CN108913685B - The method for removing primer dimer - Google Patents
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Abstract
The present invention relates to field of nucleic acid purification, provide a kind of method for removing primer dimer, including 1) are purified using ultrafiltration centrifugal process to PCR product；2) 1) the middle product obtained is purified using paramagnetic particle method, further to remove primer dimer；Wherein, the PCR product and the magnitude difference of primer dimer are 20bp~170bp.It is combined in addition, the present invention also provides magnetic bead reagents in the application and a kind of reagent for removing primer dimer gone in primer dimer.
Description
Technical field
The invention belongs to field of nucleic acid purification, and in particular to by removing primer dimer in PCR product.
Background technique
The natural base of polymerase chain reaction,PCR PCR (United States Patent (USP) US 4,683,202) technical modelling that twentieth century end is born
Because of geometric progression reproduction process, the even individual molecule amplification of dozens of target molecule can be made with its unique geometric index amplification mode
Millions of times until more than one hundred million times, PCR shirtsleeve operation becomes bio-century and uses most wide basic core technology in addition.
Primer dimer is often generated during PCR amplification, and these primer dimers lead to non-specific amplification, and
It can participate in library construction and subsequent sequencing.In sequencing, excessive primer dimer can occupy sequencing throughput, thus
Increase sequencing cost, therefore remove primer dimer after PCR amplification to be very important.
At present the method for removal primer dimer mainly using paramagnetic particle method, the methods of be tapped and recovered.Currently, in high pass
Paramagnetic particle method (for example, CN107236726A and CN106701737A), principle are as follows: add is usually taken when measuring the library construction of sequence
The magnetic bead reagent for entering different volumes ratio can adsorb the DNA of different fragments size, when be added magnetic bead volume it is fewer when, absorption it is big
Segment is more.Therefore, when PCR product segment differs larger (such as 180bp) with primer dimer size, paramagnetic particle method is being removed
Also primer dimer can be removed while the impurity such as protein, salt ion；And when PCR product is differed with primer dimer size
When little, although paramagnetic particle method still is able to removal impurity, but can not distinguish well PCR product and primer dimer,
Thus purification effect is bad.
In view of this, effectively being divided in the presence of the PCR product little to size gap with primer dimer in the art
From tight demand.
Summary of the invention
Currently, usually used in the little nucleic acid fragment of differentiation size gap is ultrafiltration centrifugal process, for example,
It is given in the scheme 3 of " Molecular Cloning:A Laboratory guide (third edition) " the 8th chapter and few nucleosides in DNA cloning product is removed by ultrafiltration
The method of acid points out that filter membrane can penetrate the expansion core of 48bp when using micro- inspissator of trapped molecular weight > 100,000
Thuja acid primer, but the PCR product greater than 125bp is trapped.This actually conveyed a kind of information, i.e., can using ultrafiltration centrifugation
It is separated so that size is effectively differed lesser nucleic acid fragment, and it is true really not so.
After further study, inventors have surprisingly discovered that even with micro- concentration with suitable trapped molecular weight
Device, the separating effect of primer dimer are still undesirable (Examples 1 to 3 as described below).Although that is, being theoretically used only
Size can be differed lesser nucleic acid fragment and is effectively separated by ultrafiltration centrifugal process, but the experimental results showed that, only use ultrafiltration
Centrifugation can not effectively remove primer dimer.Therefore, it is necessary to find remaining primer dimerization after a kind of removal ultrafiltration centrifugation
The method of body.
On the basis of by extensive work, the wondrous discovery of the present inventor, if successively using ultrafiltration centrifugal process and magnetic
Pearl method, even if PCR product and the size gap of primer dimer are smaller, using also capable of further being removed through ultrafiltration after paramagnetic particle method
Residual primers dimer in the product of centrifugation.
Accordingly, the present invention provides a kind of methods of primer dimer in removal PCR product:
1) ultrafiltration centrifugation is carried out to PCR product；
2) 1) the middle product obtained is purified using paramagnetic particle method, further to remove primer dimer；
Wherein, the PCR product and the magnitude difference of primer dimer are about 20bp~170bp.
