CN113514297A - Magnetic control molecular combing method for stretching single molecular DNA - Google Patents
Magnetic control molecular combing method for stretching single molecular DNA Download PDFInfo
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- CN113514297A CN113514297A CN202110683152.6A CN202110683152A CN113514297A CN 113514297 A CN113514297 A CN 113514297A CN 202110683152 A CN202110683152 A CN 202110683152A CN 113514297 A CN113514297 A CN 113514297A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The invention discloses a magnetic control molecular combing method for stretching monomolecular DNA, which drives magnetic particles doped in DNA liquid drops to drive the DNA liquid drops and gas-liquid-solid interfaces to move through a magnetic field, and when the gas-liquid-solid interfaces stride over DNA which is adsorbed on the surface of a substrate at the middle end of the DNA liquid drops, the DNA monomolecular is stretched into a straight line by interfacial force so as to realize the fluorescent observation of information sites on the monomolecular DNA. Compared with the prior art, the DNA molecule stretching method provided by the invention has the advantages of less DNA sample demand, convenience and rapidness in operation, high stretching rate, good stretching uniformity, convenience in integration with DNA sample treatment and detection links and the like.
Description
Technical Field
The invention is applied to the field of micro-nano biology, and particularly relates to a magnetic control molecular combing method for stretching single molecular DNA.
Background
Currently, the detection and analysis of genetic and epigenetic information in DNA molecules is an important basis for both bioscience and medical applications. The direct observation of fluorescence labeling information sites on DNA stretched in a straight line by DNA optical mapping technology is an efficient method for detecting epigenetic information, and has been increasingly widely used, such as gene screening, base methylation analysis, DNA-protein interaction analysis, and DNA damage detection. The primary step of DNA optical mapping technology is to stretch DNA molecules from a freely relaxed coiled form into a linear form, enabling direct observation of information sites. For example, a freely relaxed bulk lambda DNA (lambda phage DNA, 48.5kbp in length) with a radius of gyration of only 0.73 μm in solution could theoretically be stretched into a 32 μm straight form. The DNA stretching link directly and obviously affects the efficiency and the precision of the DNA optical map detection: (1) lower stretch ratios will result in lower spatial resolution of the detection; (2) the uneven stretching rate causes serious errors in measurement and analysis such as the relative position of information sites, the linear density of the information sites, the transverse contrast of DNA samples and the like; (3) expensive and complicated stretching instruments and procedures will limit the widespread use of DNA optical mapping techniques. With the wide development of biology, chemistry and informatics related to DNA optical mapping technology, the mechanical problem of stretching DNA molecules efficiently (conveniently, rapidly, with high stretching rate and uniform stretching) becomes the key of high-efficiency and high-precision DNA optical mapping analysis.
The current main DNA stretching methods comprise a nano-channel electrophoresis stretching method, an atomic force microscope manipulation method, an optical clamp manipulation method, a magnetic clamp manipulation method, a micro-flow field stretching method and the like. The nano-channel electrophoresis stretching method (Nat Biotechnol,2012,30(8),762- & 763; Nucleic Acids Res,2019,47(15), e89.) drives DNA molecules into a nano-channel (usually the diameter of the channel is less than 30nm) by electrophoretic force, and DNA extends to be linear under the restriction of the nano-channel. The method has high stretching ratio (about 0.8-1) and stretching uniformity. However, the nanochannel processing and instrumentation required for this method is very complex and expensive. Individual DNA molecules can be stretched accurately by atomic force microscopy, optical and magnetic clamping (Nucleic Acids Res,2016,44(9), 3971-7988), however, this type of method stretches only one or a small number of DNA molecules at a time and is very expensive in terms of equipment. The micro-flow field stretching method (Soft Matter,2015,11(16), 3105-. The molecular combing and stretching method (ACS Nano,2013,7: 9724-.
In summary, so far, a unimolecular DNA stretching method with the advantages of less DNA sample demand, convenient operation, high stretching rate, convenient integration with DNA sample processing and detection links, etc. needs to be constructed, and the method has important significance for DNA optical map application.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a magnetic control molecular combing method for stretching single-molecule DNA aiming at the defects of the prior art.
