CN-121994898-A - Device and method for eluting, purifying and recovering oversized DNA (deoxyribonucleic acid) based on pulsed field gel electrophoresis

CN121994898ACN 121994898 ACN121994898 ACN 121994898ACN-121994898-A

Abstract

The invention relates to a device and a method for eluting, purifying and recovering oversized DNA based on pulsed field gel electrophoresis, which are used for efficiently recovering hundred kb-level to Mb-level complete DNA molecules. The device comprises an electrophoresis protection chamber and a sample recovery chamber of a polygonal container structure, wherein a semi-permeable membrane is arranged on the side edge of the protection chamber to pass current, the recovery chamber is detachably arranged in the center and contains the semi-permeable membrane, a detachable replaceable magnesium sheet is arranged in the protection chamber to ionize magnesium ions so as to promote DNA compression, the method comprises the steps of preparing a biological sample into agarose gel blocks, cracking and cleaning the agarose gel blocks, placing the agarose gel blocks into the device, adding recovery protection liquid containing PEG, and under the condition of pulsed field gel electrophoresis, the DNA migrates out the gel blocks, combines with the magnesium ions to compress the gel blocks into spheres, and collecting the gel blocks after neutralizing charges. Compared with the prior art, the method overcomes the defects of low product purity and long steps of the traditional recovery method, realizes high-purity and high-efficiency integrated recovery, and is suitable for genomics research and biotechnology application.

Inventors

XUE XIAOLI
HE FAN
WANG YUXUAN
LI WEI

Assignees

上海交通大学

Dates

Publication Date: 20260508
Application Date: 20260116

Claims (10)