In one preferred embodiment, the PCR product and the magnitude difference of primer dimer be about 26bp~
167bp。
In a preferred embodiment, the magnitude difference of the PCR product and primer dimer be about 30bp~
100bp。
In a particular embodiment, ultrafiltration centrifugation used in centrifugal column molecular cut off be about 50kD~
100kD
In a preferred embodiment, parameter of noncentricity used in ultrafiltration centrifugation is about 8000 × g~15000 × g, and 40
Second~5 minutes；It is highly preferred that parameter of noncentricity used in ultrafiltration centrifugation is about 10000 × g~14000 × g, 1~4 minute；
Especially preferably, parameter of noncentricity used in ultrafiltration centrifugation is about 11000 × g~13000 × g, and 1.5~2.5 minutes.
On the other hand, the present invention provides magnetic bead reagents in the application gone in primer dimer, wherein the magnetic bead reagent
It is used to handle the PCR product after ultrafiltration is centrifuged, and the PCR product and the magnitude difference of primer dimer are about 20bp
~170bp.
In one preferred embodiment, the PCR product and the magnitude difference of primer dimer be about 26bp~
167bp。
In a preferred embodiment, the magnitude difference of the PCR product and primer dimer be about 30bp~
100bp。
In a preferred embodiment, parameter of noncentricity used in ultrafiltration centrifugation is about 8000 × g~15000 × g, and 40
Second~5 minutes；It is highly preferred that parameter of noncentricity used in ultrafiltration centrifugation is about 10000 × g~14000 × g, 1~4 minute；
Especially preferably, parameter of noncentricity used in ultrafiltration centrifugation is about 11000 × g~13000 × g, and 1.5~2.5 minutes.
In a particular embodiment, ultrafiltration centrifugation used in centrifugal column molecular cut off be about 50kD~
100kD
Another aspect, the reagent combination that the present invention provides a kind of for removing primer dimer, wherein the primer two
The magnitude difference of aggressiveness and PCR product is about 20bp~170bp, and the reagent combination includes ultra-filtration centrifuge tube and magnetic bead reagent.
In one preferred embodiment, the PCR product and the magnitude difference of primer dimer be about 26bp~
167bp。
In a preferred embodiment, the magnitude difference of the PCR product and primer dimer be about 30bp~
100bp。
In a particular embodiment, ultrafiltration centrifugation used in centrifugal column molecular cut off be about 50kD~
100kD。
Method of the invention can be in the lesser situation of magnitude difference of nucleic acid amplification product and primer dimer, effectively
Ground removes primer dimer.
Detailed description of the invention
Fig. 1 is the gel electrophoresis figure measured by Qsep100, wherein sample 1: not purified, sample 2: pure through paramagnetic particle method
Change, sample 3: purifying (10000 × g, 1min) through centrifugal process, sample 4: purifying (14000 × g, 4min) through centrifugal process, sample 5:
Centrifugal process (10000 × g, 1min) and paramagnetic particle method purifying, sample 6: centrifugal process (14000 × g, 4min) and paramagnetic particle method purifying；
Fig. 2 is the gel electrophoresis figure measured by Qsep100, wherein sample 7: centrifugal process (10000 × g, 1min) and magnetic
The purifying of pearl method, sample 8: centrifugal process (12000 × g, 2min) and paramagnetic particle method purify, sample 9: be centrifuged (14000 × g, 4min) and
Paramagnetic particle method purifying；
The gel electrophoresis figure that Fig. 3 is that treated in embodiment 5 each sample measures through Qsep100.
Specific embodiment
Below in conjunction with specific embodiment and embodiment, it is specifically described the present invention, advantages of the present invention and various effects
It thus will clearly present.It will be understood by those skilled in the art that these specific embodiments and embodiment are for illustrating
The present invention is not intended to limit the present invention.
Throughout the specification, unless otherwise specified, terms used herein are interpreted as usual in this field
Used meaning.Therefore, unless otherwise defined, all technical and scientific terms used herein has leads with belonging to the present invention
The identical meaning of the general understanding of field technique personnel.Contradiction if it exists, this specification are preferential.
In the present invention, " removing primer dimer ", " removal primer dimer " and " removing primer dimer " can be mutual
Substitution uses.