In order to solve the technical problem, the magnetic control molecular combing method for stretching the single-molecule DNA comprises the following steps:
dripping a DNA solution containing magnetic particles to a substrate;
magnetic control is utilized to drive the magnetic particles to drive the DNA liquid drops to move;
and in the process of moving the DNA liquid drop, the moving gas-liquid-solid interface stretches the monomolecular DNA adsorbed on the substrate.
As a possible embodiment, further, the magnetic particles are one or more of iron powder, ferroferric oxide magnetic microspheres, urea resin magnetic microspheres, polystyrene magnetic microspheres, silica magnetic microspheres, and g-ferric oxide microspheres.
As a possible embodiment, further, the substrate is one or more of a hydrophobic modified substrate and a positively charged modified substrate.
As a possible embodiment, further, the hydrophobic modification method of the substrate is one or more of silanized molecule fumigation modification and hydrophobic polymer spin coating modification.
As a possible embodiment, further, the silanized molecule in the fumigation modification of the silanized molecule is one or more of trimethylchlorosilane, 3-aminopropyltriethoxysilane and trimethylchlorosilane; the hydrophobic polymer in the hydrophobic polymer spin-coating modification is one or more of polymethyl methacrylate, polydimethylsiloxane, polystyrene, highly oriented pyrolytic graphite, graphene oxide, polytetrafluoroethylene and ZEONEX.
As a possible embodiment, further, the positive charge modification method of the positive charge modified substrate is polylysine coating, APES silicidation, positive charge adsorption (Zn)2+、Mg2+、Co3+Etc.).
As a possible implementation manner, further, the magnetic particles driven by magnetron are specifically: driving magnetic particles mixed in the DNA liquid drops by using a moving magnetic field to enable the liquid drops to move directionally, so that the DNA is directionally stretched into a straight line by utilizing a moving gas-liquid-solid interface; wherein the moving magnetic field is one or more of moving magnetic fields generated by moving a permanent magnet and a magnetic induction coil.
As a possible embodiment, further, the single-molecule DNA adsorbed on the substrate is adsorbed by one or more of binding adsorption of hydrophobic bases exposed by uncoiling of the end of the DNA to a hydrophobic substrate or electrostatic adsorption of a negatively charged phosphate backbone of the DNA to a positively charged substrate.
As a possible embodiment, further, the method for uncoiling to expose the hydrophobic base at the end of DNA comprises using one or more of DNA helicase, heating, adjusting the pH of the solution; wherein the pH value is 3-11, and preferably 5.5.
As a possible implementation mode, the moving speed of the gas-liquid-solid interface is 100 mu m/s-1 cm/s.
By adopting the technical scheme, the invention has the following beneficial effects:
the invention only needs a small amount of DNA samples and can be used for stretching trace DNA samples and detecting fluorescence signals. And the instrument is simple and easy, easy operation, and is with low costs, the commonality is strong. According to the technical scheme, multiple DNA samples can be stretched simultaneously in a magnetic control mode; meanwhile, the movement regulation and control of the gas-liquid-solid interface is more accurate, and the stretching rate and the stretching uniformity of the DNA are better. The integration of the DNA single molecule stretching link, the DNA sample pretreatment, the subsequent DNA single molecule information detection and other links can be realized.
Drawings
The invention is described in further detail below with reference to the following figures and embodiments:
FIG. 1 is a schematic diagram of the principle of the present invention, in which reference numeral 1 is a droplet containing DNA molecules and magnetic particles, reference numeral 2 is a magnet, reference numeral 3 is a DNA single molecule, and reference numeral 4 is a magnetic particle;
FIG. 2 is a fluorescence diagram of a DNA molecule stretched into a linear form by the method of the present invention;
FIG. 3 is a graph showing the stretch ratio distribution of DNA stretched by the method of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described in detail and completely with reference to the accompanying drawings.