1. A device for purifying and recovering oversized DNA based on pulsed field gel electrophoresis, comprising: The electrophoresis protection chamber (1) is of a polygonal container structure, a semipermeable membrane is arranged on the side edge of the electrophoresis protection chamber, and the electrophoresis protection chamber (1) is used for containing electrophoresis protection liquid and enabling current to pass through the semipermeable membrane in the process of pulsed field gel electrophoresis; The sample recovery chamber (2) is of a polygonal container structure, a semipermeable membrane is arranged in the middle of the side edge of the sample recovery chamber (2), the sample recovery chamber (2) is detachably arranged in the center position inside the electrophoresis protection chamber (1), the side edge of the sample recovery chamber is parallel to the corresponding side edge of the electrophoresis protection chamber, so that current uniformly passes, and the sample recovery chamber (2) is used for placing oversized DNA sample gel blocks to be recovered and adding electrophoresis protection liquid; And the detachable replaceable magnesium sheet (3) is arranged in the electrophoresis protection chamber (1), and the detachable replaceable magnesium sheet (3) is parallel to the side edge of the sample recovery chamber and keeps a preset distance, and is used for ionizing magnesium ions in the electrophoresis process so as to promote the compression and protection of oversized DNA.
2. The device for purifying and recovering oversized DNA based on pulsed field gel electrophoresis according to claim 1, wherein the semipermeable membranes of the electrophoresis protection chamber (1) and the sample recovery chamber (2) are of a material that allows ions and small molecules to pass through but blocks the permeation of oversized DNA, thereby allowing the oversized DNA to remain in the sample recovery chamber (2) during electroelution.
3. The device for purifying and recovering oversized DNA based on pulsed field gel electrophoresis according to claim 1, wherein the electrophoresis protection chamber (1) is a container with a hexagonal bottom surface, and the sample recovery chamber (2) is a container with a trapezoidal bottom surface; The electrophoresis protection chamber (1), the sample recovery chamber (2) and the detachable replaceable magnesium sheet (3) are all independent components, and when in use, the components are assembled and then the oversized DNA sample is recovered; The electrophoresis protection chamber (1) and the sample recovery chamber (2) are soaked in ultrapure water for long-term storage after being used, so that the electrophoresis protection chamber is repeatedly used.
4. The method for eluting, purifying and recovering the oversized DNA based on the pulsed field gel electrophoresis is characterized by comprising the following steps of: S1, culturing and concentrating a biological sample containing an oversized DNA fragment, preparing a pulsed field gel electrophoresis gel block by using low-melting-point agarose, and performing cracking treatment and cleaning treatment on the gel block to expose the oversized DNA; S2, assembling the device according to any one of claims 1 to 3, placing the treated gel block in a sample recovery chamber (2) and placing the treated gel block close to a side semipermeable membrane, adding recovery protection liquid containing PEG high polymer into the sample recovery chamber (2) and an electrophoresis protection chamber (1) to enable the liquid level to submerge the pulse field gel electrophoresis gel block, then placing the whole device in a pulse field gel electrophoresis tank, and adding electrophoresis buffer solution; S3, electrophoresis is carried out under the condition of pulsed field gel electrophoresis, so that oversized DNA migrates out of a gel block and is combined with ionized magnesium ions of a detachable replaceable magnesium sheet (3), and the gel is compressed into spheres under the action of a recovery protection liquid, so that charges are neutralized and migration is stopped, and the gel is collected in a sample recovery chamber; s4, sucking the solution containing the compressed oversized DNA from the sample recovery chamber (2).
5. The method for recovering oversized DNA based on pulsed-field gel electrophoresis elution purification of claim 4 wherein in S1 the cleavage treatment is digestion of the gel cake at 50℃for 12 to 48 hours with a cleavage buffer comprising proteinase K, the cleavage buffer comprising 500 mM EDTA at pH 8.0, 1wt% sodium lauroyl sarcosine and 200 μg/mL proteinase K; The washing treatment is to wash the digested gel block with TE buffer solution with pH of 8.0 at room temperature for 30 to 60 minutes each time.
6. The method for eluting, purifying and recovering oversized DNA based on pulsed field gel electrophoresis of claim 4 wherein in S3, the conditions of pulsed field gel electrophoresis include an electrophoresis temperature controlled at 7-14 ℃, an electric field intensity set at 2-6V/cm, a pulsed electric field switching time of 30-500 seconds, a pulsed electric field switching angle of 106-120 DEG, and a total electrophoresis time adjusted between 1-12 hours according to the size of the targeted DNA fragment, so that oversized DNA fragments can migrate out of the gel block and undergo strand-sphere transformation.
7. The method for recovering oversized DNA based on pulsed-field gel electrophoresis elution purification of claim 6, wherein in S3, the pulsed-field switching time is specifically adjusted according to the size of the oversized DNA fragment to be recovered, specifically: When the recovered DNA fragment was of the order of hundred kb, the pulse diversion time was set to 30-90 seconds; When the recovered DNA fragment is of Mb grade, the pulse steering time is set to 90-500 seconds to adapt to the migration characteristics of DNA fragments of different sizes in a pulse field, and the separation efficiency and recovery yield are optimized.
8. The method for recovering oversized DNA based on pulsed-field gel electrophoresis elution purification of claim 4 wherein in S2 the recovered protecting solution comprises 15wt% of PEG polymer with a degree of polymerization ranging from 3000 to 20000; the PEG polymer is dissolved in 0.5 XTBE buffer, and is used for promoting the combination of oversized DNA and magnesium ions to generate chain-ball conversion in the electroelution process, forming a stable spherical structure, and neutralizing the negative charge on the surface of the DNA, so that the DNA stops migrating and stably exists in a sample recovery chamber.
9. The method for recovering oversized DNA based on pulsed-field gel electrophoresis elution purification of claim 6 wherein in S2 the oversized DNA is a DNA fragment ranging in size from 100 kb to several Mb.
10. The method for recovering oversized DNA based on pulsed-field gel electrophoresis elution purification of claim 6 wherein in S1 the biological sample is derived from a prokaryote or eukaryote including E.coli, saccharomyces cerevisiae or mammalian cells.

