Patent Application
20090286241
This application claims benefit of priority under 35 USC 119 based on Japanese Patent Application No. P2008-131243, filed on May 19, 2008, the entire contents of which are incorporated by reference herein.
1. Field of the Invention
The present invention relates to an examination technology for a gene mutation, and particularly relates to a system for detecting a gene mutation and a method for detecting a gene mutation.
2. Description of the Related Art
In the anti-HIV therapy, drug resistance of the HIV virus has become a serious problem. In general, an examination of drug-resistant virus has been performed by a direct sequencing method for a resistance-related gene. However, it has been heretofore impossible to detect the resistance-related gene unless such resistant virus is not present in a ratio of 1/3 or more in all viruses. As opposed to this, if it is made possible to detect and quantify drug-resistant virus in a minority group, then it is expected to become possible to acquire important information in grasping a transmission state of the drug-resistant HIV-1 and in selecting a curative medicine in the salvage therapy. In this connection, Hance et.al succeeded in measuring the resistant virus present in a ratio of 0.1% in all the viruses by using an allele-specific real-time PCR (see, for example, J. Virol. 2001 75:6410-6417.). Thereafter, the technology of Hance et.al has been used for researching quantitative changes of the resistant virus in untreated periods in a variety of time periods, maternal infections, strategic treatment interruptions and the like. However, such a method of Hance et.al has had problems that it cannot measure three types of point mutations simultaneously, and that it cannot measure a consecutive two-base mutation (for example, T215Y). Therefore, it has been desired that a novel system for detecting a gene mutation be established.
It is an object of the present invention to provide a system for detecting a gene mutation and a method for detecting a gene mutation, which are capable of detecting the gene mutation, and particularly, the gene mutation in the minority group.
An aspect of the present invention inheres in a system for detecting a gene mutation, encompassing (a) a spectrum generation mechanism configured to acquire a first amplified product containing a specific site of a nucleotide sequence so that the specific site is amplified by a first polymerase chain reaction by using a specific site-oriented primer, and a second amplified product containing the specific site sandwiched by recognition sites of a restriction enzyme so that the first amplified product is amplified by a second polymerase chain reaction by using a recognition site introduction-oriented primer containing the recognition sites of the restriction enzyme, and to generate a mass spectrum of an oligonucleotide fragment containing the specific site, the oligonucleotide fragment being cut out from the second amplified product by using the restriction enzyme, (b) an area ratio calculation mechanism configured to calculate a calculated value of an area ratio of a peak of a wild-type sequence of the specific site and a peak of a mutation-type sequence of the specific site in the mass spectrum, (c) a relation storage device configured to store a relationship between a previously acquired area ratio and an abundance ratio of the wild-type sequence and the mutation-type sequence, and (d) an abundance ratio calculation mechanism configured to obtain the abundance ratio of the wild-type sequence and the mutation-type sequence from the calculated value of the area ratio based on the relationship.
Another aspect of the present invention inheres in a method for detecting a gene mutation encompassing (a) acquiring a first amplified product containing a specific site of a nucleotide sequence so that the specific site is amplified by a first polymerase chain reaction by using a specific site-oriented primer, (b) acquiring a second amplified product containing the specific site sandwiched by recognition sites of a restriction enzyme so that the first amplified product is amplified by a second polymerase chain reaction by using a recognition site introduction-oriented primer containing the recognition sites of the restriction enzyme, (c) cutting out an oligonucleotide fragment containing the specific site from the second amplified product by using the restriction enzyme, (d) obtaining a mass spectrum of the cut-out oligonucleotide fragment by a mass spectrometry method, (e) calculating a calculated value of an area ratio of a peak of a wild-type sequence of the specific site and a peak of a mutation-type sequence of the specific site in the mass spectrum, and (f) obtaining an abundance ratio of the wild-type sequence and the mutation-type sequence from the calculated value of the area ratio based on a relationship between a previously acquired area ratio and the abundance ratio of the wild-type sequence and the mutation-type sequence.
A description will be made below of an embodiment of the present invention. In the following description made with reference to the drawings, the same or similar portions are denoted by the same or similar reference numerals. Note that the drawings are schematic. Hence, specific dimensions and the like should be determined with reference to the following description. Moreover, it is a matter of course that portions different in dimensional relationship and ratio from one another are also included in the drawings.
