NUCLEIC ACID QUANTIFICATION METHOD USING STABLE ISOTOPE-LABELLED NUCLEIC ACID AS INTERNAL STANDARD AND USE OF THE SAME

Abstract

In order to quantitatively analyze nucleic acids present in a sample or a complex medium, a nucleic acid extraction or purification process is required. However, the yield of nucleic acid extraction and purification is greatly variable depending on the purification principle and the characteristics of kit and sample used. Hence, efficient normalization of nucleic acid extraction and purification yield is a prerequisite for accurate quantitative analysis of nucleic acid based on the original sample. The present invention relates to a quantitative analysis method of a nucleic acid present in a sample or a complex medium without amplification of a target nucleic acid.

Claims

1. A quantitative analysis method of a nucleic acid comprising: 1) preparing a nucleic acid (SILD) substituted with stable isotopes of .sup.13C and/or .sup.15N; 2) adding the substituted nucleic acid (SILD) as an internal standard to an analyte sample and a control sample in the same amount; 3) obtaining a nucleic acid from the analyte sample and a nucleic acid from the control sample; 4) hydrolyzing the nucleic acids obtained in the step 3) to a single nucleoside level; 5) attaining detection values of a normal nucleoside and a nucleoside derived from the substituted nucleic acid (SILD) from the nucleosides obtained in the step 4) in mass spectrometric analysis; and 6) normalizing an amount of the nucleic acid in the analyte sample by utilizing a characteristic that the detection value of the nucleoside derived from the substituted nucleic acid (SILD) is the same in the analyte sample and the control sample.

2. The quantitative analysis method of a nucleic acid according to claim 1, wherein the nucleic acid is DNA or RNA, and the sample is at least one or more of whole blood, plasma, serum, urine, saliva, sweat, milk, animal extract, plant extract, cell extract, cell culture, drinking water, service water, sewage, river water, or seawater.

3. The quantitative analysis method of a nucleic acid according to claim 2, wherein the nucleic acid is DNA and the sample is serum.

4. The quantitative analysis method of a nucleic acid according to claim 1, wherein the substituted nucleic acid (SILD) is derived from one of Escherichia coli, a human, a mouse, yeast, a plant, a fruit fly, or Caenorhabditis elegans.

5. The quantitative analysis method of a nucleic acid according to claim 4, wherein the substituted nucleic acid (SILD) is derived from Escherichia coli.

6. The quantitative analysis method of a nucleic acid according to claim 1, wherein the obtaining step is extraction and purification.

7. The quantitative analysis method of a nucleic acid according to claim 1, wherein the hydrolysis is at least one or more of an enzymatic reaction, an acid treatment, a heat treatment, a radiation treatment, or an ultrasonic treatment.

8. The quantitative analysis method of a nucleic acid according to claim 1, wherein the single nucleoside level is that 99.5% (by weight) or more of an entire nucleic acid is hydrolyzed to a single nucleoside.

9. The quantitative analysis method of a nucleic acid according to claim 1, wherein the normalization step is calculation by the following equation:
nucleic acid (analyte)=detection value of nucleic acid (analyte)nucleic acid (control)/detection value of nucleic acid (control)detection value of SILD (control)/detection value of SILD (analyte) where the nucleic acid (analyte) denotes an amount of a nucleic acid in an analyte sample, the detection value of nucleic acid (analyte) denotes a detection value of a nucleic acid in an analyte sample in mass spectrometric analysis, the nucleic acid (control) denotes an amount of a nucleic acid in a control sample, the detection value of nucleic acid (control) denotes a detection value of a nucleic acid in a control sample in mass spectrometric analysis, the detection value of SILD (control) denotes a detection value of a substituted nucleic acid (SILD) in a control sample in mass spectrometric analysis, and the detection value of SILD (analyte) denotes a detection value of a substituted nucleic acid (SILD) in an analyte sample in mass spectrometric analysis.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1 is a schematic diagram illustrating a process for quantifying a nucleic acid in a medium by using SILD as an internal standard. denotes a stable isotope-labelled substance.

[0032] FIG. 2 is the mass spectrometric analysis results for Escherichia coli genomic DNA produced to use SILD as an internal standard.

[0033] FIG. 3 is a diagram illustrating the results attained by normalizing the DNA extraction and purification efficiency by using SILD as an internal standard.

[0034] FIG. 4 is the results attained by measuring the amount of free nucleic acids (cell free DNA) in human serum by using SILD as an internal standard.

