Long fragment DNA digestion-free size exclusion chromatography-inductively coupled plasma mass spectrometry detection method

Abstract

The application belongs to the technical field of plasma mass spectrometry, and particularly relates to a long fragment DNA digestion-free size exclusion chromatography-inductively coupled plasma mass spectrometry detection method. A long fragment DNA sample purified through gel electrophoresis is cyclically concentrated and purified through a 50kDa molecular weight cutoff ultrafiltration centrifuge tube to remove short fragment oligonucleotide impurities. A Tris-HCl-EDTA buffer mobile phase is prepared, and the purified long fragment DNA is chromatographically separated through an isocratic elution mode. Chromatographic effluent components are directly introduced into an inductively coupled plasma mass spectrometer, and a time-resolved analysis mode is used to collect phosphorus element mass spectrometry signals. A series of inorganic phosphorus standard solutions are prepared, and a linear standard curve is fitted with the phosphorus concentration as the abscissa and the mass spectrometry peak area as the ordinate. The phosphorus element content in the sample is inversely deduced based on the standard curve, and the long fragment DNA concentration is calculated according to the fixed stoichiometric ratio of the DNA phosphorus element, and the DNA does not need to be digested throughout the whole process.

Description

Technical Field

[0001] This invention belongs to the field of plasma mass spectrometry detection technology, and particularly relates to a long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method without digestion. Background Technology

[0002] Traditional methods for quantifying long DNA fragments often rely on ultraviolet spectrophotometry, fluorescent dye quantification, and digestion-ICP-MS. Ultraviolet and fluorescent detection are easily interfered with by impurities such as short oligonucleotides, proteins, and salt ions. Short nucleic acid impurities can significantly increase the detection value, making it impossible to accurately distinguish between intact long fragments and degraded fragments. Conventional digestion-coupled ICP-MS technology requires strong acid and microwave high-temperature digestion to destroy the DNA molecular structure in order to release phosphorus. The digestion process is time-consuming and the operation procedure is complex. The strong oxidative digestion system can also cause long DNA fragmentation and loss. It is not suitable for quantitative analysis scenarios that require the preservation of the intact DNA macromolecular structure and cannot meet the requirements of long-fragment sequencing, gene vector construction, and other experiments that require accurate quantification of intact DNA.

[0003] Size exclusion chromatography (SEC) can separate nucleic acid fragments of different lengths based on molecular size. However, most existing SEC-mass spectrometry detection schemes still require digestion pretreatment, lacking an integrated digestion-free detection system suitable for long DNA fragments. At the same time, conventional purification methods can only roughly remove large molecular impurities and cannot efficiently remove short oligonucleotides below 750bp. Trace amounts of short fragment impurities will continue to interfere with phosphorus signal acquisition, leading to a decrease in the repeatability and accuracy of long DNA quantification. Currently, there is still a lack of an integrated digestion-free quantitative detection scheme for long DNA fragments that combines efficient impurity removal, non-destructive injection, high linearity, and low detection bias. Summary of the Invention

[0004] In view of the aforementioned problems, and in conjunction with the first aspect of the present invention, embodiments of the present invention provide a method for detecting long DNA fragments without digestion, using size exclusion chromatography-inductively coupled plasma mass spectrometry, the method comprising: Step 1: The long DNA fragments purified by gel electrophoresis were cyclically concentrated and purified using ultrafiltration centrifuge tubes with a molecular weight cutoff of 50 kDa to remove short oligonucleotide impurities. Step 2: Prepare a Tris-HCl-EDTA buffer mobile phase and perform chromatographic separation of the purified long DNA fragments using isocratic elution mode; Step 3: The chromatographic eluent is directly introduced into an inductively coupled plasma mass spectrometer, and the phosphorus mass spectrum signal is acquired using time-resolved analysis mode; Step 4: Prepare a series of inorganic phosphorus standard solutions, and fit a linear standard curve with phosphorus concentration as the x-axis and mass spectrum peak area as the y-axis; Step 5: Based on the standard curve, infer the phosphorus content in the sample, and calculate the concentration of long-fragment DNA according to the fixed stoichiometric ratio of phosphorus in DNA. No DNA digestion is required throughout the process.

