Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-09-05
2004-06-15
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200
Reexamination Certificate
active
06750018
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to detection of specific sequences contained in DNAs of test subjects, detection of polymorphisms of genes, analysis of single nucleotide polymorphisms (SNPs), and the like.
The progress of the human genome mapping project is prompting a trend towards applying DNA sequence information to medical diagnoses and various industries, for example, in producing new drugs. Particularly, in a medical field, noticeable is a trend towards revealing and applying gene functions. In particular, analyses of gene expression profiles, in which genes active in a body are examined, and analyses of single nucleotide polymorphisms (SNPs) in genomes, which may be a cause of variations in gene expression, are drawing attention.
In genomes, single nucleotide polymorphisms are said to be present approximately one per 1000 bases, which means every individual has a huge number of single nucleotide polymorphisms. These single nucleotide polymorphisms are considered to be associated with characteristics of individuals so that the analysis of these single nucleotide polymorphisms is expected to provide a therapy guide or the like for the treatment of diseases for the individuals.
The number of single nucleotide polymorphisms present in genomes of one human being is enormous, and the type of single nucleotide polymorphisms in genomes of many people are polymorphic, in which two kinds of bases appear in particular sites. It is necessary to examine single nucleotide polymorphisms occurring at sites of mutation and further to reveal their correlation with diseases or the like, which requires the accumulation of enormous amounts of DNA data. Thus, development of appropriate methods to meet this requirement is in need.
At present, various methods for analyzing base substitutions are available. They are generally divided into two types, i.e., methods for detecting unknown base substitutions and methods for examining, for example, whether certain known base substitutions are found in many people or whether these known substitutions have causal relationships with certain diseases.
Examples of the methods for detecting unknown base substitutions include a method in which a base sequence is determined using a DNA sequencer, and a method in which hybridization of a DNA probe and a target DNA is detected using a DNA probe array (DNA chip or gene chip).
On the other hand, in order to detect single nucleotide polymorphisms at known positions, methods, such as an SSCP (single strand configuration polymorphism) method, an invader assay (Nature Biotechnology, 17, 292-296 (1999)), and DASH (Nature Biotechnology, 17, 87-88 (1999)), have been developed and implemented for practical use. All of these methods use laser or the like as a light source.
In synthesizing a DNA complementary strand using a primer, complementary strand extension may or may not proceed depending on whether the 3′-end of the primer is complementary to the target or not. Accordingly, a method, in which a PCR amplification is performed using a primer having the 3′-end at an accordant position of mutation and products are analyzed using gel electrophoresis or the like (ARMS: amplified refractory mutation system; Nucleic Acids Research, 17, 2503-2515 (1989)), has also been used. In this case, a key point is how exclusively the complementary strand synthesis occurs in the presence of a target of interest.
In the SSCP method, single, nucleotide polymorphisms can be detected based on the observation that the configurations of single-stranded DNAs with and without mutation during electrophoresis are different, and these configurations influence electrophoretic mobility.
In the invader assay, a triple-stranded chain is formed with a DNA probe of a DNA probe array, a target DNA and a DNA probe, and whether a part of the probe is cleaved with an enzyme is detected.
In DASH, a target DNA and a probe DNA are hybridized under the presence of an intercalator, and light generated from the intercalator by irradiation is observed while changing a temperature. The double stranded chain is dissociated and the emission ceases if mutation is present in the target DNA.
On the other hand, anew technique is about to be implemented, in which a short DNA base sequence is determined in a short time and then mutations are examined using the determined DNA base sequence.
For example, Nyren et al. have proposed a method for determining a DNA base sequence (pyrosequencing), in which a target DNA is hybridized with a primer, pyrophosphate produced by a complementary strand extension reaction is converted into ATP, the ATP is reacted with luciferin to emit light, and this chemiluminescence is detected to discriminate the substrate (dNTP) incorporated during the complementary strand extension reaction. The pyrosequencing has drawn an attention as a simple and easy method for determining a DNA sequence without the need for gel electrophoresis. Recently, detection of SNPs using this pyrosequencing has been reported (Anal Biochemistry, 280, 103-110 (2000)). This method does not require the use of a new light source because it utilizes chemiluminescence.
SUMMARY OF THE INVENTION
Mutations which appear in genome DNAs are associated with characteristics of organisms, sensitivity to diseases and medicines, and the like, and thus a huge number of subjects have to be analyzed. Since individuals to be tested are numerous, there is a demand for a method and an apparatus which suit for a simple and large scale operation at a low running cost.
Further, SNP measurements are necessary for individual genome samples, as well as for samples from a patient group and a healthy subject group for comparison. Namely, in order to study a causative relationship between particular SNPs and a certain disease it is necessary to compare the incidence of the SNPs (allele frequency) in the two groups. This comparison study can be most efficiently and easily carried out by combining DNAs of each group and quantitatively analyzing the SNPs contained in the combined DNAs. However, a quantitative method accurate enough for such analyses remains to be developed. Further problems arise upon measurement of SNPs even with individual samples, as described below.
In various methods which have been proposed previously, a targeted DNA region is amplified by PCR and the resulting DNA fragments are used as a target. Accordingly, the use of PCR is inevitable.
In the DASH, it is necessary to use a detector equipped with a laser light source and a filter to remove scattering light, which requires a large-scale apparatus.
In a pyrosequencing for detecting chemiluminescence, pyrophosphate is converted into ATP to generate chemiluminescence and the resulting photo-emission is detected. However, the pyrosequencing has disadvantages such that four kinds of dNTP (dATP, dTTP, dGTP and dCTP) have to be added independently in a designated order to a reaction part, that the photo-emission intensity is weak, that a system for dNTP injection is necessary, and that a considerably large-scale apparatus is required.
Further problems in this method are that because reagents are injected in a designated order, the time required for the analysis is 10-20 minutes although it is shorter than that with the use of electrophoresis, and that sample DNAs have to be PCR-amplified to increase the number of DNA copies of the target region because the method is not sensitive enough without amplification.
Furthermore, in pyrosequencing, if a multiple number of base substitutions exist in a sample to be tested, the resulting spectrum may be too complicated to perform accurate analyses.
In order to apply the pyrosequencing for the detection of DNA mutations, it is important to further improve the sensitivity so that the DNA mutations can be readily detected and DNA strands containing multiple kinds of mutations can be analyzed.
An object of the present invention is to provide a tool necessary to create a data base applicable for genetic diagnoses, new drug manufacturing using genes, and gene therap
Kamahori Masao
Kambara Hideki
Okano Kazunori
Zhou Guohua
A. Marquez, Esq. Juan Carlos
Fisher Esq. Stanley P.
Hitachi , Ltd.
Horlick Kenneth R.
Reed Smith LLP
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