In the present invention, " size is not much different " and " magnitude difference is smaller " can be substituted for each other use, refer to that nucleic acid expands
The length difference of primer dimer generated when volume increase object and amplification away between 20bp between 170bp.The length difference is situated between away from preferred
It between 26bp~167bp, is more preferably between 30bp~100bp, particularly preferably between 39bp~89bp.
In the present invention, " centrifugal process " or " centrifugal purification " refers in such a way that ultrafiltration is centrifuged and is purified.
Suitable ultra-filtration centrifuge tube can be selected according to the size of primer dimer and PCR product.Ultra-filtration centrifuge tube has
The ability of certain molecular cut off, the molecular size range of primer dimer obtained by calculation select the super of suitable size
Centrifuge tube is filtered, enables ultra-filtration centrifuge tube to filter primer dimer, retains PCR product.In the present invention, general to choose
The ultra-filtration centrifuge tube of 50kD~100kD.
For estimating the calculation formula of double chain DNA molecule amount for example are as follows:
DsDNA molecular weight (Da)=base pairs (bp) × 650
In the present invention, magnetic bead reagent refer to surface of the nanotechnology to superparamagnetic nano particle carry out improvement and
After surface modification, be prepared into superparamagnetism silica nanometer magnetic bead, pan coating carboxylic group.Such as it can be Backman
The Agencourt Ampure Beads (article No. A63881) or BioMag hundred of Coulter steps the two generations sequencing DNA of lattice biology
Segment screening purifying magnetic bead (article No. BMSX-50), however, the present invention is not limited thereto.
In the present invention, can be selected according to the operation instruction of magnetic bead reagent using the volume ratio of sample and magnetic bead reagent
It selects, when the PCR product and primer dimer being not much different to size separate, volume ratio for example 1:1.0~1.8.
In the present invention, ultrafiltration centrifugal process can be any for removing the ultrafiltration centrifugal process of primer dimer, exemplary
Step may is that
1. rinse ultra-filtration centrifuge tube；
2. centrifugal column is inverted in collecting pipe, low-speed centrifugal (500 × g~800 × g, 20~60s), it then will centrifugation
Column is suitable to be placed in collecting pipe；
3. Tris-HCl (9~11mM) and PCR product (so that total volume is 500 μ l) are added into centrifugal column, 10000 ×
G~14000 × g room temperature is centrifuged 1~4min；
4. suck partially liq in collecting pipe (450 μ l), then into centrifugal column plus same volume Tris-HCl (9~
11mM), 10000 × g~14000 × g room temperature is centrifuged 1~4min；Repeatable step is 4. one to three times；
5. centrifugal column is inverted in new collecting pipe, low-speed centrifugal (500 × g~800 × g, 20~60s), draws and receive
All liquid is into new pipe in collector.
In the present invention, paramagnetic particle method can be the magnetic bead reagent by the way that certain volume is added into nucleic acid, and removal nucleic acid is molten
Primer dimer and protein, salt ion in liquid etc., reach the paramagnetic particle method of purifying purpose, and illustrative steps may is that
1. magnetic bead (mix, wink from) is added into the pipe containing sample to be processed by the volume of 1:1.0~1.8, one is reacted at room temperature
The section suitable time (5~10 minutes)；
2. separating magnetic bead, solution is sucked；
3. washing magnetic bead (80% ethyl alcohol), discards supernatant, repeat this step 1~2 time；
4. separating magnetic bead again, residual solution is removed, it is dry；
5. the eluent (Tris-HCL or nuclease-free water etc.) of proper volume is added, oscillation is mixed, and is eluted on magnetic bead
DNA；
6. separating magnetic bead, supernatant is transferred in new pipe, obtains product nucleic acid after purification.
The present invention is described in more detail by the following examples, but the present invention is not limited to these Examples.
Embodiment 1 generates the lesser primer dimer of magnitude difference using PCR
Amplified production length is made to be 100bp according to the EGFR gene design primer on 20 exon of the mankind,
The primer dimer of generation is about 61bp, carries out six groups of PCR reactions according to following experiment condition, then acquired PCR product is put down
6 parts are divided into, is denoted as sample 1~6 respectively.