As shown in
The PCR device 151 is used for acquiring a second amplified product containing the specific site sandwiched by recognition sites of a type-I restriction enzyme so that the first amplified product is amplified by a second polymerase chain reaction by using a recognition site introduction-oriented primer containing the recognition sites of the type-I restriction enzyme. The solid-phase column 153 is used for purifying the second amplified product. The mass spectrometer 152 is used for analyzing an oligonucleotide fragment containing the specific site, which is cut out from the second amplified product by using the type-I restriction enzyme. As the mass spectrometer 152, a liquid chromatograph and the like are usable.
A system for detecting a gene mutation according to the embodiment includes a central processing unit (CPU) 300. The CPU 300 encompasses a spectrum generation mechanism 301 configured to generate a mass spectrum of the oligonucleotide fragment analyzed by the mass spectrometer 152, and an area ratio calculation mechanism 302 configured to calculate a calculated value of an area ratio of a peak of a wild-type sequence of the specific site and a peak of a mutation-type sequence of the specific site in the mass spectrum. To the CPU 300, a relationship storage device 201 is connected, which stores a previously acquired relationship between an area ratio and abundance ratio of the wild-type sequence and the mutation-type sequence. Moreover, the CPU 300 further includes an abundance ratio calculation mechanism 303 configured to obtain a measurement value of the abundance ratio of the wild-type sequence and the mutation-type sequence from the calculated value of the area ratio based on the relationship stored in the relationship storage device 201.
To the CPU 300, there are further connected an input device 312, an output device 313, a program storage device 330, and a temporary storage device 331. As the input device 312, for example, a keyboard, a pointing device such as a mouse, and the like are usable. As the output device 313, an image display device such as a liquid crystal display and a monitor, a printer, and the like are usable. The program storage device 330 stores an operating system for controlling the CPU 300, and the like. The temporary storage device 331 sequentially stores results of arithmetic operations by the CPU 300. As each of the program storage device 330 and the temporary storage device 331, for example, a recording medium for recording the program, such as a semiconductor memory, a magnetic disk, an optical disk, a magneto-optical disk and a magnetic tape, is usable.
Next, a description will be made of a method for detecting a gene mutation according to the embodiment by using a flowchart shown in
(a) In Step S101, a sample containing a cDNA derived from a wild strain and a cDNA derived from a mutant strain is prepared. In comparison with the cDNA derived from the wild strain, a point mutation occurs in the cDNA derived from the mutant strain. Note that a ratio of the cDNA derived from the wild strain and the cDNA derived from the mutant strain in the sample is unknown. Next, a specific site in which a point mutation of the cDNA derived from the wild strain can occur and a specific site in which the point mutation of the cDNA derived from the mutant strain has occurred are amplified by using the specific site-oriented primer and the PCR device 151 shown in
(b) In Step S102, the first amplified product is amplified by using the recognition site introduction-oriented primer containing the recognition sites of the type-I restriction enzyme and the PCR device 151, whereby the second amplified product containing the specific site sandwiched by the recognition sites of the type-I restriction enzyme is obtained. Next, in Step S103, the oligonucleotide fragment containing the specific site is cut out from the second amplified product by using the type-I restriction enzyme. Thereafter, in Step S104, the oligonucleotide fragment is dissolved by ammonium acetate, and in Step S105, the oligonucleotide fragment is purified by the solid-phase column 153. Note that, by dissolving the oligonucleotide fragment by the ammonium acetate, the oligonucleotide fragment becomes likely to be protonated in the subsequent mass spectrometry using electrospray ionization (ESI).
(c) In Step S106, the oligonucleotide fragment is subjected to the mass spectrometry by the mass spectrometer 152. Note that it is possible to perform the mass spectrometry for a plurality of the oligonucleotide fragments at a time. A result of the mass spectrometry is transmitted to the spectrum generation mechanism 301. The spectrum generation mechanism 301 generates the mass spectrometry based on the result of the mass spectrometry. The spectrum generation mechanism 301 transmits the generated mass spectrum to the area ratio calculation mechanism 302,
(d) In Step S107, the area ratio calculation mechanism 302 detects, in the mass spectrum, the peak corresponding to the wild-type 35 sequence of the specific site, and the peak corresponding to the mutation-type sequence of the specific site in which the point mutation has occurred. Next, the area ratio calculation mechanism 802 calculates a peak area of the peak corresponding to the wild-type sequence, and a peak area of the peak corresponding to the mutation-type sequence, and calculates the calculated value of the area ratio of the peak area of the mutation-type sequence with respect to the peak area of the wild-type sequence. The area ratio calculation mechanism 302 transmits the calculated area ratio to the abundance ratio calculation mechanism 303.