[0005] Preferably, in step one, the initial concentration of the DNA sample purified by gel electrophoresis is 150 ng / μL, the single processing volume is 15 mL, and the single concentration target volume is 10 mL; the ultrafiltration centrifugation parameters are 5500×g and centrifugation for 15-20 min. After concentrating to 1 mL, add ultrapure water to make up to 10 mL, and repeat the concentration and purification twice.

[0006] Preferably, in step one, the ultrafiltration purification can remove short nucleic acid fragments of 750bp and below. After secondary purification, the proportion of oligonucleotide impurities in 2000bp long DNA fragments is reduced to 0.88%, and the proportion of oligonucleotide impurities in 4000bp long DNA fragments is reduced to 0.81%.

[0007] Preferably, in step two, the mobile phase is a 5mM Tris-HCl + 1mM EDTA mixed solution, and the pH is adjusted to 7.2 with ammonia. The chromatographic flow rate was 0.8 mL / min, and the injection volume was 2 μL.

[0008] Preferably, in step three, the ICP-MS instrument parameters are as follows: RF power 1550W, RF voltage 1.80V, sampling depth 8.0mm, nebulizer gas flow rate 0.8L / min, quadrupole vacuum degree 0.000847Pa, nebulizer chamber temperature 2.0℃, carrier gas flow rate 0.8L / min, feedback power 12W, peristaltic pump speed 0.4rps, and integration time 1.00s.

[0009] Preferably, in step four, the concentration gradient of the inorganic phosphorus standard solution is 1 ppm, 2 ppm, 5 ppm, 10 ppm, and 20 ppm, the standard curve regression equation is y = 45859x - 70.273, and the linear correlation coefficient R² ≥ 0.9999.

[0010] Preferably, in step five, the long DNA fragment to be detected is a 2000bp fragment or a 4000bp fragment; the RSD of six parallel tests of 2000bp DNA is ≤0.19%, and the RSD of six parallel tests of 4000bp DNA is ≤1.21%.

[0011] Preferably, the method also includes a chromatography column linearity verification step: using gradient-valued long fragment DNA sample injection detection, fitting a linear curve with DNA concentration as the abscissa and phosphorus signal peak area as the ordinate, the linear equation for the 2kb fragment is y=60273x+28127, R²=0.9993; the linear equation for the 4kb fragment is y=49736x+30647, R²=0.9992.

[0012] Preferably, the sample short-term stability verification step is also included: after purification, the DNA sample is stored at 4°C in the dark for 0-14 days, and samples are taken at intervals for testing. The RSD of 2000bp DNA is ≤0.84%, and the RSD of 4000bp DNA is ≤2.61%.

[0013] Preferably, the long DNA fragment is present in its polymerized macromolecular form and is directly detected by ICP-MS via SEC, without the need for pretreatment such as acid digestion or microwave digestion to destroy the DNA molecular structure.

[0014] Based on the above, short oligonucleotide impurities of 750bp and below can be efficiently removed by cyclic concentration and purification using 50kDa ultrafiltration centrifuge tubes. After two cycles of purification, the proportion of oligonucleotide impurities in 2000bp and 4000bp long DNA fragments is reduced to 0.88% and 0.81%, respectively, significantly eliminating the interference of short nucleic acid fragments on phosphorus signals. The entire detection process does not require acid digestion or microwave digestion. Long DNA fragments are directly introduced into ICP-MS after separation by SEC in their complete polymerized macromolecular form, avoiding the problems of DNA breakage and target analyte loss caused by digestion steps. Quantification is completed by relying on the inorganic phosphorus standard curve and the stoichiometry of DNA-fixed phosphorus. The RSD of six parallel detections of 2000bp is as low as 0.19%, and the RSD of parallel detection of 4000bp is only 1.21%, demonstrating excellent detection precision.