Description

Device and method for eluting, purifying and recovering oversized DNA (deoxyribonucleic acid) based on pulsed field gel electrophoresis Technical Field The invention relates to a nucleic acid separation and purification technology in the field of biotechnology, in particular to a device and a method for recovering oversized DNA based on pulsed field gel electrophoresis elution and purification. Background With the rapid development of genome sequencing technology and synthetic genomics, in vitro recovery of ultra-large DNA, purified hundred kb to Mb, has become a key link connecting genome "blueprints" and "functions" enabling researchers to manipulate genetic material directly on a larger scale. Currently, the mainstream methods rely on Pulsed Field Gel Electrophoresis (PFGE) techniques, such as separation of DNA by PFGE followed by digestion recovery with agarase (a two-dimensional electrophoresis method as described in CN102586227 a), or field reversed gel electrophoresis recovery using an electroelution apparatus. However, the methods have the remarkable limitation in practical application that although CN102586227A optimizes the separation effect through two-way PFGE (FIGE and CHEF modes), the recovery step needs to digest the low-melting agarose gel by using agarase, so that the product contains high-concentration salt ions, polysaccharide and enzyme residues, the purity is low, the process is long (about 3 days are required for recovery), and the subsequent operation is not convenient. Further analyzing the prior art, CN1479790a discloses a method for rapid typing of microorganisms, which shortens the electrophoresis time to 2.5-7 hours using PFGE micro-device and emphasizes the enzyme-free preparation of DNA samples to reduce costs. However, the method focuses on microbial typing, does not solve the purification problem in the recovery of oversized DNA, still depends on the traditional recovery mode after electrophoresis separation, and cannot avoid the problem of product pollution. Meanwhile, although the field inversion gel Electrophoresis method (as mentioned in CN102586227 a) attempted to improve recovery efficiency, DNA was easily blocked in the gel block, resulting in a low recovery amount and detection only by Southern hybridization, and poor practicality (Viovy et al., electrophorsis, 1992). The above references are ：Viovy, J.L., et al., Irreversible Trapping of DNA during Crossed-Field Gel-Electrophoresis. Electrophoresis, 1992. 13(1-2): p. 1-6. In summary, the core problem of the prior art is that the method of combining pulsed field gel electrophoresis with enzyme digestion or electroelution has the defects of low recovery efficiency, insufficient product purity and complicated process when recovering oversized DNA. This directly restricts the application of oversized DNA in functional genomics, and there is a need for a recovery and purification technique that can be efficient, pure and simplified in flow. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide a device and a method for purifying and recovering oversized DNA based on pulsed field gel electrophoresis, the method for purifying and recovering the oversized DNA in vitro is simple in operation, short in time required by the whole recovery process, large in total amount of oversized DNA fragments obtained by one-time recovery, high in integrity and capable of being stably stored for a long time. The aim of the invention can be achieved by the following technical scheme: The first aspect of the invention provides a device for purifying and recovering oversized DNA based on pulsed field gel electrophoresis, which comprises an electrophoresis protection chamber, a sample recovery chamber and a detachable replaceable magnesium sheet The electrophoresis protection chamber is of a polygonal container structure, a semipermeable membrane is arranged on the side edge of the electrophoresis protection chamber, and the electrophoresis protection chamber is used for containing electrophoresis protection liquid and enabling current to pass through the semipermeable membrane in the process of pulsed field gel electrophoresis; the sample recovery chamber is of a polygonal container structure, a semipermeable membrane is arranged in the middle of the side edge of the sample recovery chamber, the sample recovery chamber is detachably arranged in the center of the inside of the electrophoresis protection chamber, the side edge of the sample recovery chamber is parallel to the corresponding side edge of the electrophoresis protection chamber, so that current uniformly passes, and the sample recovery chamber is used for placing an oversized DNA sample gel block to be recovered and adding electrophoresis protection liquid; And the detachable replaceable magnesium sheet is arranged in the electrophoresis protection chamber, is parallel to the side edge of the sample recovery chamber and keeps a preset distanc