Reaction system (25 μ l):
Response procedures:
Embodiment 2 purifies PCR product using distinct methods
Experimental method
Ultrafiltration centrifugal process:
To the 10mM Tris- that 500 μ l are added in ultra-filtration centrifuge tube (Millipore, article No. UFC505096,50kD)
HCl, 10000 × g~14000 × g room temperature are centrifuged 1~4min, exhaust the liquid in collecting pipe with liquid-transfering gun；Centrifugal column is inverted
In collecting pipe, 700 × g is centrifuged 30 seconds, then by centrifugal column along being placed in collecting pipe.10mM Tris- is added into centrifugal column
HCl and sample to be processed, so that total volume is 500 μ l；10000 × g~14000 × g room temperature is centrifuged 1~4min；Use liquid-transfering gun
450 μ l liquid in collecting pipe is sucked, 450 μ l 10mM Tris-HCl, 10000 × g~14000 × g are then added into centrifugal column
Room temperature is centrifuged 1~4min, repeats twice；Centrifugal column is inverted in new collecting pipe, 700 × g is centrifuged 30 seconds, is inhaled with liquid-transfering gun
Take liquid all in collecting pipe into a clean 1.5mL low adsorption EP pipe.
Paramagnetic particle method:
Body step are as follows: sample to be processed is added in 1.5mL low adsorption EP pipe, and presses 1:1.4~1:1.8 volume ratio
It is added magnetic bead reagent (BeckMan Coulter, Agencourt AMPure XP 60mL Kit)；Mixing, wink be vortexed from room temperature
Reaction 5 minutes；EP pipe is put on magnetic frame, completely to magnetic bead absorption, solution becomes after clarification, sucks solution；Use 80%
Ethanol washing magnetic bead twice；Remove EP pipe, wink from；EP pipe is put on magnetic frame again, after magnetic bead absorption completely, is used
Rifle sucks residual solution；EP pipe lid is opened, it is 5 minutes dry；22 μ l 10mM Tris-HCl are added, be vortexed mixing, wink are from room
Temperature is incubated for 5 minutes；EP pipe is put on magnetic frame, completely to magnetic bead absorption, 20 μ l supernatants is drawn with liquid-transfering gun and is managed in new EP
In.
Sample 1~6 obtained in embodiment 1 is handled:
Any purifying is not carried out to sample 1, is used as control；
Paramagnetic particle method (volume ratio of sample and magnetic bead reagent is 1.8) purifying is only carried out to sample 2；
Ultrafiltration centrifugal process (10000 × g, 1min) purifying is only carried out to sample 3；
Ultrafiltration centrifugal process (14000 × g, 4min) purifying is only carried out to sample 4；
Ultrafiltration centrifugal process (10000 × g, 1min) purifying is first carried out to sample 5, then carries out paramagnetic particle method (sample and magnetic bead examination
The volume ratio of agent is 1.8) to purify；
Ultrafiltration centrifugal process (14000 × g, 4min) purifying is first carried out to sample 6, then carries out paramagnetic particle method (sample and magnetic bead examination
The volume ratio of agent is 1.8) to purify.
3 DNA concentration of embodiment and primer dimer purification result
Using Qubit3.0 fluorescent quantitation instrument (Life Invitrogen) to sample 1~6 processed in embodiment 2 into
The measurement of row DNA concentration, as a result as shown in table 1 below:
Table 1
Segment detection is carried out to above-mentioned sample using Qsep100 full-automatic foranalysis of nucleic acids system (Bioptic) simultaneously, as a result
As shown in Figure 1.
In conjunction with table 1 and Fig. 1 it is found that compared with not purified sample 1, the sample 2 purified through paramagnetic particle method does not make primer
The amount of dimer is reduced；It is also surplus although the amount of primer dimer significantly reduces in the sample 3 and 4 purified through ultrafiltration centrifugal process
Remaining part point fails to effectively remove；And primer two is substantially not present in the sample 5 and 6 item successively purified through ultrafiltration centrifugal process and paramagnetic particle method
Aggressiveness.