DESCRIPTION OF EMBODIMENTS

[0035] The present invention is a quantification method of a nucleic acid in a medium including 1) adding SILD as an internal standard to an analyte sample and a comparison target sample (control or standard) in the same amount, 2) extracting or purifying a nucleic acid from each sample, 3) hydrolyzing the purified nucleic acid to a single nucleoside level through an enzymatic reaction, 4) separating, detecting, and quantifying each nucleoside and a stable isotope-substituted nucleoside by liquid chromatography-mass spectrometry (LC-MS), and 5) normalizing a difference in efficiency of the whole steps by utilizing a signal value of the internal standard and quantitatively calculating an amount of the nucleic acid in the analyte sample.

[0036] FIG. 1 is a schematic diagram illustrating a process for quantifying a nucleic acid in a medium by using SILD as an internal standard. SILD is added to the analyte sample and the comparison target sample (or standard sample) in the same amount, and then the two samples are sequentially subjected to extraction and purification, hydrolysis by enzymatic reaction, and mass spectrometric analysis. Finally, the signal value of SILD which has been added to the two samples in the same amount in the mass spectrometric analysis results is a measure of the overall reaction efficiency and yield for the two samples. The absolute or relative amount of a nucleic acid in a medium can be calculated by the formula depicted in the figure, exactly the equation described below.

Nucleic acid (analyte)=detection value of nucleic acid (analyte)nucleic acid (control)/detection value of nucleic acid (control)detection value of SILD (control)/detection value of SILD (analyte)

[0037] Here, the nucleic acid (analyte) denotes the amount of nucleic acid in the analyte sample, the detection value of nucleic acid (analyte) denotes the detection value of nucleic acid in the analyte sample in mass spectrometric analysis, the nucleic acid (control) denotes the amount of nucleic acid in the control sample, the detection value of nucleic acid (control) denotes the detection value of nucleic acid in the control sample in mass spectrometric analysis, the detection value of SILD (control) denotes the detection value of substituted nucleic acid (SILD) in the control sample in mass spectrometric analysis, and the detection value of SILD (analyte) denotes the detection value of substituted nucleic acid (SILD) in the analyte sample in mass spectrometric analysis.

[0038] In order to implement the present invention, it is first required to produce SILD.

[0039] 1. Production and Verification of SILD

[0040] The production of SILD was conducted according to the method described in the reference (Appl Microbiol Biotechnol (2010) 88: 771-779). Briefly, (NH.sub.4).sub.2SO.sub.4 substituted with .sup.15N was used in the composition of the LMR medium (176 mM KH.sub.2PO.sub.4, 25 mM NaOH, 10 l H.sub.2SO.sub.4, 12.6 mM (NH.sub.4).sub.2SO.sub.4, 2 mM MgSO.sub.4, 10 micromole FeSO.sub.4, 0.2% trace metal solution) composed only of essential inorganic elements (Cambridge Isotope Laboratory), and a medium to which glycerol substituted with 0.2% of .sup.13C as a carbon source was used. As Escherichia coli, a standard strain KCTC11 was used. Genomic DNA extraction from Escherichia coli cultured in stable isotope medium was conducted using Genelute Bacterial genomic DNA kit (Sigma-Aldrich). In order to verify that the extracted genomic DNA is favorably labelled with stable isotopes, about 500 ng of DNA was hydrolyzed to a nucleoside (dNMP) level using DNase I (Takara) and Phosphodiesterase I (Affymetrics) and each nucleoside was detected using LC-Quadrupole-TOF (AB SCIEX 5600) mass spectrometer (see FIG. 2).

[0041] As can be seen from FIG. 2, the difference in molecular weight between Escherichia coli DNA cultured in a normal medium and Escherichia coli DNA cultured in a stable isotope medium is 12 in the case of dCMP and TMP and is 15 in the case of dAMP and dGMP. This difference corresponds to the difference based on the assumption that both carbon and nitrogen in each nucleoside are substituted. In addition, normal nucleosides having a small molecular weight are not detected in DNA cultured in a stable isotope medium. Hence, according to the method implemented in the present invention, it has been verified that Escherichia coli DNA is labelled with stable isotopes at a level close to 100%.

[0042] 2. Normalization of Extraction and Purification Efficiency of DNA in Medium and Quantitative Analysis of DNA

