA is considered to be associated in practically every disease, and diagnosis on the gene level has become indispensable.
It has also been revealed that substantially all of the various enzyme deficiencies which have been known as congenital dysbolism for quite some time are gene diseases, and detection of mutation in the nucleotide sequence of the gene is quite effective in the gene diagnosis of such diseases.
Gene diagnosis of the diseases induced by acquired dysbolism, for example, cancer have encountered with an extreme difficulty in collecting the cancer cell from the cancer lesion, and normal cells were always included in the collected specimen. Accordingly, there is a need for a method by which only the mutant gene in the cancer cell can be specifically detected under the conditions where both the normal cells and the cancer cells are present. In the current process, detection of the mutant gene is possible when the amount of the mutant gene is about one tenth of the normal gene, namely, when the amount of the cancer cells is about one fifth to one tenth of the normal cells. Detection of the mutant gene of smaller amount, however, has been difficult, and the gene diagnosis is not yet effectively utilized in the early stage detection and early stage treatment of cancer.
Recent development in gene amplification methods such as PCR (polymerase chain reaction) enabled increase of the amount of gene. Not only such mere increase in the amount of the gene but also increase in the percentage of the mutant gene in the sample is required for identifying cancer cell from normal cell, or mutation of the gene responsible for the particular genetic disease of a patient from the gene of a normal donor. In such a case, the particular gene can be selectively concentrated by subtraction method (In Current Protocol in Molecular Biology, (1992), John Wiley & Sons, Inc.) when the particular gene of either cell includes a deletion or an insertion of a considerable length or when the particular gene is expressed only in either cell.
Detection of the mutant gene by the subtraction method, however, was substantially difficult when the mutation of the gene responsible for the disease was minute, namely, when the size of the mutation through deletion, addition or substitution is very small, or when no substantial difference in the gene expression level is found between the genes.
SUMMARY OF THE INVENTION
The present invention has been made in view of the situation as described above, and an object of the present invention is to provide a nucleic acid concentration method which is capable of readily and reliably concentrating a mutant nucleic acid of a sample even if the sample contains both the normal nucleic acid wherein the particular segment of the target nucleic acid is normal and the mutant nucleic acid of minute amount, and which is capable of detecting the mutant nucleic acid of minute amount and identifying the mutant nucleic acid; as well as an assay kit for nu
REFERENCES:
patent: 5200314 (1993-04-01), Urdea
patent: 5273882 (1993-12-01), Snitman et al.
patent: 5484699 (1996-01-01), Bouma et al.
Oka et al NAR vol. 22, No. 9 pp. 1541-1547, 1994.
Horlick Kenneth R.
Siew Jeffrey
Wakunga Seiyaku Kabushiki Kaisha
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