A kind of nucleic acid purification reagent based on paramagnetic particle method
Technical field
The present invention relates to biology field, more particularly to a kind of nucleic acid purification reagent based on paramagnetic particle method.
Background technology
At present, the purifying of nucleic acid, many reactions such as sequencing of two generations, TA clones are all be unable to do without in biomedical every field
Etc. need by after enzyme reaction nucleic acid purification recycle, there are many isolation and purification methods of nucleic acid:Exist if any using DNA and RNA
Different characteristics are dissolved in salting liquid to detach the strong salty method of the two, has and protein denaturation is inhibited into nucleic acid simultaneously using organic solvent
The method of organic solvent extraction of enzyme degradation has the density gradient centrifugation using the different principle separation nucleic acid of different contents density,
The characteristic that different sorbing materials (such as siliceous material, anion exchange resin and magnetic bead) and nucleic acid combine also is utilized to obtain core
The sorbing material combined techniques of acid.Paramagnetic particle method, can be very safe and easy with fast separating and purifying nucleic acid under the action of magnetic force is added outside
In automation.Magnetic bead nucleic acid purification method refers to using superparamagnetism silica nano-magnetic microsphere as carrier, by magnetic bead in height
Adsorb nucleic acid in salting liquid, and the method that the principle that nucleic acid is detached from from magnetic bead surfaces is subjected to nucleic acid purification in low salt solutions.
The existing nucleic acid purification reagent based on paramagnetic particle method, recovery of nucleic acid only have 90% hereinafter, and can only adsorb
The segment of more than 100bp is very serious for the segment loss of 100bp or so.If encountered, operation system step is various, waits to locate
The problems such as the rare sampling of sample is difficult, pending sample initial amount is low are managed, how efficiently to carry out experimentation amplifying nucleic acid purifying
Recycling has vital effect, and there are many this kind of clip sizes of cfDNA in liquid biopsy for the result of experimental implementation
In 100bp or so or hereinafter, the existing nucleic acid purification reagent based on paramagnetic particle method is very low for the rate of recovery of this kind of segment.
Invention content
The technical problems to be solved by the invention be to provide a kind of purification efficiency it is higher be more suitable for trace sample and
There is the nucleic acid purification reagent based on paramagnetic particle method of good recovery efficiency for DNA small pieces segment molecule.
The technical solution that the present invention solves above-mentioned technical problem is as follows:A kind of nucleic acid purification reagent based on paramagnetic particle method, by
Each material composition of following mass fraction:1%~5% carboxyl modified magnetic bead, 11.78%~23.56% sodium chloride, 10%~
20% polyethylene glycol, 0.01%~0.05%N- sodium lauroyl sarcosines, 0.16%~0.79%mM Tris-HCl, 0.5%~
2%EDTA and 0.5%~2% polysorbas20, surplus are water.The molecular weight of the polyethylene glycol is 6000-10000, enhances institute
The effect of the leading nanometer magnetic bead absorption nucleic acid of high salt concentration in nucleic acid purification reagent (mainly sodium chloride) is stated, the nucleic acid is pure
The pH value for changing reagent is 7.0.
The carboxylated modification magnetic bead has carried out carboxylated modification for superparamagnetism silica nano-magnetic bead surface, adopts
Carboxylated modification is carried out with the method for polymeric acrylic acid, a diameter of 600nm~1000nm has superparamagnetism, quick magnetic response
It the features such as property, high-carboxyl-content, monodispersity, can be under the action of special chemical reagent by polypeptide, albumen and oligonucleotides
The bio-ligands such as acid are coupled to microsphere surface, and the carboxylated magnetic bead is two layers of magnetosphere, magnetic larger, in the effect of externally-applied magnetic field
Under, gather that speed is fast, and polystyrene kernel is small, and quality is small, under the action of no magnetic field, sinking speed is slow, suspends
Time is longer, more effectively adsorbs nucleic acid.
A kind of nucleic acid purification QIAquick Gel Extraction Kit based on paramagnetic particle method, including the nucleic acid purification reagent, washing buffer and
Elution buffer, the washing buffer are the ethyl alcohol that volume fraction is 80%, and the elution buffer is TE buffer solution or surpasses
Pure water.