[0015] The linear range of the chromatography column established in this method is adapted to the detection of 2kb and 4kb long DNA fragments. The correlation coefficients of the two linear curves are both higher than 0.999, indicating a good linear relationship. The purified DNA samples can be stably stored for 14 days under light-protected conditions at 4℃. The RSD of the stability detection of 2000bp and 4000bp samples is only 0.84% ​​and 2.61%, respectively, with no significant shift in detection results after short-term storage. The entire detection system has a simplified operation process and fewer pretreatment steps. The isocratic elution of chromatography combined with stable ICP-MS time-resolved acquisition parameters, the phosphorus standard curve R²≥0.9999, and the high quantitative accuracy, can be stably adapted to scientific research scenarios such as long-fragment sequencing and plasmid vector construction for the precise quantification of complete long DNA fragments. Attached Figure Description

[0016] Figure 1The original DNA fragment sample after alcohol precipitation is obtained from the long fragment DNA non-digestion size exclusion chromatography-inductively coupled plasma mass spectrometry detection method provided in this embodiment of the invention, and is analyzed by size exclusion coupled inductively coupled plasma mass spectrometry 2k spectrum. Figure 2 The original DNA fragment sample after alcohol precipitation is obtained from the long fragment DNA non-digestion size exclusion chromatography-inductively coupled plasma mass spectrometry detection method provided in this embodiment of the invention, and is analyzed by size exclusion coupled inductively coupled plasma mass spectrometry 4k spectrum. Figure 3 The sample purified by ultrafiltration is obtained by the long fragment DNA non-digestion size exclusion chromatography-inductively coupled plasma mass spectrometry detection method provided in the embodiments of the present invention, and is then detected by size exclusion coupled inductively coupled plasma mass spectrometry. Figure 4 The sample purified by ultrafiltration is obtained by the long fragment DNA non-digestion size exclusion chromatography-inductively coupled plasma mass spectrometry detection method provided in the embodiments of the present invention, and is then detected by size exclusion coupled inductively coupled plasma mass spectrometry. Figure 5 This refers to the peak area of ​​the long DNA fragment detection method provided in this embodiment of the invention, which involves size exclusion chromatography-inductively coupled plasma mass spectrometry (ICP-MS). Figure 6 This refers to the peak area of ​​the long DNA fragment detection method provided in this embodiment of the invention, which involves size exclusion chromatography-inductively coupled plasma mass spectrometry (ICP-MS). Figure 7 This is the inorganic phosphorus standard curve of the long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method provided in the embodiments of the present invention; Figure 8 The long DNA fragment detection method provided in this embodiment of the invention, which involves size exclusion chromatography-inductively coupled plasma mass spectrometry without digestion, allows the DNA fragments to directly enter the mass spectrometer in polymeric form through chromatography. Figure 9 This invention relates to a linear 2k fragment on a SEC-4000 column for long DNA fragment detection using size exclusion chromatography-inductively coupled plasma mass spectrometry (ICP-MS) without digestion, as provided in this embodiment. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings.

[0018] DNA sample concentration and purification; A nucleic acid fragment of approximately 750 bp was removed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 50 kDa.

[0019] Take 15 mL of DNA sample purified by gel electrophoresis (concentration 150 ng / μL) and set the concentration target volume to 10 mL.

[0020] Centrifugation conditions: 5500×g, 15-20min.

[0021] After each concentration to 1 mL, replenish with ultrapure water to a final volume of 10 mL. Repeat the concentration process twice to complete the purification.

[0022] Liquid chromatography conditions; Mobile phase: 5 mM Tris-HCl + 1 mM EDTA (pH 7.2, adjusted with ammonia).

[0023] Elution method: isocratic elution (constant salt concentration).

[0024] Flow rate: 0.8 mL / min.

[0025] Injection volume: 2 μL.

[0026] Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Parameter Settings; RF power: 1550W, RF voltage: 1.80V, sampling depth: 8.0mm, atomizing gas flow rate: 0.8L / min, quadrupole vacuum: 0.000847Pa, atomization chamber temperature: 2.0°C, carrier gas flow rate: 0.8L / min, feedback power: 12W, peristaltic pump speed: 0.4rps, data acquisition mode: TRA (time-resolved analysis), integration time: 1.00s.