Parameter of noncentricity advanced optimizes in 4 ultrafiltration centrifugation of embodiment and magnetic bead method for combined use
The preparation and processing of sample 7~9
Three groups of PCR reactions are carried out according to the method in embodiment 1, then acquired PCR product is equally divided into 3 parts, respectively
It is denoted as sample 7~9.
Ultrafiltration centrifugal process purifying is first carried out to sample 7~9 according to method in embodiment 2, then carries out paramagnetic particle method (sample and magnetic
The volume ratio of pearl reagent is 1.8) to purify, and wherein the parameter of noncentricity of sample 7,8,9 is respectively 10000 × g, 1min；12000 × g,
2min；14000 × g, 4min.
The purification result of sample 7~9 measures
DNA concentration is carried out to processed sample 7~9 using Qubit3.0 fluorescent quantitation instrument (Life Invitrogen)
Measurement, as a result as shown in table 2 below.
Table 2
Segment detection is carried out to above-mentioned sample using Qsep100 full-automatic foranalysis of nucleic acids system (Bioptic) simultaneously, as a result
As shown in Figure 2.
In conjunction with table 2 and Fig. 2 it is found that primer is substantially not present in the sample 7~9 successively purified through ultrafiltration centrifugal process and paramagnetic particle method
Dimer, and parameter of noncentricity is 12000 × g, DNA output is relatively higher when 2min (sample 8).
Influence of the magnitude difference of 5 primer dimer of embodiment and PCR product to purification effect
The preparation and processing of PCR product
Amplified production length is made to be point according to the EGFR gene template design primer on 20 exon of the mankind
Not Wei 87bp, 100bp, 150bp, 228bp, the primer dimer of generation is each about 61bp.It is carried out according to the method in embodiment 1
PCR reacts, wherein 2 parts of 87bp product amplification, and 3 parts of 228bp product amplification, 100bp product and 150bp product respectively expand 1 part.
Referring to the method in embodiment 2, above-mentioned product is purified, concrete mode are as follows:
87bp is only centrifuged: only carrying out ultrafiltration centrifugal process (12000 × g, 2min) purifying；
87bp centrifugation+magnetic bead: ultrafiltration centrifugal process (12000 × g, 2min) purifying is first carried out, then carries out paramagnetic particle method purifying (sample
The volume ratio of product and magnetic bead reagent is 1:1.8)；
100bp centrifugation+magnetic bead: ultrafiltration centrifugal process (12000 × g, 2min) purifying is first carried out, then carries out paramagnetic particle method purifying
(volume ratio of sample and magnetic bead reagent is 1:1.6)；
150bp centrifugation+magnetic bead: first carrying out ultrafiltration centrifugal process (12000 × g, 2min) purifying, is carrying out paramagnetic particle method purifying
(volume ratio of sample and magnetic bead reagent is 1:1.6)；
228bp centrifugation+magnetic bead: first carrying out ultrafiltration centrifugal process, (12000 × g, 2min, centrifugal column is purchased from Millipore and cuts
Staying molecular weight is 100kD) purifying, then carry out paramagnetic particle method purifying (volume ratio of sample and magnetic bead reagent is 1:1.4)；
228bp is only centrifuged: only carrying out ultrafiltration centrifugal process (12000 × g, 2min, the molecular cut off of centrifugal column are 100kD)
Purifying；
228bp only magnetic bead: only carry out paramagnetic particle method purifying (volume ratio of sample and magnetic bead reagent is 1:1.4).
Purification result measurement
DNA concentration survey is carried out to processed each product using Qubit3.0 fluorescent quantitation instrument (Life Invitrogen)
It is fixed, as a result as shown in table 3 below.
Table 3
| Sample | Finally obtained DNA volume (μ l) | Finally obtained DNA concentration (ng/ μ l) | DNA total amount (ng) |
| 87bp is only centrifuged | 60 | 0.91 | 54.6 |
| 87bp centrifugation+magnetic bead | 20 | 2.55 | 51 |
| 100bp centrifugation+magnetic bead | 20 | 3.54 | 70.8 |
| 150bp centrifugation+magnetic bead | 20 | 5.42 | 108.4 |
| 228bp centrifugation+magnetic bead | 20 | 3.63 | 72.6 |
| 228bp is only centrifuged | 60 | 1.3 | 78 |
| 228bp only magnetic bead | 20 | 3.68 | 73.6 |
Segment detection is carried out to above-mentioned sample using Qsep100 full-automatic foranalysis of nucleic acids system (Bioptic) simultaneously, as a result
As shown in Figure 3.