The beneficial effects of the invention are as follows:The nucleic acid purification reagent based on paramagnetic particle method and kit of the present invention is for nucleic acid
The more existing magnetic bead reagent of organic efficiency and kit organic efficiency higher are lower and right for the recycling loss of trace sample
It is more preferable in 100bp or so or smaller nucleic acid fragment organic efficiency.It can wait to locate by adjusting nucleic acid purification reagent simultaneously
The volume ratio of DNA is managed, so as to reach Piece Selection effect.
Description of the drawings
Fig. 1 purifies organic efficiency figure for 1 amplifying nucleic acid of the embodiment of the present invention;
Fig. 2 purifies organic efficiency figure for 2 amplifying nucleic acid of the embodiment of the present invention;
Fig. 3 purifies organic efficiency figure for 3 amplifying nucleic acid of the embodiment of the present invention;
Fig. 4 is to analyze glue figure to the agilent 2200 of different fragments organic efficiency in the embodiment of the present invention 4, and wherein A0 is
DNA marker, A1, B1, C1, D1 be respectively 1X nucleic acid purifications reagent and Ampure XP Beads reagents recycling sample, E1,
F1, G1, H1 are respectively 1.5X nucleic acid purifications reagent and Ampure XP Beads reagents recycling sample, and A2, B2, C2, D2 are respectively
1.8X nucleic acid purifications reagent and Ampure XP Beads reagents recycling sample;
Fig. 5 is to the organic efficiency of the mixing nucleic acid fragment containing small fragment in the embodiment of the present invention 4;
Fig. 6 is to the organic efficiency of the mixing nucleic acid fragment containing small fragment in the embodiment of the present invention 5;
Fig. 7 is to the organic efficiency of the mixing nucleic acid fragment containing small fragment in the embodiment of the present invention 6.
Specific embodiment
The principle of the present invention and feature are described below in conjunction with drawings and the specific embodiments, example is served only for solving
The present invention is released, is not intended to limit the scope of the present invention.
Embodiment 1
The PCR purified products that concentration is taken to be respectively 16.6ng/ μ L (concentration one) and 22.8ng/ μ L (concentration two), contain respectively
There is the segment that size is 127bp and 306bp, using the nucleic acid purification reagent and kit of the present invention, measure two kinds of differences respectively
The rate of recovery of concentration samples.
1) 20 μ L PCR purified products is taken to be placed in centrifuge tube, 38 μ L of nucleic acid purification reagent are added in into centrifuge tube, fully
Mixing is stored at room temperature 5min, then centrifuge tube is placed in magnetic frame and is clarified up to supernatant, and supernatant is abandoned in suction.
Wherein, the nucleic acid purification reagent is made of each raw material original of following mass fraction:5% carboxyl modified magnetic bead,
19% sodium chloride, 18% polyethylene glycol, 0.04%N- sodium lauroyl sarcosines, 0.16%Tris-HCl, 2%EDTA and 0.5%
Polysorbas20, remaining ingredient are water.
2) 200 μ L washing buffers are added in from another side wall of magnetic bead, gently blown and beaten 5 times, it is impossible to dispel magnetic bead, suction is abandoned
Clearly, 200 μ L washing buffers are added, are gently blown and beaten 5 times, supernatant is abandoned in suction, and centrifuge tube from magnetic frame is removed, uncaps and dries in the air
5min or so is dried to magnetic bead, and surface is without washmarking or small crack, unsuitable overdrying.
3) 20 μ L elution buffers are added in, piping and druming is resuspended magnetic bead, is stored at room temperature 5min.Centrifuge tube is placed on magnetic frame
1min is drawn in supernatant to new centrifuge tube, measures DNA concentration after recycling.
4) contrast agents select Ampure XP Beads reagents to compare, and each concentration are done 3 repetitions, according to statistics
Data are mapped, and the results are shown in Figure 1.
As a result the rate of recovery for showing the nucleic acid purification reclaim reagent of the present invention is respectively 96.39% and 95.48%, to impinging upon
The rate of recovery under two concentration is respectively 87.35% and 88.60%, illustrates the nucleic acid purification reclaim reagent and kit of the present invention
It is very high for the organic efficiency of more than 100bp DNA fragmentations, due to highest magnetic bead reagent A mpure XP of utilization rate on the market
Beads, and no significant difference between experimentai batches, repeatability are preferably, small by ectocine.
Embodiment 2
The PCR purified products that concentration is taken to be respectively 16.6ng/ μ L (concentration one) and 22.8ng/ μ L (concentration two), contain respectively
There is the segment that size is 127bp and 306bp, using the nucleic acid purification reagent and kit of the present invention, measure two kinds of differences respectively
The rate of recovery of concentration samples.
