CN109765289A - A kind of organic phosphorus and Phos method in quantitative detection DNA - Google Patents
A kind of organic phosphorus and Phos method in quantitative detection DNA Download PDFInfo
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- CN109765289A CN109765289A CN201910103264.2A CN201910103264A CN109765289A CN 109765289 A CN109765289 A CN 109765289A CN 201910103264 A CN201910103264 A CN 201910103264A CN 109765289 A CN109765289 A CN 109765289A
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Abstract
The present invention provides a kind of organic phosphorus in quantitative detection DNA and Phos method, separation and quantitative detection have been carried out to the organic phosphorus and Phos in DNA molecular by the separating capacity of combination HPLC and the quantitation capabilities of ICP-MS, optimize the conditional parameter of LC-MS, each each condition of step cooperates, organic phosphorus and Phos cannot be distinguished when solving the problems, such as common ICP-MS fixing phosphorus method to DNA accurate quantitative analysis, DNA fixing phosphorus method is greatly improved to quantify the accuracy of DNA concentration and ensured the magnitude tracing of DNA concentration to world SI unit, it has broad application prospects and huge market value.
Description
Technical field
The invention belongs to biochemistry crossing domain, it is related to organic phosphorus and inorganic in a kind of quantitative detection DNA
The method of phosphorus.
Background technique
DNA (DNA) is the organic compound of molecular structure complexity, as chromosome an ingredient and deposit
It is in nucleus, function is to store hereditary information, including oligonucleotide, long chain DNA, Plasmid DNA, genomic DNA etc.,
Genetic engineering field has wide and deep application.
Oligonucleotides is general name (including the DNA DNA of a kind of short nucleotide for there was only 20 or less bases
Or the nucleotide in Ribonucleic RNA), oligonucleotides can easily link with their complementary pair, so being commonly used to make
The structure that DNA or RNA are determined for probe is frequently used for during genetic chip, electrophoresis, fluorescence in situ hybridization etc.；Genomic DNA
It is made of the aggregate of biological all genes, is analyzed from the structure of cell, gene is present in nucleus, gene
Group DNA and histone combine closely to form chromatin；Plasmid DNA be free on extrachromosomal small-sized (1-200kb) it is covalent,
Closure, cricoid double chain DNA molecule can independently replicate and can stablize the gene of heredity.
DNA is quantitative to have extensive demand in molecular biology field, such as carries out digestion and connection when clone library building
When operating, and DNA being sequenced, good subsequent experimental is as a result, require to carry out accurately the concentration of DNA in order to obtain
Measurement.
Commonly quantitatively DNA method mainly has ultraviolet spectrophotometry (UV), fluorescent dye determination, qPCR
(Quantitative PCR), dPCR (Digital PCR), first two method need to be mentioned in terms of sensitivity and stability
It is high；Latter two method is there are the risk of false positive is larger, and for the above DNA quantitative approach, magnitude tracing problem is still
It is very big challenge.
With the development of elemental analysis method, fixing phosphorus method becomes a kind of feasible DNA quantitative detecting method, due to for spy
Fixed DNA molecular comes, and says, phosphorus content is a preset parameter, passes through the phosphorus content in detection DNA molecular, so that it may calculate
The sensitivity of the content of DNA molecular, this method is higher, can be traceable to international unit, and testing cost is lower.
Since DNA molecular length has from several hundred a bases to a bases thousands of or even up to ten thousand, usual molecular weight is very big,
Common performance liquid chromatographic column is difficult to separate DNA, and when common ICP-MS fixing phosphorus method quantifies DNA exists
Organic phosphorus and Phos problem cannot be distinguished.
Therefore, it researches and develops and a kind of efficiently separate organic phosphorus and Phos and realize to content of inorganic phosphorus quantitative detection in DNA
Method has broad application prospects and huge market value.
Summary of the invention
In view of the deficiencies of the prior art and actual demand, the present invention provides has in a kind of quantitative detection DNA
The method of machine phosphorus and Phos, by the separating capacity of combination HPLC and the quantitation capabilities of ICP-MS to organic in DNA molecular
Phosphorus and Phos have carried out separation and quantitative detection, and solving when common ICP-MS fixing phosphorus method quantifies DNA molecular cannot be distinguished
The problem of organic phosphorus and Phos, DNA molecular is quantified and is effectively connected to international SI unit, have broad application prospects with it is huge
Big market value.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides a kind of organic phosphorus in quantitative detection DNA and Phos method, institute
Method is stated to be combined using liquid chromatogram and inductivity coupled plasma mass spectrometry；
Wherein, the chromatographic column of the liquid chromatogram includes molecular-exclusion chromatography column.