(e) In Step S108, the abundance ratio calculation mechanism 303 reads out a linear function indicating a previously acquired relationship between the area ratio of the peak area of the mutation-type sequence with respect to the peak area of the wild-type sequence and the abundance ratio of the mutation-type sequence with respect to the wild-type sequence in the sample. Here, the previously acquired relationship is stored in the relationship storage device 201. Next, the abundance ratio calculation mechanism 303 assigns the calculated value of the area ratio to the linear function, calculates a measurement value of the abundance ratio of the wild-type sequence and the mutation-type sequence, outputs the measurement value to the output device 313, and then ends the method for detecting a gene mutation according to the embodiment.
In accordance with the system for detecting a gene mutation and the method for detecting a gene mutation according to the embodiment described above, it becomes possible to measure the abundance ratio of a trace quantity of the mutation-type sequence contained in the sample.
First, a wild strain IIIB and a drug-resistant mutant strain K2901 separated from blood collected, with the consent, from one of HIV-1 carriers who visit the Keio University Hospital were prepared. The wild strain IIIB does not have a resistance mutation to a reverse transcriptase inhibitor or a protease inhibitor. As opposed to this, the drug-resistant mutant strain K2901 has mutations of M41L, K103N, M184V and T215Y in a reverse transeriptase gene. For example, owing to the mutation of K103N, drug resistance to Lamivudine as a reverse transcriptase inhibitor occurs. Moreover, owing to the mutation of M184V, drug resistance to Efavirenz as a reverse transcriptase inhibitor occurs. Furthermore, owing to the mutation of T215Y, drug resistance to Zidovudine as a reverse transcriptase inhibitor occurs. Moreover, the drug-resistant mutant strain K2901 has mutations of 154V, G73S, V82A and L90M in a protease gene. For example, owing to the mutation of L90M, drug resistance to most of the protease inhibitors occurs.
Next, 60 μl of RNA extract was extracted from 50 pg (containing viruses of approximately 25,000 copies), in conversion to p24 antigen level, of a culture supernatant of each of the wild strain IIIB and the drug-resistant mutant strain K2901 by using QIAamp UltraSens (registered trademark) Virus Kit (made by QIAGEN GmbH). Moreover, to 6 μl of the RNA extract, there were added 2 μl of a random hexamer with 50 μ mmol/l, and 1 μl of dNTPs with 10 mmol/l. Then, an obtained mixture was denatured at 65° C. for 5 minutes, and was thereafter allowed to still stand for 1 minute or more on ice. Furthermore, to the RNA extract, there were added 4 μl of 5×first strand buffer attached to SuperScript (registered trademark) III (made by Invitrogen Corporation), 1 μl of DTT with 0.1 mol/l, 1 μl of SuperScript III with 200 U/ul, and 1 μl of RNasin with 40 U/μl. Then, an obtained mixture was allowed to still stand at room temperature for 5 minutes, at 50° C. for 10 minutes, and at 70° C. for 15 minutes. In such a way cDNA was created.
Next, 5 μl of 10×PCR Buffer attached to Platinum (registered trademark) Taq (made by Invitrogen Corporation), 3 μl of MgCl2 with 50 mmol/l, 0.4 μl of dNTPs with 25 mmol/l, 0.5 μl of a specific site-oriented forward primer with 20 μ mmol/l, 0.5 μl of a specific site-oriented reverse primer with 20 μ mol/l, both of which are shown in
Next, 5 μl of the 10×PCR Buffer, 3 μl of MgCl2 with 50 mmol/l, 0.4 μl of dNTPs with 25 mmol/l, 5 μl of a recognition site introduction-oriented forward primer with 20 μ mol/l, 5 μl of a recognition site introduction-oriented reverse primer with 20 μmol/l, both of which are shown in
Next, 5 μl of 10×NE Buffer 2 attached to the restriction enzyme (made by New England Biolabs Japan, Inc.), 2.5 μl of S-adenosyl methionine with 1.6 mmol/l, 7 μl of MgCl2 with 50 mmol/l, 5 μl of DTT with 10 mmol/l, 28.5 μl of distilled water and 2 μl of the restriction enzyme AcuI were mixed together, whereby a restriction enzyme reaction-oriented premix was prepared. Thereafter, 50 μl of the purified second PCR amplified product was added to 50 μl of the restriction enzyme reaction-oriented premix, and a resultant was allowed to still stand at 37° C. for 1 hour, and at 65° C. for 20 minutes. Then, an oligonucleotide fragment containing the K103 site, an oligonucleotide fragment containing the M184 site, an oligonucleotide fragment containing the T215 site, and an oligonucleotide fragment containing the L90 site, were individually cut out.