1) 20 μ L PCR purified products is taken to be placed in centrifuge tube, 38 μ L of nucleic acid purification reagent are added in into centrifuge tube, fully
Mixing is stored at room temperature 5min, then centrifuge tube is placed in magnetic frame and is clarified up to supernatant, and supernatant is abandoned in suction.
Wherein, the ingredient of addition is respectively in the nucleic acid purification reagent:3% carboxyl modified magnetic bead, 11.78% chlorination
Sodium, 20% polyethylene glycol, 0.05%N- sodium lauroyl sarcosines, 0.79%Tris-HCl, 0.5%EDTA, 1% polysorbas20,
Remaining ingredient is water.
2) 200 μ L washing buffers are added in from another side wall of magnetic bead, gently blown and beaten 5 times, it is impossible to dispel magnetic bead, suction is abandoned
Clearly, 200 μ L washing buffers are added, are gently blown and beaten 5 times, supernatant is abandoned in suction, and centrifuge tube from magnetic frame is removed, uncaps and dries in the air
5min or so is dried to magnetic bead, and surface is without washmarking or small crack, unsuitable overdrying.
3) 20 μ L elution buffers are added in, piping and druming is resuspended magnetic bead, is stored at room temperature 5min.Centrifuge tube is placed on magnetic frame
1min is drawn in supernatant to new centrifuge tube, measures DNA concentration after recycling.
Contrast agents select Ampure XP Beads reagents to compare, and each concentration are done 3 repetitions, according to statistical number
According to mapping, the results are shown in Figure 2.
As a result the rate of recovery for showing the nucleic acid purification reclaim reagent of the present invention is respectively 94.21% and 95.38%, to impinging upon
The rate of recovery under two concentration is respectively 87.35% and 88.60%, illustrates the nucleic acid purification reclaim reagent and kit of the present invention
It is very high for the organic efficiency of more than 100bp DNA fragmentations, due to highest magnetic bead reagent A mpure XP of utilization rate on the market
Beads, and no significant difference between experimentai batches, repeatability are preferably, small by ectocine.
Embodiment 3
The PCR purified products that concentration is taken to be respectively 16.6ng/ μ L (concentration one) and 22.8ng/ μ L (concentration two), contain respectively
There is the segment that size is 127bp and 306bp, using the nucleic acid purification reagent and kit of the present invention, measure two kinds of differences respectively
The rate of recovery of concentration samples.
1) 20 μ L PCR purified products is taken to be placed in centrifuge tube, 38 μ L of nucleic acid purification reagent are added in into centrifuge tube, fully
Mixing is stored at room temperature 5min, then centrifuge tube is placed in magnetic frame and is clarified up to supernatant, and supernatant is abandoned in suction.
Wherein, the ingredient of addition is respectively in the nucleic acid purification reagent:1% carboxyl modified magnetic bead, 23.56% chlorination
Sodium, 10% polyethylene glycol, 0.01%N- sodium lauroyl sarcosines, 0.5%Tris-HCl, 1.5%EDTA, 2% polysorbas20, remaining
Ingredient is water.
2) 200 μ L washing buffers are added in from another side wall of magnetic bead, gently blown and beaten 5 times, it is impossible to dispel magnetic bead, suction is abandoned
Clearly, 200 μ L washing buffers are added, are gently blown and beaten 5 times, supernatant is abandoned in suction, and centrifuge tube from magnetic frame is removed, uncaps and dries in the air
5min or so is dried to magnetic bead, and surface is without washmarking or small crack, unsuitable overdrying.
3) 20 μ L elution buffers are added in, piping and druming is resuspended magnetic bead, is stored at room temperature 5min.Centrifuge tube is placed on magnetic frame
1min is drawn in supernatant to new centrifuge tube, measures DNA concentration after recycling.
Contrast agents select Ampure XP Beads reagents to compare, and each concentration are done 3 repetitions, according to statistical number
According to mapping, the results are shown in Figure 3.
As a result the rate of recovery for showing the nucleic acid purification reclaim reagent of the present invention is respectively 93.89% and 93.12%, to impinging upon
The rate of recovery under two concentration is respectively 87.35% and 88.60%, illustrates the nucleic acid purification reclaim reagent and kit of the present invention
It is very high for the organic efficiency of more than 100bp DNA fragmentations, due to highest magnetic bead reagent A mpure XP of utilization rate on the market
Beads, and no significant difference between experimentai batches, repeatability are preferably, small by ectocine.