[0027] Preparation of standard curves; Prepare inorganic phosphorus standard solutions (1, 2, 5, 10, 20 ppm) with an injection volume of 2 μL.

[0028] A standard curve was plotted with phosphorus concentration (ppm) on the x-axis and peak area of ​​the mass spectrometry signal on the y-axis. A linear equation was fitted and the regression coefficient (R²) was calculated. 2 ).

[0029] Example 1; 2kb DNA fragment molecular weight cutoff aids in purification; The original DNA fragment sample, after alcohol precipitation, was subjected to size exclusion coupled inductively coupled plasma mass spectrometry (ICP-MS) as follows: Figure 1 and 2 As shown Figure 1 For 2k, Figure 2 It is 4k.

[0030] like Figure 3 and Figure 4 The sample purified by ultrafiltration was then analyzed by size exclusion coupled inductively coupled plasma mass spectrometry, as shown in the figure below.

[0031] like Figure 5 and 6As shown, secondary purification further reduces the impurity content. Under these conditions, the purity is further improved, and direct quantification is performed based on the peak area of ​​the DNA fragment.

[0032] Table 1 shows the quantitative characterization of the purification effect;

[0033] Example 2 Preparation of the standard curve for phosphorus; Using a 1000 ppm inorganic phosphorus standard solution as the stock solution, inorganic phosphorus solutions of five concentrations (20 ppm, 10 ppm, 5 ppm, 2 ppm, and 1 ppm) were prepared by gravimetric method. Each solution was measured three times, and the average peak area was calculated. The regression equation and standard curve were obtained by using linear regression with inorganic phosphorus concentration as the x-axis and peak area as the y-axis.

[0034] As shown in Table 1;

[0035] Example 3; Determination of fragment DNA content; The DNA fragments were purified using the protocol described in Example 1. After purification, no digestion was required. The DNA fragment samples were directly passed through size exclusion chromatography followed by mass spectrometry, and then identified by inductively coupled plasma mass spectrometry. The fragment lengths were 2000 bp and 4000 bp, and the identification results are shown in the figure below. The DNA fragments entered the mass spectrometer directly in polymeric form. This process is significantly different from digestion into phosphorus before injection.

[0036] The regression equation and standard curve obtained by the above method can be used to calculate the content of inorganic phosphorus in the DNA macromolecule. Based on the stoichiometric ratio of phosphorus fixed in DNA, the DNA concentration can be calculated.

[0037] As shown in Table 2;

[0038] Example 4; Verification of linear separation by chromatography column; DNA fragments were tested using a series of samples of the same type at different concentrations. Each injection was repeated three times and the average value was taken. The original DNA fragments with pre-determined concentrations were diluted by weighing to obtain accurate concentration values. The concentration was plotted on the x-axis and the peak area on the y-axis using linear regression analysis. The corresponding regression equation and correlation coefficient were fitted, and a relatively good fitting result was obtained, proving that the chromatography column used in this method has good linearity.

[0039] Example 5: Repeatability verification of the separation method; To verify the short-term stability of the isolated and purified DNA fragments, one sample was randomly selected and placed at 4°C for 0, 1, 3, 7, and 14 days. The DNA concentration was determined using the method described above. Each sample was measured three times, and the average value was calculated. The results are shown in Table 3 below.

[0040] According to the table above, the DNA fragments purified by this method have good short-term stability. The samples are stable at 4℃, and the RSDs after the test are 0.84% ​​(2000bp) and 2.61% (4000bp), which meet the requirements of short-term stability for scientific research.

[0041] It should be noted that, in order to simplify the description of the present invention and thus help to understand one or more embodiments of the invention, multiple features may sometimes be grouped into one embodiment, drawing or description thereof in the foregoing description of the embodiments of the present invention.

