This invention relates to a method for examining nucleotide sequences for DNA tests and genetic diagnosis, and specifically to a method for simultaneous examination of multiple mutations contained in the genome DNA.
With recent development of human genome projects, the relation between disease- or drug-sensitivity and genes is being elucidated. It has been clarified that the genes related to disease- or drug-sensitivity do not operate solely but several genes function in combination with each other. Single-nucleotide polymorphism (SNP) in the human genome has been documented to occur once per 1000 bases and to be related to individual""s constitution, drug sensitivity and resistance to diseases, and the relation between loci of SNPs and these phenomena is being elucidated. Genetic diagnosis for actual diseases and consultation based on genetic information are now starting, and rapid development of a method for examining SNPs in multiple genomic sites efficiently and at low cost is essential.
Under these conditions, various analytical methods have been proposed; (a) Taqman assay, detecting an increase in fluorescent intensity accompanied by degradation of marker probes during PCR amplification (Ref. 1: Proc. Natl. Acad. Sci. USA: 88, 7276-7280 (1991)), (b) Invader assay, detecting fluorescence caused by degradation of fluorescence-labeled probes, for which formation of triple-strand DNA and an enzyme recognizing mismatch are combined (Ref. 2: Nature Biotech.: 17, 292-296 (1999)), (c) single-strand conformation polymorphism (SSCP), detecting the difference in electrophoretic mobility of DNA with or without mutations by gel electrophoresis (Ref. 3: Genomics: 5, 874-879 (1989)), and (d) DNA chips, or DNA probe arrays in which probes are immobilized to surface of a plane (Ref. 4: Genomics Res.: 10, 853-860 (2000)), and (e) a method in which DNA probes are immobilized to colorcoded beads and these are collected to be used as probe arrays (Ref. 5: Science: 287, 451-452 (2000)). All these five methods utilize detection of fluorescence by laser excitation. In addition, several other methods such as pyrosequencing using chemiluminescence (Ref. 6: Anal. Biochem.: 280, 103-110 (2000)) have been reported.
Since any of the above-mentioned methods does not satisfy all the basic points such as low running costs, simple and brief operation and high reliability, a new method has been greatly desired. Inventors and others have already proposed a convenient and low-cost detection method for DNA mutations (bioluminometric assay with modified primer extension reaction, BAMPER) (Ref. 7: Nucl. Acid Res.: 29, e93 (2001)) and have obtained excellent results. However, this method also has several problems; amplification of the copy number of DNA by PCR is necessary in prior to each measurement, and sample preparation is necessary for every subject of examination.
In detection of chemiluminescence, pyrophosphate is reacted with APS and converted to ATP, which then reacts with luciferin to produce chemiluminescence. Although chemiluminescence is believed to have a high sensitivity, the normal detection limit for the number of DNA copies is only in the order of femtomole (fmol) because APS that converts pyrophosphate to ATP can also react with luciferin. For this reason, amplification of target DNA is essential in prior to examination.
On the other hand, examination of multiple test sites has been desired in clinical diagnosis related to diseases. For this purpose, multiple test sites should be amplified. However, amplifying every test site one by one is not realistic because of its high cost. DNA probe arrays (DNA chips) recently used for analysis of genetic expression profiles are known to be suitable for examining multiple target regions, but it is difficult to recognize one-base mutation since the method involves hybridization of targets and probes. A new method solving these problems is desired.
Most of the methods that have been developed so far or are currently used require amplification of the copy number and/or sample preparation for every test subject, which often causes problems of handiness, time and cost especially for the subjects with many test sites.
The purpose of this invention is to provide a method for examining nucleotide sequences, including an efficient and low-cost method for preparing samples with many test sites, based on a method utilizing chemiluminescence, for which low-priced equipment and test reagents are available.
To solve the above problems, this invention discloses a means, comprising;
a sample solution containing multiple targets (DNA fragments) subjected to examination is equally added to multiple subcells set in a reaction vessel. The sequences designed to hybridize with different targets are used to hybridize different DNA probes and targets in each of the subcells. Under the conditions in which the solution in the subcells is not mixed, complementary strand synthesis and chemiluminescence resulting from pyrophosphate are induced to examine SNPs at any of the test sites easily.
The invention also discloses a method, comprising;
following the synthesis of strands complementary to subject DNA fragments by using anchor primers, all of the DNA fragments are PCR-amplified en bloc using a pair of universal primers common to all the DNA fragments. Complementary DNA strands including test sites are synthesized en bloc by means of the anchor primers, and complementary strand synthesis is repeatedly conducted in the subcells by using PCR products of the DNA fragments and the complementary DNA strands including test sites. With these procedures, more pyrophosphate is produced and reacted with chemiluminescent reagents so that multiple mutations in DNA can be detected simultaneously with optically high sensitivity. Furthermore, the invention discloses a method for producing a large amount of pyrophosphate, a chemiluminescent substance, by using DNA strands specifically prepared for signal amplification in addition to target DNA.