In the present invention, inventor during long-term research practice, for the prior art fixing phosphorus method there are the shortcomings that, by
Have in DNA molecular length from several hundred a bases to a bases thousands of or even up to ten thousand, usual molecular weight is very big, and common is efficient
Liquid-phase chromatographic column is difficult to separate DNA, and by the suitable liquid-phase chromatographic column of extensive experiment screening, innovative uses molecule
Exclusion chromatography column divides DNA by the relativeness between the pore size of gel pore and the coil dimension of DNA molecular
From the P elements in DNA skeleton being separated with the P elements in inorganic salt solution, so that realizing has in DNA
The quantitative detection of machine phosphorus and content of inorganic phosphorus.
Preferably, the pore size of the molecular-exclusion chromatography column be 10nm-50nm, such as can be 10nm, 15nm,
20nm, 25nm, 30nm, 35nm, 40nm, 45nm or 50nm.
Technical solution provided by the invention can not only detect Plasmid DNA, real by adjusting the aperture of size exclusion chromatograph
Now DNA different types of to the overwhelming majority is detected.
The partial size of the filler of the molecular-exclusion chromatography column is 3 μm.
Preferably, the mobile phase of the liquid chromatogram includes common biological buffer, for example, phosphate buffer and/or
Tris buffer, preferably Tris buffer；
Preferably, the pH of the Tris buffer is 6.0-8.0, such as can be 6.0,6.5,7.0,7.4 or 8.0.
Preferably, the concentration of the Tris buffer be 5mM-100mM, such as can be 5mM, 10mM, 20mM, 30mM,
40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM.
Preferably, the Tris buffer the preparation method comprises the following steps: prepared using ultrapure water, and used membrane filtration；
Preferably, the aperture of the filter membrane is 0.22 μm.
Preferably, the resistivity of the ultrapure water is 18M Ω/cm.
Preferably, the flow velocity of the mobile phase be 0.5mL/min-2.0mL/min, such as can be 0.5mL/min,
0.9mL/min, 1.0mL/min, 1.5mL/min or 2.0mL/min.
Preferably, the sampling volume of the liquid chromatogram be 5 μ L-50 μ L, such as can be 5 μ L, 10 μ L, 15 μ L, 20 μ L,
25 μ L, 30 μ L, 35 μ L, 40 μ L, 45 μ L or 50 μ L.
Preferably, the testing conditions of the liquid chromatogram are as follows: the ultraviolet absorption value at detection 230nm-280nm, such as can
To be 230nm, 240nm, 250nm, 260nm or 280nm.
Preferably, the radio-frequency emission power of the inductivity coupled plasma mass spectrometry is 1400W-1600W, such as be can be
1400W, 1500W, 1550W or 1600W.
Preferably, radio-frequency voltage 1.7V-1.8V, for example, can be 1.7V, 1.72V, 1.74V, 1.76V, 1.78V or
1.8V。
Preferably, the carrier gas flux of the inductivity coupled plasma mass spectrometry is 0.5L/min-0.6L/min, such as can be with
It is 0.5L/min, 0.52L/min, 0.54L/min, 0.56L/min, 0.58L/min or 0.6L/min.
Preferably, compensation current amount be 0.25-0.35L/min, such as can be 0.25L/min, 0.27L/min,
0.29L/min, 0.30L/min, 0.32L/min, 0.34L/min or 0.35L/min.
Preferably, crash response throughput is 0.8L/min-1.2L/min, such as can be 0.8L/min, 0.85L/
Min, 0.9L/min, 0.95L/min, 1L/min, 1.1L/min or 1.2L/min.
In the present invention, inventor carries out DNA points using method associated with liquid chromatogram and inductivity coupled plasma mass spectrometry
Organic phosphorus in DNA molecular and Phos is distinguished using fixing phosphorus method, avoids Phos to DNA by the accurate quantitative analysis detection of son
DNA concentration is directly traceable to international SI unit by the interference of organic phosphorus accurate quantitative analysis in skeleton, ensures that the accurate of measurement result can
By property.The DNA such as Plasmid DNA bigger for number molecular weight in DNA, genomic DNA etc., conventional liquid phase chromatographic column can not be right
It is separated, and can be blocked since its volume is big using conventional chromatographic column, and inventor optimizes the condition of LC-MS, from
Mobility, chromatographic column and Mass Spectrometry Conditions set out, and each each condition of step cooperates, synergistic, finally realize accurate stabilization
Detection.