In conjunction with table 3 and Fig. 3 it is found that when PCR product and the magnitude difference of primer dimer are between 20bp~170bp,
Ultrafiltration centrifugal process and paramagnetic particle method combination can efficiently remove primer dimer.
Claims (13)
1. a kind of method for removing primer dimer in PCR product, comprising:
1) PCR product is purified using ultrafiltration centrifugal process, wherein parameter of noncentricity used in ultrafiltration centrifugal process is 8000
× g ~ 15000 × g, 40 seconds ~ 5 minutes；The molecular cut off of used ultra-filtration centrifuge tube is 50kD ~ 100kD；
2) 1) the middle product obtained is purified using paramagnetic particle method, further to remove primer dimer, wherein used magnetic
Bead surface is coated with carboxylic group, and the volume ratio of the PCR product and magnetic bead reagent is 1:1.0 ~ 1.8；
Wherein, the PCR product and the magnitude difference of primer dimer are 20bp ~ 170bp.
2. according to the method described in claim 1, wherein, the magnitude difference of the PCR product and primer dimer be 26bp ~
167bp。
3. according to the method described in claim 2, wherein, the magnitude difference of the PCR product and primer dimer be 30bp ~
100bp。
4. according to the method described in claim 1, wherein, parameter of noncentricity used in ultrafiltration centrifugal process be 10000 × g ~
14000 × g, 1 ~ 4 minute.
5. according to the method described in claim 4, wherein, parameter of noncentricity used in ultrafiltration centrifugal process be 11000 × g ~
13000 × g, 1.5 ~ 2.5 minutes.
6. the application described in claim 1 gone in PCR product in the method for primer dimer of magnetic bead reagent, wherein described
Magnetic bead reagent is used to handle the PCR product after ultrafiltration is centrifuged, and the magnitude difference of the PCR product and primer dimer
For 20bp ~ 170bp.
7. application according to claim 6, wherein the PCR product and the magnitude difference of primer dimer be 26bp ~
167bp。
8. application according to claim 7, wherein the PCR product and the magnitude difference of primer dimer be 30bp ~
100bp。
9. application according to claim 6, wherein ultrafiltration centrifugation used in parameter of noncentricity be 10000 × g ~
14000 × g, 1 ~ 4 minute.
10. application according to claim 9, wherein ultrafiltration centrifugation used in parameter of noncentricity be 11000 × g ~
13000 × g, 1.5 ~ 2.5 minutes.
11. reagent combines the application described in claim 1 gone in PCR product in the method for primer dimer, wherein described
Primer dimer is reacted through PCR to be generated, and the magnitude difference of the primer dimer and PCR product is 20bp ~ 170bp, the examination
Agent combination includes ultra-filtration centrifuge tube and magnetic bead reagent.
12. the magnitude difference of application according to claim 11, the PCR product and primer dimer be 26bp ~
167bp。
13. the magnitude difference of application according to claim 12, the PCR product and primer dimer be 30bp ~
100bp。
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| US5605798A (en) * | 1993-01-07 | 1997-02-25 | Sequenom, Inc. | DNA diagnostic based on mass spectrometry |
| EP2931899A1 (en) * | 2012-12-12 | 2015-10-21 | The Broad Institute, Inc. | Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof |
| CN106701737B (en) * | 2015-11-17 | 2020-07-31 | 安诺优达基因科技(北京)有限公司 | High-efficiency DNA purification magnetic bead reagent with fragment selective purification capability |
| CN107236726A (en) * | 2017-05-23 | 2017-10-10 | 北京创新乐土基因科技有限公司 | Clean CL purification kits and its application |
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| Title |
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| 3种PCR产物纯化方法的比较;潘尚领 等;《广西医科大学学报》;20011031;第18卷(第5期);第768-769页 |
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