As shown in FIGS. 1 to 3, the present invention provides a magnetic controlled molecular combing method for stretching single-molecule DNA, comprising the following steps:
will containDripping a DNA solution of magnetic particles to the substrate; the magnetic particles are one or more of iron powder, ferroferric oxide magnetic microspheres, urea-formaldehyde resin magnetic microspheres, polystyrene magnetic microspheres, silicon dioxide magnetic microspheres and g-ferric oxide microspheres. Wherein the substrate is one or more of a hydrophobic modified substrate and a positively charged modified substrate. Further, the hydrophobic modification method of the substrate is one or more of silanized molecule fumigation modification and hydrophobic polymer spin coating modification. The silanization molecules in the fumigation modification of the silanization molecules are one or more of trimethylchlorosilane, 3-aminopropyl triethoxysilane and trimethylchlorosilane; the hydrophobic polymer in the hydrophobic polymer spin-coating modification is one or more of polymethyl methacrylate, polydimethylsiloxane, polystyrene, highly oriented pyrolytic graphite, graphene oxide, polytetrafluoroethylene and ZEONEX. The positive charge modification method of the positive charge modified substrate comprises the steps of coating polylysine, APES silicification and positive charge adsorption (Zn)2+、Mg2+、Co3+Etc.).
Magnetic control is utilized to drive the magnetic particles to drive the DNA liquid drops to move; an operator drives magnetic particles mixed in the DNA liquid drops by using a moving magnetic field to make the liquid drops move directionally, so that the DNA is directionally stretched into a straight line by using a moving gas-liquid-solid interface; wherein the moving magnetic field is one or more of moving magnetic fields generated by moving a permanent magnet and a magnetic induction coil. The moving speed of the gas-liquid-solid interface is preferably 100 μm/s to 1 cm/s.
And in the process of moving the DNA liquid drop, the moving gas-liquid-solid interface stretches the monomolecular DNA adsorbed on the substrate. The adsorption mode of the monomolecular DNA adsorbed on the substrate is one or more of the combination adsorption of hydrophobic bases exposed by uncoiling at the end of the DNA and the hydrophobic substrate or the electrostatic adsorption of a phosphate skeleton with negative electricity of the DNA and the substrate with positive electricity. The method for exposing the hydrophobic base at the end of the DNA by uncoiling comprises one or more of DNA helicase, heating and regulating the pH value of the solution; wherein the pH value is 3-11.
Example (b):
(1) preparing a hydrophobic substrate. Glass coverslips (22X 22mm) were soaked in fume hood with 70% w/w nitric acid and 37% w/w hydrochloric acid at a ratio of 2:1 and soaked overnight to erode all fluorescent contaminants. Clean glass coverslips were carefully washed from both sides with deionized water and ethanol. The glass coverslip was dried with N2 gas. Zeonex (480R, Zeon, Japan) was dissolved in chlorobenzene overnight to prepare a 1.5% w/v solution. Polymethyl methacrylate (PMMA) was dissolved in chloroform to prepare a 1.5% w/v solution. A hydrophobic substrate for DNA molecule stretching and immobilization was prepared by dropping 60. mu.l of Zeonex or PMMA solution with a glass pipette at the center of a dry glass cover slip and spin-coating at 2500rpm for 90 minutes (WS-650HZB-223NPP, Laurell, USA) to coat a hydrophobic polymer film on the surface of the glass slip.
(2) And preparing a DNA solution. Dropping NaOH solution into DNA solution to regulate pH value of DNA solution, unwinding the ends of DNA molecules in the solution and exposing hydrophobic nucleotide bases, adding magnetic particles into the DNA solution, and dropping the DNA solution onto hydrophobic surface with liquid-moving gun. The hydrophobic nucleotide bases exposed by the uncoiling of the DNA molecule are bound to the non-polar hydrophobic substrate by van der waals forces.
(3) The driving droplet stretches the DNA. The magnetic particles in the DNA droplets are driven by the moving magnet, which in turn drives the movement of the DNA droplets on the hydrophobic surface. Along with the retrogression of the liquid drop gas-liquid-solid interface, when the gas-liquid-solid interface strides over a section of DNA molecules fixed on the substrate, the DNA molecules are stretched into a straight line by the gas-liquid interfacial force.
The foregoing is directed to embodiments of the present invention, and equivalents, modifications, substitutions and variations such as will occur to those skilled in the art, which fall within the scope and spirit of the appended claims.
Claims (10)
1. A magnetic control molecular combing method for stretching single molecular DNA is characterized by comprising the following steps:
dripping a DNA solution containing magnetic particles to a substrate;
magnetic control is utilized to drive the magnetic particles to drive the DNA liquid drops to move;
and in the process of moving the DNA liquid drop, the moving gas-liquid-solid interface stretches the monomolecular DNA adsorbed on the substrate.