As optimal technical scheme, a kind of organic phosphorus in quantitative detection DNA and Phos method, the method uses liquid
Phase chromatography and inductivity coupled plasma mass spectrometry are combined, and are specifically comprised the following steps:
(1) liquid chromatogram is carried out to DNA using molecular-exclusion chromatography column first；
The mobile phase of the liquid chromatogram is the Tirs for the 5mM-100mM that pH is 6.0-8.0, flow velocity 0.5mL/min-
2.0mL/min, sampling volume are 5 μ L-50 μ L, detect the ultraviolet absorption value at 230nm-280nm；
(2) efflux of the liquid chromatogram of step (1) is continued into inductivity coupled plasma mass spectrometry, wherein described
The radio-frequency emission power of inductivity coupled plasma mass spectrometry is 1400W-1600W, radio-frequency voltage 1.7V-1.8V, carrier gas flux
For 0.5L/min-0.6L/min, compensation current amount is 0.25L/min-0.35L/min, and crash response throughput is 0.8L/min-
1.2L/min。
Compared with prior art, the invention has the following beneficial effects:
The separating capacity of method combination HPLC provided by the invention and the quantitation capabilities of ICP-MS to organic phosphorus in DNA and
Phos has carried out separation and quantitative detection, solve common ICP-MS fixing phosphorus method it is quantitative to DNA molecular when cannot be distinguished it is organic
The problem of phosphorus and Phos separates the P elements in DNA skeleton with the P elements in inorganic salt solution, to realize
To quantitative detection organic phosphorus and content of inorganic phosphorus in DNA molecular, as a result accurate stable, simple and efficient to handle, greatly improves
DNA fixing phosphorus method quantifies the accuracy of DNA concentration and has ensured the magnitude tracing of DNA concentration to world SI unit.
Detailed description of the invention
Fig. 1 is the HPLC testing result figure of Plasmid DNA plw07 sample of the invention；
Fig. 2 is the HPLC chromatogram of pure EDTA solution of the invention；
Fig. 3 is the HPLC-ICP-MS Analysis of test results figure of Plasmid DNA plw07 sample of the invention；
The HPLC figure that Fig. 4 is volume exclusion column mobile phase of the invention when being water；
Fig. 5 is ion chromatography-ICP-MS combination detection Phos standard solution figure of the invention；
Fig. 6 is ion chromatography-ICP-MS combination detection TE buffer figure of the invention；
Fig. 7 is ion chromatography-ICP-MS combination detection Plasmid DNA figure of the invention；
Fig. 8 is ion chromatography-ICP-MS combination of the invention continuous detection Plasmid DNA figure for a long time；
Fig. 9 is detection figure when having Phos in Plasmid DNA of the invention；
Figure 10 is the datagram of HPLC-ICP-MS combination detection DNA molecular range of the invention.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with attached drawing and by specific real
Mode to further illustrate the technical scheme of the present invention is applied, but the present invention is not limited in scope of embodiments.
Embodiment 1
Simple liquid chromatogram determines Plasmid DNA, and water, Tris-EDTA and Phos go out peak position.
Liquid phase chromatogram condition is as follows:
1, chromatographic column: size exclusion chromatograph column is used
2, liquid-phase condition:
Mobile phase: 10mM Tirs (pH=7.4) prepares (18M Ω/cm resistivity) using ultrapure water, and with 0.22 μm of mistake
Membrane filtration.
Flow velocity: 1mL/min
Sampling volume: 50 μ L
Detection method: UV absorption at 260nm
Liquid chromatogram is used alone to be tested, by taking plw07 Plasmid DNA as an example, that estimates Phos goes out peak position, as a result
See Fig. 1 and Fig. 2；
From figure 1 it appears that the appearance time of DNA is in 5min or so, and 10min or so appearance is proved to be EDTA
(see Fig. 2).Therefore, it according to the molecular size range relationship of Phos and EDTA, can extrapolate, if having in Plasmid DNA inorganic
Phosphorus, then it goes out peak position after 10min.
Embodiment 2
HPLC-ICP-MS is combined P elements in quantitative detection DNA plasmid (plw07) standard substance
Liquid chromatogram and inductivity coupled plasma mass spectrometry are combined, condition is as follows:
1, chromatographic column: size exclusion chromatograph column is used
2, liquid-phase condition:
Mobile phase: 10mM Tirs (pH=7.4) prepares (18M Ω/cm resistivity) using ultrapure water, and with 0.22 μm of mistake
Membrane filtration.