Embodiment 4
Take size be respectively 50bp, 100bp, 150bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp,
The marker of 500bp is sample, recycled respectively with 1X, 1.5X, 1.8X nucleic acid purification reagent and kit (1X, 1.5X,
1.8X refers to the volume ratio of sample and the nucleic acid purification reagent).
1) 20 μ L samples is taken to be placed in three centrifuge tubes respectively, be separately added into centrifuge tube 20 μ L of nucleic acid purification reagent,
30 μ L, 38 μ L, abundant mixing are stored at room temperature 5min, then centrifuge tube are placed in magnetic frame and is clarified up to supernatant, and supernatant is abandoned in suction.
2) 200 μ L washing buffers are added in from another side wall of magnetic bead, gently blown and beaten 5 times, it is impossible to dispel magnetic bead, suction is abandoned
Clearly, 200 μ L washing buffers are added, are gently blown and beaten 5 times, supernatant is abandoned in suction, and centrifuge tube from magnetic frame is removed, uncaps and dries in the air
5min or so is dried to magnetic bead, and surface is without washmarking or small crack, unsuitable overdrying.
3) 20 μ L elution buffers are added in, piping and druming is resuspended magnetic bead, is stored at room temperature 5min.Centrifuge tube is placed on magnetic frame
1min is drawn in supernatant to new centrifuge tube, measures the DNA concentration after recycling and by the DNA solution agilent after recycling
2200 analyses, shown in gained glue Fig. 4, the rate of recovery is as shown in Figure 5.
Contrast agents select Ampure XP Beads reagents to compare, and 1X, 1.5X, 1.8X purified reagent are done 3 times respectively
It repeats.
Experimental result is shown:When using 1X magnetic beads for purifying, the present invention can effectively recycle more than 200bp segments, 150bp
Segment only a little remains, and Ampure XP magnetic bead 200bp fragment recovery efficiencies decline, and 150bp segments are substantially not visible item
Band;When using 1.5X magnetic beads for purifying, the present invention can effectively recycle more than 150bp segments, and the loss of 150bp segments is tighter
Weight, and Ampure XP magnetic bead 150bp fragment recovery efficiencies decline, 100bp segments are substantially not visible band;Using 1.8X magnetic
When pearl purifies, the present invention can effectively recycle more than 100bp segments, 50bp fragment loss, and Ampure XP magnetic bead 100bp pieces
Section only a little remains, 50bp fragment loss.
The present invention is better than Ampure XP to the recyclability of small fragment DNA, exists for this kind of Partial Fragment of dissociative DNA
The library construction of the sample type of 100bp or so has bigger advantage.But since the segment for being less than 100bp is removed big portion
Point so organic efficiency is compared to for embodiment 1 reduces, but be compared to for Ampure XP, magnetic bead examination of the present invention
Agent is better than Ampure XP.
The nucleic acid purification reagent used in the present embodiment 4 with it is used in embodiment 1 identical.
Embodiment 5
Take size be respectively 50bp, 100bp, 150bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp,
The marker of 500bp is sample, recycled respectively with 1X, 1.5X, 1.8X nucleic acid purification reagent and kit (1X, 1.5X,
1.8X refers to the volume ratio of sample and the nucleic acid purification reagent).
1) 20 μ L samples is taken to be placed in three centrifuge tubes respectively, be separately added into centrifuge tube 20 μ L of nucleic acid purification reagent,
30 μ L, 38 μ L, abundant mixing are stored at room temperature 5min, then centrifuge tube are placed in magnetic frame and is clarified up to supernatant, and supernatant is abandoned in suction.
2) 200 μ L washing buffers are added in from another side wall of magnetic bead, gently blown and beaten 5 times, it is impossible to dispel magnetic bead, suction is abandoned
Clearly, 200 μ L washing buffers are added, are gently blown and beaten 5 times, supernatant is abandoned in suction, and centrifuge tube from magnetic frame is removed, uncaps and dries in the air
5min or so is dried to magnetic bead, and surface is without washmarking or small crack, unsuitable overdrying.