[0043] In order to verify that the extraction and purification efficiency of DNA in a medium is properly normalized when stable isotope-labelled Escherichia coli DNA is added as an internal standard, a buffer (hGH buffer: 2.25% mannitol, 0.5% glycine, 0.15% sodium phosphate, 5 mg/mL bovine serum albumin) for protein drug storage was selected as a representative medium. DNA as an analyte sample was added to the hGH buffer in a known amount of 100 ng and SILD as an internal standard was added to the hGH buffer in an amount corresponding to about 100 ng. The same amount of SILD was also added to human placental DNA and dNMP samples with values already known as standards for quantitation. The SILD-added samples were extracted and purified using four different kinds of kits of PCR purification kit (QPK, Qiagen), QiaAmp DNA Blood mini kit (QBD, Qiagen), Serum/plasma cell free DNA midi kit (Sigma, Sigma-Aldrich), and QiaAmp circulating nucleic acid kit (QC, Qiagen) (FIG. 3). DNA was hydrolyzed to a nucleotide (dNMP) level using DNase I (Takara) and Phosphodiesterase I (Affymetrics) and further hydrolyzed to nucleoside (dN) using Shrimp alkaline phosphatase (Takara). The four kinds of hydrolyzed nucleosides were quantitatively analyzed using LC-Quadrupole-TOF (AB SCIEX 5600) mass spectrometer. The amount of DNA in the medium was calculated by applying the following equation based on the peak areas of normal nucleosides and SILD-derived nucleosides calculated from each purified sample. The following equation can be applied only when the analyte sample and the internal standard were used in the same amount of 100 ng.

DNA (sample)=(DNA (standard)SILD (standard)/SILD (sample)

[0044] The quantification results for nucleic acid before and after the normalization for every kit are compared with each other in FIG. 3. The quantification results attained without normalization using SILD show quantitative values to be 20% to 70% of the initial reference values depending on the kit. On the other hand, quantitative values to be 90% to 105% of the reference values were attained in the results attained by conducting normalization of purification and hydrolysis reaction using SILD proposed in the present invention. These results indicate that the accuracy of quantification can be dramatically increased by using a SILD internal standard in the quantitative analysis of nucleic acids in a medium. In addition, it has been verified that the use of SILD internal standard enables highly accurate nucleic acid quantification even when the nucleic acid purification process is omitted.

[0045] As can be seen from FIG. 3, the peak area of nucleoside is about 50% of the reference value when normalization is not conducted, and thus it can be seen that the efficiency of enzymatic hydrolysis and the ionization efficiency in mass spectrometric analysis are lower than those in the case of purified nucleic acids. It is interpreted that the efficiency is decreased because the impediments contained in the hGH buffer have not been removed by purification. However, when SILD is used, even such low hydrolysis and ionization efficiencies are normalized, thus the final nucleic acid quantification value is 101.5% of the reference value, and significantly accurate quantification is possible. Based on these observation results, it can be concluded that the amount of nucleic acid in a medium can be measured significantly accurately regardless of the kind of extraction and purification kit and further even when purification is not conducted when SILD is used as an internal standard.

[0046] 3. Quantitative Analysis of Free DNA in Human Serum

[0047] In order to verify that the extraction and purification efficiency of DNA in a medium is properly normalized when stable isotope-labelled Escherichia coli DNA as an internal standard is added to the medium, free DNA in human serum was quantified. About 50 ng of SILD was added to 0.5 ml of each of 16 human serum samples prior to the purification of free DNA in serum. In addition, SILD as an internal standard was also added to a dNMP standard mixture with a known amount (four individual nucleotides each having a concentration of 20 ng/mL) as a reference for quantification in the same amount as the above.

[0048] The SILD-added samples were subjected to DNA extraction using Circulating cell free DNA purification kit (Qiagen). The extracted DNA was subjected to hydrolysis in the same manner as described above and then quantitatively analyzed by LC-MS. The results are illustrated in FIG. 4. The range of the measured values illustrated in FIG. 4 is 50 to 500 ng/ml, and these values are significantly higher than 20 to 100 ng/ml generally calculated in experiments in which DNA extraction and purification efficiency is not normalized. Considering that the purification efficiency of the kit used for DNA purification is about 40%, it is judged that a measured value as high as about two times is attained since the measurement method used in the present invention completely normalizes the purification efficiency. Through the results described above, it has been verified that the measurement method of DNA in a medium using a stable isotope-labelled DNA as an internal standard, which is proposed in the present invention is a method which enables accurate quantification by collectively normalizing the purification efficiency of DNA, the efficiency of enzymatic hydrolysis, and the variability in LC-MS.

INDUSTRIAL APPLICABILITY

[0049] The present invention is to normalize the difference in yield occurring in the extraction and purification process of a nucleic acid in a medium by using SILD as an internal standard. In addition, the added internal standard also normalizes the efficiency of enzymatic reaction after purification and mass spectrometric analysis. For example, when some impurities remain, the efficiency of enzymatic reaction or the ionization efficiency in mass spectrometric analysis may change, and the interference effect received by the analyte can be normalized using the signal value ratio of the internal standard since the internal standard also receives this effect to the same extent. Overall, the use of SILD as an internal standard makes it possible to improve the accuracy of quantitative analysis of nucleic acids in a medium by normalizing the efficiency of all the procedures and reactions conducted to quantitatively analyze nucleic acids in a medium sample.