Flow velocity: 1mL/min
Sampling volume: 50 μ L
Detection method: UV absorption at 260nm
3, inductivity coupled plasma mass spectrometry condition:
Radio-frequency emission power 1500W, radio-frequency voltage 1.74V, carrier gas flux 0.58L/min, compensation current amount 0.30L/
Min, crash response throughput 1.0L/min.
Obtained data are shown in Fig. 3, from figure 3, it can be seen that DNA skeleton P element goes out peak position in Plasmid DNA standard substance
In 5min or so, and Phos standard solution appearance, in 10min or so, the DNA standard substance of the present embodiment is in Phos standard
Going out at peak position for solution does not have appearance, it was demonstrated that Phos is not contained in the Plasmid DNA standard substance of the present embodiment, by right
The quantitative detection of Phos confirmed the influence when DNA standard substance definite value of the present embodiment without considering Phos.
Embodiment 3
Mobile phase is Tris, the not no Phos of condition and embodiment 2 it is identical；The map for having Phos in DNA is not purified
As shown in Figure 4；
It as can be seen from Figure 4 is to have apparent nothing in 10min or so containing Phos in unpurified DNA molecular
Machine phosphorus peak occurs, and pure TE buffer is not no appearance, it was demonstrated that is that there are Phos in the unpurified situation of Plasmid DNA
, this may be the Phos that buffer etc. during the extraction process is brought into.
Embodiment 4
Condition is same as Example 2, using the datagram of volume exclusion post detection DNA molecular range, as shown in Figure 5；
As shown in Figure 5, the range of HPLC-ICP-MS combination detection DNA, from mononucleotide molecule to thousands of a bases
DNA molecular may detect that, and can obviously distinguish with the peak position that goes out of Phos, show technical side provided by the invention
Case can not only detect Plasmid DNA, by adjusting the aperture of size exclusion chromatograph, realize DNA different types of to the overwhelming majority
It is detected.
Comparative example 1
Compared with Example 1, in addition to mobile phase is changed to water, other conditions are constant, flow velocity 1mL/min, as a result see Fig. 6 institute
Show, when using pure water as mobile phase isolated plasmid dna, it is found that the effect of DNA and EDTA separation is bad, there are also other miscellaneous peaks
Occur, it was demonstrated that the selection of mobile phase is very important for the separation of sample.
Comparative example 2
Ion chromatographic column and ICP-MS combination are selected, organic phosphorus in Plasmid DNA and Phos detection, mobile phase are carried out
For buffer solution A: 100mM Tris, 100mM NaCl and buffer B:100mM Tris, 1M NaCl；
Phos standard solution is detected first, determines the appearance time of Phos in 0.5min or so, such as Fig. 7
It is shown, change the concentration of Phos standard solution, the peak height of Phos can be with variation, it was demonstrated that the spike occurred in 0.5min
Really Phos appearance.
In addition, being detected to the buffer TE of Plasmid DNA storage, as shown in Figure 8, it was demonstrated that TE is in 0.5min or so
It will appear spike, the appearance of such TE may interfere with Phos detection.
Plasmid DNA is detected, as shown in figure 9, discovery has apparent spike to go out in 0.5min and 5min or so
It is existing, ion chromatographic column is analyzed, the peak of 0.5min or so may be TE or Phos, and 5min or so is that DNA skeleton is organic
Phosphorus goes out peak position.
Therefore, ion chromatographic column is unsuitable does condition associated with ICP-MS, cannot distinguish between in Phos and buffer from
Sub- appearance；It is also, very high due to being required using salinity of the ion chromatographic column to mobile phase, it is necessary to could to be allowed with high salt concentration
DNA is separated, and high salt concentration is bigger to the damage of the detecting instrument of ICP-MS, for a long time using inspection result can be seriously affected,
As shown in Figure 10, after the multiple Plasmid DNA of follow-on test, discovery instrument cannot normal appearance, further prove ion color
Composing column is not optimal selection.
In conclusion the present invention provides a kind of organic phosphorus in quantitative detection DNA and Phos method, lead to
It crosses and the organic phosphorus and Phos in DNA is separated and quantified in conjunction with the separating capacity of HPLC and the quantitation capabilities of ICP-MS
Detection solves the problems, such as that organic phosphorus and Phos cannot be distinguished when common ICP-MS fixing phosphorus method is quantitative to DNA, has wide
Application prospect and huge market value.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (10)
1. a kind of organic phosphorus in quantitative detection DNA and Phos method, which is characterized in that the method uses
Liquid chromatogram and inductivity coupled plasma mass spectrometry are combined；
Wherein, the chromatographic column of the liquid chromatogram includes molecular-exclusion chromatography column.
