JP6366053B2 - Standard sample manufacturing method - Google Patents

Description

  The present invention relates to a method for producing a standard sample having the same target sequence as a DNA fragment having a target sequence having a specific copy number.

  The polymerase chain reaction (PCR) is used as a nucleic acid analysis technique in a wide range of fields such as clinical diagnosis, forensic examination, and food analysis. In recent years, it has been required to guarantee the reliability of analysis results for various analyzes including PCR tests, and international standards such as ISO17025 have been created. In international commercial activities, quality control of products through analysis that meets these international standards may lead to the possibility of transactions, and technology related to assurance of analysis reliability is also industrially applicable. The importance is increasing.

  In ISO 17025, the validity of the analysis method is required to manage the quality of the analysis. Validation of an analytical method means to objectively evaluate the performance of the analytical method used and confirm that the standard is satisfied according to the purpose of use. Items for performance evaluation include specificity and detection limit. The PCR method enables nucleic acid amplification from one molecule because of its technical characteristics, but individual analysis methods using PCR detect from one molecule depending on the composition of the reaction solution and the performance of the PCR device. However, it cannot always be said that it is possible, so it is necessary to evaluate the detection limit of PCR analysis for each analysis method. Conventionally, nucleic acid samples obtained by diluting a plasmid nucleic acid or a genomic nucleic acid containing a target sequence to a certain concentration have been used for evaluation of the detection limit of PCR analysis. Specifically, the detection limit concentration is evaluated by analyzing a nucleic acid solution sample diluted to a level that theoretically contains 1 to several molecules of plasmid nucleic acid or genomic nucleic acid by PCR and examining the presence or absence of detection or the detection rate. Yes. However, when a sample diluted to the level of 1 to several molecules is used, the number of target nucleic acid molecules finally added to the PCR reaction solution is not constant due to the chance of sampling and varies greatly from reaction solution to reaction solution. Will have. For this reason, it has been extremely difficult to accurately evaluate the detection limit concentration in PCR analysis.

  Real-time PCR is a technology that detects fluorescence corresponding to nucleic acid amplification in a PCR process in a timely manner and enables quantitative measurement of the number of initial template nucleic acid molecules in a reaction solution. Have been used. In quantitative analysis by real-time PCR, a calibration curve that associates the measurement result with the number of molecules of the target nucleic acid is created. Conventionally, a solution sample group in which a plasmid nucleic acid or a genomic nucleic acid containing a PCR target base sequence is diluted stepwise has been used as a standard sample for preparing a calibration curve. However, when a sample having a low molecular number of about 1 to several molecules is used, the number of molecules of the target nucleic acid finally added to the PCR reaction solution varies greatly. For this reason, it has been difficult to stably create a calibration curve that can accurately define the number of molecules down to the low molecular number side.

  In addition, there are three processes in the gene-related test for detecting the base sequence of a gene and the like using the PCR method, a process before measurement (pre-analysis), a measurement process, and a process after measurement. In gene-related measurement processes, various gene analysis techniques, including PCR, are used, but the measurement accuracy is the largest factor in the measurement process (pre-analysis) from sample collection to pretreatment of test materials. It is known to affect. For this reason, in the pre-measurement process (pre-analysis), among several issues, the physical properties, chemical properties, presence of interfering substances that are growth inhibitors, etc., especially the properties of the specimens are diverse. In addition, it is known that the absence of an objective evaluation method affects the nucleic acid extraction step prior to measurement, resulting in a decrease in measurement accuracy (Non-Patent Document 1). Therefore, a nucleic acid solution sample in a state as close to an actual sample as possible is desired as a nucleic acid solution sample with a specified number of molecules for determining the qualitative limit and the quantitative limit.

  As described above, a nucleic acid solution sample in which the number of molecules of nucleic acid that is the target of PCR is strictly defined is necessary to accurately evaluate the detection limit in the analysis method using PCR and to create a calibration curve for real-time PCR. Has been.