Next, 360 μl of 99.5% ethanol was added to 90 μl of a solution containing the cut-out oligonucleotide fragments, and a mixed solution was subjected to centrifugal separation at 14000 rpm for 2 minutes. A supernatant was removed from a resultant, and 10 μl of ammonium acetate with 5 mmol/l was added thereto, whereby the resultant was dissolved. Then, the resultant was purified by using a solid-phase column (ZipTip (registered trademark), made by Nihon Millipore K.K.), and was thereafter dried for 5 minutes in a vacuum dryer. 10 μl of ammonium acetate with 5 mmol/l was added to the dried oligonucleotide fragments, whereby the oligonucleotide fragments were dissolved. Then, a resultant was subjected to the centrifugal separation at 14000 rpm for 2 minutes, whereby 9 μl of a supernatant was collected. The oligonucleotide fragments are protonated by ammonium acetate. 8 μl of the collected supernatant was analyzed by a liquid chromatography-mass spectrometry method (LC-MS). As a liquid chromatograph, HP1100 series HPLC system (made by Agilent Technologies) was used. Moreover, as a mass spectrometer, Q-STAR pulsar i (made by Applied Biosystems) as a quadrupole time-of-flight mass spectrometry (TOF-MS) was used.
Next, an area surrounded by a plot showing the peak at 779.5 amu and a base line when a detected intensity was 0 in each of
Next, also in the case of calculating a peak area of the chromatogram, which corresponds to the K103N mutation of the reverse transcriptase gene, as the specific peak area of the drug-resistant mutant strain K2901, as shown in
As described above, the oligonucleotide fragments obtained by cutting the PCR amplified products by the type-I restriction enzyme are analyzed by LC-MS, whereby it becomes possible to detect and quantify the trace quantity of drug-resistant mutant viruses. In particular, it also becomes possible to measure an abundance ratio of the virus having T215Y as an important mutation to the nucleotide reverse transcriptase inhibitor.
The description has been made as above of the present invention based on the embodiment; however, it should not be understood that the description and the drawings, which compose a part of this disclosure, limit this invention. For example, though the oligonucleotide fragment is purified by the solid-phase column in Step S105 of
Moreover, in Example, by using the specific site-oriented primer, the specific site of the nucleotide sequence was amplified by the first PCR, whereby the first amplified product containing the specific site was acquired. Thereafter, by using the recognition site introduction-oriented primer, the first amplified product was amplified by the second PCR, whereby the second amplified product containing the specific site sandwiched by the recognition sites of the restriction enzyme was acquired. The reason for the above is as follows. Specifically, a mutated gene derived from an HIV-1 carrier has a plurality of the mutations, and accordingly, if the first PCR is omitted, then there occurs a difference in amplification factor of the PCR between the nucleotide derived from the mutant strain and the nucleotide derived from the wild strain owing to a difference therebetween in homology to the recognition site introduction-oriented primer. However, in the case where there is no difference in homology to the recognition site introduction-oriented primer, the first PCR may be omitted, and the specific site of the nucleotide sequence may be amplified by using the recognition site introduction-oriented primer from the beginning.
Moreover, in Example, the detection example of the mutant strain carried by the HIV-1 carrier has been shown; however, it is a matter of course that the system for detecting a gene mutation and the method for detecting a gene mutation according to the embodiment of the present invention are applicable not only to the analysis of the ratio of the cDNA mutation but also to detection of a ratio of a DNA mutation of virus or bacteria, and further, to detection of duplication, deletion and the like of SNPs and genes of a cell.
From this disclosure as described above, a variety of alternative embodiments, examples and application technologies will be obvious for those skilled in the art. Hence, the technical scope of the present invention is defined only by items which specify the invention, and are according to the scope of claims reasonable from the above description.
Sequence numbers 1 to 32 described in a sequence table of this specification denote the following sequences.
In the case of displaying the bases by abbreviations in this specification, abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature, or idiomatic abbreviations in the field concerned are used. Examples of the abbreviations are shown below. a: adenine, t: thymine, g: guanine, c: cytosine, u: uracil, m: adenine or cytosine, r: guanine or adenine
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-131243 | May 2008 | JP | national |