2. The magnetic control molecular combing method for stretching the single-molecule DNA, according to claim 1, is characterized in that: the magnetic particles are one or more of iron powder, ferroferric oxide magnetic microspheres, urea-formaldehyde resin magnetic microspheres, polystyrene magnetic microspheres, silicon dioxide magnetic microspheres and g-ferric oxide microspheres.
3. The magnetic control molecular combing method for stretching the single-molecule DNA, according to claim 1, is characterized in that: the substrate is one or more of a hydrophobic modified substrate and a positive charge modified substrate.
4. The magnetic control molecular combing method for stretching the single-molecule DNA, according to claim 3, is characterized in that: the hydrophobic modification method of the substrate is one or more of silanized molecule fumigation modification and hydrophobic polymer spin coating modification.
5. The magnetically controlled molecular combing method for stretching single-molecule DNA as set forth in claim 4, characterized in that: the silanization molecules in the fumigation modification of the silanization molecules are one or more of trimethylchlorosilane, 3-aminopropyl triethoxysilane and trimethylchlorosilane; the hydrophobic polymer in the hydrophobic polymer spin-coating modification is one or more of polymethyl methacrylate, polydimethylsiloxane, polystyrene, highly oriented pyrolytic graphite, graphene oxide, polytetrafluoroethylene and ZEONEX.
6. The magnetic control molecular combing method for stretching the single-molecule DNA, according to claim 3, is characterized in that: the positive charge modification method of the positive charge modified substrate is to coat morePolylysine, APES silicidation, positive charge adsorption (Zn)2+、Mg2 +、Co3+Etc.).
7. The magnetic control molecular combing method for stretching the single-molecule DNA, according to claim 1, is characterized in that: the magnetic particles driven by the magnetic control are specifically as follows: driving magnetic particles mixed in the DNA liquid drops by using a moving magnetic field to enable the liquid drops to move directionally, so that the DNA is directionally stretched into a straight line by utilizing a moving gas-liquid-solid interface; wherein the moving magnetic field is one or more of moving magnetic fields generated by moving a permanent magnet and a magnetic induction coil.
8. The magnetic control molecular combing method for stretching the single-molecule DNA, according to claim 1, is characterized in that: the adsorption mode of the monomolecular DNA adsorbed on the substrate is one or more of the combination adsorption of hydrophobic bases exposed by uncoiling at the end of the DNA and the hydrophobic substrate or the electrostatic adsorption of a phosphate skeleton with negative electricity of the DNA and the substrate with positive electricity.
9. The magnetically controlled molecular combing method for stretching single-molecule DNA as set forth in claim 8, characterized in that: the method for exposing the hydrophobic base at the end of the DNA by uncoiling comprises one or more of DNA helicase, heating and regulating and controlling the pH value of the solution; wherein the pH value is 3-11.
10. The magnetic control molecular combing method for stretching the single-molecule DNA, according to claim 1, is characterized in that: the moving speed of the gas-liquid-solid interface is 100 mu m/s-1 cm/s.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1144540A (en) * | 1994-02-11 | 1997-03-05 | 巴斯德研究所 | Method for ordering macromolecules by means of moving meniscus, and uses thereof |
| CN1376801A (en) * | 2002-03-04 | 2002-10-30 | 中国科学院长春应用化学研究所 | Horizontal power molecular comb |
| CN101371124A (en) * | 2006-02-13 | 2009-02-18 | 新加坡科技研究局 | Method for processing biological sample and/or chemical example |
-
2021
- 2021-06-21 CN CN202110683152.6A patent/CN113514297A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1144540A (en) * | 1994-02-11 | 1997-03-05 | 巴斯德研究所 | Method for ordering macromolecules by means of moving meniscus, and uses thereof |
| CN1376801A (en) * | 2002-03-04 | 2002-10-30 | 中国科学院长春应用化学研究所 | Horizontal power molecular comb |
| CN101371124A (en) * | 2006-02-13 | 2009-02-18 | 新加坡科技研究局 | Method for processing biological sample and/or chemical example |
Non-Patent Citations (1)
| Title |
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| 李美芝: "《妇科内分泌学》", 30 April 2001, 人民军医出版社 * |
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Application publication date: 20211019 |
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