  Thus, several examples of methods and apparatuses for preparing nucleic acid solution samples in which the number of molecules is strictly defined from a low concentration of one molecule have been known so far. For example, it is known that a standard substance of one molecule can be prepared by a limiting dilution method. In the limiting dilution method, most of the samples do not contain nucleic acids, and after diluting in a condition that a solution containing a single molecule of nucleic acid is produced with a slight probability, a solution containing a single molecule of nucleic acid is selected. is there. A method using a gating nanopore device has also been developed by a group including the inventors.

Genetic-related test Specimen quality control manual (approval document) JCCLS Japan Clinical Laboratory Standards Association

  However, all of them use a method of diluting synthetic nucleic acids or dispensing them by measuring the number of molecules, and they are originally retained in cells (especially in the nucleus) like the nucleic acids used in normal testing, so that cell division and metabolism Creating a nucleic acid sample containing the contents of a cell that surrounds a molecule of the desired number of molecules (one molecule if it is an intracellular molecule) that has undergone biological modification in the course of It was impossible.

  From such a background, there has been a strong demand for a method for producing a standard sample that is in a state close to that held in a cell and in which the number of molecules is strictly defined.

  In quantitative analysis by real-time PCR capable of quantitative measurement of the number of initial template nucleic acid molecules in a reaction solution, a calibration curve that associates the measurement result with the number of molecules of the target nucleic acid is created. Conventionally, a solution sample group in which a nucleic acid containing a PCR target base sequence has been diluted stepwise has been used as a standard sample for preparing a calibration curve. However, when a sample having a low molecular number of about 1 to several molecules is used. Since the number of target nucleic acid molecules finally added to the PCR reaction solution varies greatly, a calibration curve that can accurately define the number of molecules down to the low molecular number side is created stably. It was difficult. In addition, the requirements of standard samples required for quality control including measurement processes (pre-analysis) that greatly affect measurement accuracy include physical properties, chemical properties, and the presence of interfering substances that are growth inhibitors. The nucleic acid solution sample in a state that is retained in the cell, close to the real sample, and contains various factors reflecting the chemical modification and the ratio of the number of cells to nucleic acid molecules determines the qualitative limit and quantification limit For this reason, it has been desired as a nucleic acid solution sample having a defined number of molecules. However, in the methods proposed so far, it has been practically impossible to prepare a nucleic acid solution because a synthetic nucleic acid is used.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a standard sample manufacturing method capable of manufacturing a standard sample having the same target sequence as a DNA fragment having a target sequence having a specific copy number. And

  The standard sample production method of the present invention is a method for producing a standard sample having the same target sequence as a DNA fragment having a specific copy number of a target sequence, and introducing a DNA fragment having a specific copy number of the target sequence into a cell. An introduction step, a culture step for culturing the cells, and an isolation step for isolating the cultured cells.

Further, in the standard sample preparation method of the present invention, the introduction step, using budding yeast as a cell by homologous Recombination example using artificial synthetic gene by introducing DNA fragment into the chromosome of S. cerevisiae, in the culture step, budding The yeast is synchronized with the G0 / G1 phase and cultured in synchronization. In the isolation step, the budding yeast is fixed and isolated in the G1 phase. One cell of the isolated budding yeast has one copy of the target sequence. May be included.

Further, in the standard sample preparation method of the present invention may be used YER053C gene as homologous Recombination example target.

  In the standard sample manufacturing method of the present invention, in the isolation step, one cell may be sorted per well using a capillary whose tip angle is set within an angle range of 20 degrees to 30 degrees.

  In the standard sample manufacturing method of the present invention, the standard sample is a nucleic acid, and the nucleic acid may include DNA or RNA.

  According to the present invention, a standard sample having the same target sequence as a DNA fragment having a target sequence having a specific copy number can be produced.