[0042] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A digestion-free size exclusion chromatography-inductively coupled plasma mass spectrometry method for detecting long DNA fragments, characterized by: Includes the following steps: Step 1: The long DNA fragments purified by gel electrophoresis were cyclically concentrated and purified using ultrafiltration centrifuge tubes with a molecular weight cutoff of 50 kDa to remove short oligonucleotide impurities. Step 2: Prepare a Tris-HCl-EDTA buffer mobile phase and perform chromatographic separation of the purified long DNA fragments using isocratic elution mode; Step 3: The chromatographic eluent is directly introduced into an inductively coupled plasma mass spectrometer, and the phosphorus mass spectrum signal is acquired using time-resolved analysis mode; Step 4: Prepare a series of inorganic phosphorus standard solutions, and fit a linear standard curve with phosphorus concentration as the x-axis and mass spectrum peak area as the y-axis; Step 5: Based on the standard curve, infer the phosphorus content in the sample, and calculate the concentration of long-fragment DNA according to the fixed stoichiometric ratio of phosphorus in DNA. No DNA digestion is required throughout the process.

2. The long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method without digestion according to claim 1, characterized in that: In step one, the initial concentration of the DNA sample purified by gel electrophoresis is 150 ng / μL, the single processing volume is 15 mL, and the single concentration target volume is 10 mL; the ultrafiltration centrifugation parameters are 5500×g and centrifugation for 15-20 min. After concentrating to 1 mL, add ultrapure water to make up to 10 mL, and repeat the concentration and purification twice.

3. The long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method without digestion according to claim 1, characterized in that: In step one, the ultrafiltration purification can remove short nucleic acid fragments of 750bp and below. After secondary purification, the proportion of oligonucleotide impurities in 2000bp long DNA fragments is reduced to 0.88%, and the proportion of oligonucleotide impurities in 4000bp long DNA fragments is reduced to 0.81%.

4. The long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method without digestion according to claim 2, characterized in that: In step two, the mobile phase is a mixed solution of 5 mM Tris-HCl and 1 mM EDTA, and the pH is adjusted to 7.2 with ammonia. The chromatographic flow rate was 0.8 mL / min, and the injection volume was 2 μL.

5. The long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method without digestion according to claim 1, characterized in that: In step three, the parameters of the ICP-MS instrument are as follows: RF power 1550W, RF voltage 1.80V, sampling depth 8.0mm, nebulizer gas flow rate 0.8L / min, quadrupole vacuum degree 0.000847Pa, nebulizer chamber temperature 2.0℃, carrier gas flow rate 0.8L / min, feedback power 12W, peristaltic pump speed 0.4rps, and integration time 1.00s.

6. The long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method without digestion according to claim 5, characterized in that: In step four, the concentration gradient of the inorganic phosphorus standard solution is 1 ppm, 2 ppm, 5 ppm, 10 ppm, and 20 ppm. The standard curve regression equation is y = 45859x - 70.273, and the linear correlation coefficient R² ≥ 0.9999.

7. The long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method without digestion according to claim 5, characterized in that: In step five, the long DNA fragment to be tested is either a 2000bp fragment or a 4000bp fragment; the RSD of six parallel tests for 2000bp DNA is ≤0.19%, and the RSD of six parallel tests for 4000bp DNA is ≤1.21%.

8. The long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method without digestion according to claim 1, characterized in that: It also includes a chromatography column linearity verification step: using gradient-valued long-fragment DNA sample injection detection, and fitting a linear curve with DNA concentration as the x-axis and phosphorus signal peak area as the y-axis. The linear equation for the 2kb fragment is y=60273x+28127, R²=0.9993; the linear equation for the 4kb fragment is y=49736x+30647, R²=0.9992.

9. The long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method without digestion according to claim 1, characterized in that: It also includes a short-term stability verification step for the samples: after purification, the DNA samples are stored at 4°C in the dark for 0-14 days, and samples are taken at intervals for testing. The RSD of 2000bp DNA is ≤0.84%, and the RSD of 4000bp DNA is ≤2.61%.

10. The long-fragment DNA size exclusion chromatography-inductively coupled plasma mass spectrometry detection method without digestion according to claim 1, characterized in that: The long DNA fragments are present in their polymerized macromolecular form and are directly detected by ICP-MS via SEC, without the need for pretreatment such as acid digestion or microwave digestion to destroy the DNA molecular structure.

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