In a process of the invention, detection of target DNA contained in subject DNA, especially detection of one-base mutation, is achieved by detecting chemiluminescence induced by the reaction of ATP and luciferin/luciferase; ATP is made from pyrophosphate that is produced in the course of synthesis of complementary DNA strands. For higher detection sensitivity, the reaction system is constructed so that a large chain of reactions of complementary strand synthesis is triggered when targets are present in a sample and acts as a template for synthesis of complementary strands. This process of the invention has achieved four to five orders of magnitude of high sensitivity compared to the prior process, pyrosequencing, which has been known as a method for examining genetic mutations by chemiluminescence. With this process, only several hundreds of copies of DNA are enough for detection. In addition, many reaction cells are prepared in a reaction vessel to make it possible to examine multiple test sites simultaneously.
In a process of the invention, the above problems are solved by the following steps; after simultaneous amplification, target regions are hybridized with probes immobilized to different cells that are separated from each other in a reaction vessel, which is followed by complementary strand synthesis. A large amount of pyrophosphate is thus produced in each cell, where chemiluminescent reaction is performed separately. In more detail, initiation of complementary strand synthesis depends on the presence of single-base mutation when synthesis of complementary strands is performed in each cell by using a target DNA strand as a template. If the DNA contains mutations, complementary strand synthesis is allowed to be induced. The complementary strands thus acts as a trigger for the subsequent synthesis of complementary strands. A great amount of pyrophosphate is finally produced, which makes is possible to examine the presence of target DNA and the status of mutations at high sensitivity.
Representative compositions of the invention are described as follows. In the first composition of the invention, a group of primers consisting of multiple primer species is added to a solution containing a sample subjected to examination. Simultaneous synthesis of complementary strands is performed at each of the multiple regions containing target nucleotide sequences to be examined. DNA probes with specific sequences are designed so that elongation of complementary strands is affected by the presence or absence of mutations in the target nucleotide sequences. The same number of such DNA probes and the target sequences is used for elongation of complementary strands. Elongation reaction of complementary strands using the targets or the sequences complementary to the targets as a template and the following reaction, in which pyrophosphate produced during the elongation reaction is converted to ATP and reacted with chemiluminescent substrates to develop luminescence, are performed in the subcells of the reaction vessel that are compartmentalized for each said target. By detecting the luminescence, mutations present in the target nucleotide sequences are detected.
In the second composition of the invention, a group of probes consisting of multiple probe species is added to a sample solution subjected to examination. Two probes are hybridized to each of the different target sequences. The DNA probes are prepared so that the binding reaction of the two probes in ligation is affected by the presence of base mutations in DNA subjected to examination. Using a group of the probes consisting of pairs of these probes, long DNA strands are prepared by ligation reaction of the two probes. Furthermore, synthesis of complementary strands is performed at least once in subcells of a reaction vessel by using either the DNA strands or their complementary strands as a template. Pyrophosphate, the product of complementary DNA synthesis in each of the subcells, is converted to ATP and reacted with chemiluminescent substrates to develop luminescence in the subcells compartmentalized for each of the target sequences. With these processes, mutations in genes or DNA can be identified by detection of luminescence.
In the third composition of the invention, the primers with a common sequence at their 5xe2x80x2 termini can regulate synthetic reaction of complementary strands, where the target DNA strands are used as a template, depending on the presence or absence of mutations. Following the complementary strand synthesis using the primers, pyrophosphate is produced during synthesis of complementary strands using either the DNA strands obtained by amplification of the product of the complementary strand synthesis or their complementary strands. Pyrophosphate is converted to ATP, which is subsequently used for chemiluminescence. The presence of DNA mutations or the presence of target DNA is determined by luminescent intensity.
In the forth composition of the invention, using genomes or multiple target DNA as a template, multiple species of the first probes are hybridized to the templates in a single reaction vessel to prepare multiple species of the first complementary strands by the first synthesis of complementary strands. Then excess of the first probes are isolated and removed from the first complementary strands. With the first complementary strands as a template, the second synthesis of complementary strands is performed using multiple species of the second probes to obtain the second complementary strands, which partially contains the same sequence as that of said target DNA. In each compartmentalized area sorted with species of said first complementary strands, pyrophosphate is produced in the synthesis of the second complementary strands or in the complementary strand synthesis using said second complementary strands as a template, and is converted to ATP, which develops chemiluminescence for detection. With these processes, specific sequences and mutations in base sequences of the target DNA are detected.
In according to the representative compositions of the invention, a large amount of pyrophosphate (PPi) can be produced, which makes it possible to examine the presence of target DNA and the status of mutation at high sensitivity.
Other objects, features and advantages of the invention will become apparent from the following description of the embodiments of the invention taken in conjunction with the accompanying drawings.