It is explanatory drawing of the standard sample manufacturing method in one Example of this invention. It is explanatory drawing of copy number confirmation of the transgene in one Example of this invention. It is explanatory drawing of the synchronization confirmation of G0 / G1 period in one Example of this invention. It is explanatory drawing of the collection method of 1 cell by the manipulator in one Example of this invention. It is explanatory drawing of confirmation of the standard sample produced in one Example of this invention.

  Hereinafter, a standard sample manufacturing method according to an embodiment of the present invention will be described with reference to the drawings. The standard sample manufacturing method of the present embodiment is used for genetic testing to detect nucleic acids such as DNA and RNA, and is used for food testing, medical diagnosis, and the like. In addition, although the standard sample manufacturing method of embodiment of this invention is realizable with the following structures and methods, it is not restricted only to the structure and method shown here.

  According to the present embodiment, by combining introduction of a target gene into a cell and sorting of the cell, a standard sample reflecting the modification in the cell and the contents of the cell can be produced. In this case, a standard sample is prepared by introducing a DNA fragment having a gene sequence having a predetermined copy number into a cell. The cells to be used may be any cells as long as they can be cultured and introduced by gene and can control the cell cycle. However, in the case of a diploid, the target molecule is usually a diploid at a minimum. Therefore, in order to obtain a target of one molecule, the time of the haploid is in the stage that can be synchronized in the cell cycle. Some cells need to be used. The cells can be selected according to the purpose of use of the standard sample, such as those close to the biological species of the real specimen or cells such as yeast that are easy to handle. As for the culture medium for culturing cells, a medium suitable for the growth of the target cells can be selected.

In the present embodiment, the detection target for constituting the standard sample can be selected according to the use of the standard sample. The gene sequence to be treated as a target need not be single, and a nucleic acid fragment in which a plurality of gene sequences are linked in tandem can be used. In consideration of introduction into the genome of the cell, even sequence of homologous recombination of the target, it is necessary to linked in tandem, can be used artificial synthetic gene consisting of these elements. Furthermore, these may be those obtained by amplifying gene sequences existing in nature using the PCR method, or by cutting and purifying them using restriction enzymes.

  Whether or not the target of homologous recombination affects the copy number and growth in the genome for the production of recombinant cells by homologous recombination using the target gene and nucleic acid having the target gene sequence for homologous recombination It is desirable to select in consideration of Introduction of the nucleic acid into cells, that is, transformation can be performed using a commercially available reagent. In some cases, an electrical method such as an electroporation method may be used.

  Synchronization that aligns the cell cycle can be selected to suit the cell. For example, a low serum culture method, a double thymidine synchronization method, and synchronization with a tubulin polymerization inhibitor can be considered. Depending on the cell, a method specific to the cell can be used. For example, in the case of budding yeast, it is possible to synchronize with a sex hormone (pheromone) called α-factor.

  Single cell sorting can be performed with a single cell sorter (for example, PERFLOW (registered trademark) Sort: Furukawa Electric) or a micromanipulator. In the case of yeast, using a stereomicroscope, it is sucked into the tip of a capillary (ID = 10-20 μm), discharged into a culture medium for synchronous culture previously dropped on a slide glass, and together with one-cell yeast with a micropipette. It can be carried out by moving the droplet to the well of the microplate.

In this way, the work made samples are living a cell, includes the detection target of a desired number of molecules in it, the cells from one molecule to the number of molecules of the desired number, the gene-related testing It is possible to sort and provide in a form close to that of a real sample.

According to this embodiment, instead of sorting and measuring the number of molecules of a synthetic nucleic acid as in the past, one copy is introduced into a cell, and after confirming that it is one copy, the cell cycle is determined. It stopped, the cells were collected by desired several minutes to create made a standard sample by using the technique of simultaneously defining the number of molecules by defining the number of cells. Therefore, it is possible to stably supply a standard substance containing a very small amount of nucleic acid close to the state of a real sample, which has been impossible until now. If such a single molecule standard substance is put into practical use, it becomes possible to evaluate the detection ability of a commercially available PCR apparatus using a specimen. Moreover, in the PCR test, an absolute evaluation of the lower limit of detection and the lower limit of quantification in an environment close to the case where an actual sample is used can be performed.