2. the method according to claim 1, wherein the pore size of the molecular-exclusion chromatography column is 10nm-
50nm。
3. method according to claim 1 or 2, which is characterized in that the mobile phase of the liquid chromatogram includes that phosphate is slow
Fliud flushing and/or Tris buffer, preferably Tris buffer.
4. according to the method described in claim 3, it is characterized in that, the pH of the Tris buffer is 6.0-8.0；
Preferably, the concentration of the Tris buffer is 5mM-100mM.
5. the method according to claim 3 or 4, which is characterized in that the flow velocity of the mobile phase is 0.5mL/min-
2.0mL/min。
6. method according to any one of claims 1-5, which is characterized in that the sampling volume of the liquid chromatogram is 5 μ
L-50μL。
7. method according to claim 1 to 6, which is characterized in that the testing conditions of the liquid chromatogram are as follows:
Detect the ultraviolet absorption value at 230nm-280nm.
8. method according to any one of claims 1-7, which is characterized in that the inductivity coupled plasma mass spectrometry
Radio-frequency emission power is 1400W-1600W；
Preferably, radio-frequency voltage 1.7V-1.8V.
9. method according to claim 1 to 8, which is characterized in that the inductivity coupled plasma mass spectrometry
Carrier gas flux is 0.5L/min-0.6L/min；
Preferably, compensation current amount is 0.25L/min-0.35L/min；
Preferably, crash response throughput is 0.8L/min-1.2L/min.
10. method according to claim 1 to 9, which is characterized in that the method is using liquid chromatogram and electricity
Feel coupled plasma mass spectrometry combination, specifically comprises the following steps:
(1) liquid chromatogram is carried out to DNA using molecular-exclusion chromatography column first；
The Tirs buffer that the mobile phase of the liquid chromatogram is the 5mM-100mM that pH is 6.0-8.0, flow velocity 0.5mL/min-
2.0mL/min, sampling volume are 5 μ L-50 μ L, detect the ultraviolet absorption value at 230nm-280nm；
(2) efflux of the liquid chromatogram of step (1) is continued into inductivity coupled plasma mass spectrometry, wherein the inductance
The radio-frequency emission power of coupled plasma mass spectrometry is 1400-1600W, radio-frequency voltage 1.7V-1.8V, and carrier gas flux is
0.5L/min-0.6L/min, compensation current amount are 0.25L/min-0.35L/min, and crash response throughput is 0.8L/min-
1.2L/min。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008022355A2 (en) * | 2006-08-18 | 2008-02-21 | Perkinelmer Las, Inc. | Methods and reagents for detecting phosphomonoester groups |
| CN104374853A (en) * | 2013-08-14 | 2015-02-25 | 柯跃斌 | Method for high performance liquid chromatography(HPLC)-mass spectrometry (MS) detection of DNA oxidation and DNA methylation |
-
2019
- 2019-02-01 CN CN201910103264.2A patent/CN109765289A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008022355A2 (en) * | 2006-08-18 | 2008-02-21 | Perkinelmer Las, Inc. | Methods and reagents for detecting phosphomonoester groups |
| CN104374853A (en) * | 2013-08-14 | 2015-02-25 | 柯跃斌 | Method for high performance liquid chromatography(HPLC)-mass spectrometry (MS) detection of DNA oxidation and DNA methylation |
Non-Patent Citations (4)
| Title |
|---|
| ARJUN K. VENKATESAN ET AL: "Size exclusion chromatography with online ICP-MS enables molecular weight fractionation of dissolved phosphorus species inwater samples", 《WATER RESEARCH》 * |
| MARCIA J. HOLDEN ET AL: "Traceable Phosphorus Measurements by ICP-OES and HPLC for the Quantitation of DNA", 《ANAL. CHEM》 * |
| SHIN-ICHIRO FUJII ET AL: "Separation and quantification of RNA molecules using size-exclusion chromatography hyphenated with inductively coupled plasma-mass spectrometry", 《ELECTROPHORESIS》 * |
| 胡盛忠: "《石油工业新技术与标准桂法手册》", 31 December 2004, 哈尔滨地图出版社 * |
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