3) 20 μ L elution buffers are added in, piping and druming is resuspended magnetic bead, is stored at room temperature 5min.Centrifuge tube is placed on magnetic frame
1min is drawn in supernatant to new centrifuge tube, measures the DNA concentration after recycling and by the DNA solution agilent after recycling
2200 analyses.
Contrast agents select Ampure XP Beads reagents, and 1X, 1.5X, 1.8X purified reagent are done 3 repetitions respectively.
Experimental result is shown:When using 1X magnetic beads for purifying, the present invention can effectively recycle more than 200bp segments, 150bp
Segment only a little remains, and Ampure XP magnetic bead 200bp fragment recovery efficiencies decline, and 150bp segments are substantially not visible item
Band;When using 1.5X magnetic beads for purifying, the present invention can effectively recycle more than 150bp segments, and the loss of 150bp segments is tighter
Weight, and Ampure XP magnetic bead 150bp fragment recovery efficiencies decline, 100bp segments are substantially not visible band;Using 1.8X magnetic
When pearl purifies, the present invention can effectively recycle more than 100bp segments, 50bp fragment loss, and Ampure XP magnetic bead 100bp pieces
Section only a little remains, 50bp fragment loss.
The present invention is better than Ampure XP to the recyclability of small fragment DNA, exists for this kind of Partial Fragment of dissociative DNA
The library construction of the sample type of 100bp or so has bigger advantage.But since the segment for being less than 100bp is removed big portion
Point so organic efficiency is compared to for embodiment 2 reduces, but be compared to for Ampure XP, magnetic bead examination of the present invention
Agent is better than Ampure XP.
The nucleic acid purification reagent used in the present embodiment 5 is identical with being used in embodiment 2.
Embodiment 6
Take size be respectively 50bp, 100bp, 150bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp,
The marker of 500bp is sample, recycled respectively with 1X, 1.5X, 1.8X nucleic acid purification reagent and kit (1X, 1.5X,
1.8X refers to the volume ratio of sample and the nucleic acid purification reagent).
1) 20 μ L samples is taken to be placed in three centrifuge tubes respectively, be separately added into centrifuge tube 20 μ L of nucleic acid purification reagent,
30 μ L, 38 μ L, abundant mixing are stored at room temperature 5min, then centrifuge tube are placed in magnetic frame and is clarified up to supernatant, and supernatant is abandoned in suction.
2) 200 μ L washing buffers are added in from another side wall of magnetic bead, gently blown and beaten 5 times, it is impossible to dispel magnetic bead, suction is abandoned
Clearly, 200 μ L washing buffers are added, are gently blown and beaten 5 times, supernatant is abandoned in suction, and centrifuge tube from magnetic frame is removed, uncaps and dries in the air
5min or so is dried to magnetic bead, and surface is without washmarking or small crack, unsuitable overdrying.
3) 20 μ L elution buffers are added in, piping and druming is resuspended magnetic bead, is stored at room temperature 5min.Centrifuge tube is placed on magnetic frame
1min is drawn in supernatant to new centrifuge tube, measures the DNA concentration after recycling and by the DNA solution agilent after recycling
2200 analyses.
Contrast agents select Ampure XP Beads reagents, and 1X, 1.5X, 1.8X purified reagent are done 3 repetitions respectively.
Experimental result is shown:When using 1X magnetic beads for purifying, the present invention can effectively recycle more than 200bp segments, 150bp
Segment only a little remains, and Ampure XP magnetic bead 200bp fragment recovery efficiencies decline, and 150bp segments are substantially not visible item
Band;When using 1.5X magnetic beads for purifying, the present invention can effectively recycle more than 150bp segments, and the loss of 150bp segments is tighter
Weight, and Ampure XP magnetic bead 150bp fragment recovery efficiencies decline, 100bp segments are substantially not visible band;Using 1.8X magnetic
When pearl purifies, the present invention can effectively recycle more than 100bp segments, 50bp fragment loss, and Ampure XP magnetic bead 100bp pieces
Section only a little remains, 50bp fragment loss.
The present invention is better than Ampure XP to the recyclability of small fragment DNA, exists for this kind of Partial Fragment of dissociative DNA
The library construction of the sample type of 100bp or so has bigger advantage.But since the segment for being less than 100bp is removed big portion
Point so organic efficiency is compared to for embodiment 3 reduces, but be compared to for Ampure XP, magnetic bead examination of the present invention
Agent is better than Ampure XP.
The nucleic acid purification reagent used in the present embodiment 6 is identical with being used in embodiment 3.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.