  Hereinafter, the present invention will be described in detail by way of examples. However, these examples are for illustrative purposes, and the technical scope of the present invention is not limited thereto.

  In this example, a standard sample was prepared using budding yeast as an example of cells. Saccharomyces cerevisiae w303-1a (MATa, ade2-1 ura3-1 his3-11, 15 trp1-1 leu2-3, 112 can1-100) was used as a carrier cell for one copy of the target DNA sequence to produce a recombinant. (See FIG. 1). As the medium, YDP medium: 1% (w / w) bacto yeast extract, 2% (w / w) bactopeptone, 2% (w / w) glucose was used.

  An artificial gene was constructed as a model for detection. The target sequence was created by Over lapping PCR so that the homologous regions of the zeocin resistance gene, GFP gene and YER053C were arranged in tandem, and an artificial gene for homologous recombination was synthesized (see FIG. 1).

  For the production of a yeast recombinant by homologous recombination using an artificially synthesized gene, the gene of Mitochondrial phosphate transporter like protein (YER053C) was used as a target for homologous recombination. It has already been clarified that YER053C is a single copy in budding yeast, and that gene disruption does not affect the growth of the yeast (Takabatake, R., et al. J. Biochem., 129). (5): 827-33, 2001). As a target for introducing the YER053C locus into the yeast chromosome by homologous recombination, a one-copy target DNA sequence was introduced into the yeast chromosome (see FIG. 2).

  In order to prevent DNA from replicating during cell division, yeast cells were synchronized. Yeast cells cultured overnight were diluted to OD600 = 0.1 and cultured at 28 ° C. When OD600 = 0.3, α-factor (Sigma-Aldrich, St. Louis, Mo, USA) was added to 5 μg / ml, and the culture was further continued for 3 hours. The yeast was in G0 / G1 phase. Tuned to. Glycerol was added to the culture solution of cell cycle synchronized yeast to a final concentration of 20% and stored at −80 ° C.

  Confirmation of the cell cycle of synchronized cells was performed as follows. Yeast was stained with SYTOX Green Nucleic Acid Stain (Invitrogen-Molecular Probes, Eugene, OR) and analyzed by flow cytometry. Approximately 5 × 10 6 yeast cells collected were washed once with MilliQ water, suspended in 400 μl of MilliQ water, and then 950 μl of ethanol was added. The yeast fixed overnight at −20 ° C. was washed with 800 μl of 50 mM sodium citrate (pH 7.2), and 500 μl of 0.5 mg / ml RNase A (Nippon Gene, Tokyo, Japan) / 50 mM sodium citrate (pH 7. Suspended in 2) and incubated at 37 ° C. for 2 hours. Thereafter, 50 μl of 20 mg / ml proteinase K was added and incubated at 50 ° C. for 2 hours. The treated yeast was ultrasonically dispersed for 10 seconds, 6 μl of 5 mM SYTOX Green Nucleic Acid Stain was added, and the mixture was stained for 30 minutes in the dark. Using cell analyzer EC800 (Sony Biotechnology Inc. Tokyo, Japan), the cell cycle was analyzed at an excitation wavelength of 488 nm, and it was confirmed that the cell was synchronized with the G0 / G1 phase (see FIG. 3).

In order to sort one yeast cell, the following method was used. That is, yeast synchronized with the G0 / G1 phase was suspended in 1/15 M PBS (pH 7.0) and ultrasonically dispersed for 10 seconds. After washing twice with 1/15 M PBS, it was centrifuged at 4000 g for 5 minutes, and suspended in 5 mL of 5 μg / ml α-factor / 1/15 M PBS. The cell suspension was poured into a petri dish (50 × 9 mm BD Falcon Petri dishes, BD Bioscience, Franklin Lakes, NJ, USA), and was observed with a stereomicroscope (M205 FA, Leica). ID = 15 μm) and discharged onto 18 μl of 5 μg / ml α-factor / H ₂ 0 previously dropped on a slide glass, and the droplet was transferred to a well of a microplate together with 1-cell yeast with a micropipette. . In this case, it is desirable that the capillary tip angle is set within an angle range of 20 degrees to 30 degrees (see FIG. 4). This microplate becomes a standard sample.

The standard sample was confirmed by amplifying the target sequence from one cell by real-time PCR. 620 ng Zymolase T100 (105 kU / g, Nakalai Tesque, Kyoto, Japan) was added per well and incubated at 35 ° C. for 90 minutes to decompose the cell wall. Using Universal Master Mix (ABI), 16.7 × 10 ⁻⁶ μmol primer and 6.5 × 10 ⁻⁶ μmol FAM / TAMRA TaqMan probe were added, and PCR was performed with an ABI PRISM 7900HT real-time PCR system. PCR conditions are 50 ° C. for 2 minutes, 95 ° C. for 30 seconds for one cycle, 95 ° C. for 30 seconds, and 59 ° C. for 60 seconds for 50 cycles. PCR was performed using a standard plasmid pTRPWRKY diluted with a 5 ng / μl ColE1 (Nippon Gene Co., Tokyo, Japan) solution as a template and used to prepare a calibration curve. Thereby, a calibration curve could be created and the requirements as a standard sample were confirmed (see FIG. 5).

    As described in detail above, by using this configuration, a standard capable of sorting and providing the number of molecules from one molecule to a desired number of molecules in a form close to that of an actual sample of a gene-related test. The sample was shown to be feasible.

  The embodiments of the present invention have been described above by way of example, but the scope of the present invention is not limited to these embodiments, and can be changed or modified according to the purpose within the scope of the claims. is there.

  As described above, the method for producing a standard sample according to the present invention has an effect that a standard sample having the same target sequence as a DNA fragment having a target sequence having a specific copy number can be manufactured, for food inspection and medical diagnosis. It is useful for genetic testing of

1 Cells into which the target sequence has been introduced (yeast)
2 Yeast chromosome 3 Region into which the target sequence has been introduced 4 Culture vessel (petri dish)
5 Medium 6 Manipulator (capillary)
7 Collection container (micro tube)
8 Solution

Claims (5)

A method for producing a standard sample having the same target sequence as a DNA fragment having a specific copy number of the target sequence,
Introducing a DNA fragment having the specific sequence of the copy number into a chromosome of a cell capable of controlling the cell cycle ;
A culture step of culturing the cells in synchronism with the G0 / G1 phase ;
An isolation step of fixing and isolating the cultured cells in the G1 phase ;
A standard sample manufacturing method comprising: Wherein the introduction step, using budding yeast as the cells, by introducing the DNA fragment into the chromosome of the S. cerevisiae by homologous Recombination example using artificial synthetic gene,
In the culturing step, the budding yeast is synchronized with the G0 / G1 phase to perform synchronized culture,
The standard according to claim 1, wherein in the isolation step, the budding yeast is fixed in the G1 phase for isolation, and one cell of the isolated budding yeast contains one copy of the target sequence. Sample manufacturing method. Using gene of YER053C as the homologous Recombination example of a target, the standard sample preparation method according to claim 2.   The isolation step according to any one of claims 1 to 3, wherein in the isolation step, one cell is sorted per well using a capillary whose tip angle is set within an angle range of 20 degrees to 30 degrees. Standard sample manufacturing method.   The standard sample manufacturing method according to any one of claims 1 to 4, wherein the standard sample is a nucleic acid, and the nucleic acid includes DNA or RNA.