CN109609493A - Extract the method and kit of genomic DNA in whole blood - Google Patents
Extract the method and kit of genomic DNA in whole blood Download PDFInfo
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- CN109609493A CN109609493A CN201811585235.6A CN201811585235A CN109609493A CN 109609493 A CN109609493 A CN 109609493A CN 201811585235 A CN201811585235 A CN 201811585235A CN 109609493 A CN109609493 A CN 109609493A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
The present invention provides the methods and kit of genomic DNA in a kind of extraction whole blood.This method includes that leucocyte is isolated from whole blood；Leucocyte is interrupted, the leucocyte after being interrupted；The leucocyte having no progeny of fighting each other carries out cell cracking and the crosslinking of DNA- protein solution, obtains genomic DNA.The method for interrupting leucocyte processing by creative use is easy to be cleaved reconciliation crosslinking to make the site exposure of DNA- protein cross for small fragment by interrupting DNA and protein, to keep extracted DNA relatively more comprehensively complete.And obtained DNA fragmentation meet after the needs of continuing library, do not need to re-start again and interrupt.It is abnormal to leucocyte chromosome Z value caused after fixed to targetedly solve the preservation liquid of dissociative DNA heparin tube, there is the phenomenon that a large amount of fragment deletions and repetition.
Description
Technical field
The present invention relates to nucleic acid fields, in particular to the method and reagent of genomic DNA in a kind of extraction whole blood
Box.
Background technique
In the annual newborn in China, the ratio that birth defect occurs is about 5.9%, and the birth of defect infant is family's band
Huge mental burden and economic pressures are come；And with the increase of pregnant woman age, the illness rate of defect fetus is significantly improved, and
70% birth defect can be found through detection in advance.With traditional Prenatal Screening such as serological screening and puncture diagnosis method
It compares, the noninvasive prenatal gene detection carried out based on maternal blood has many advantages, such as that noninvasive, safe and accurate, detection cycle is short,
And it can effectively avoid invasive antenatal detection bring miscarriage and infection risk.
NIPT is by extracting plasma DNA to detect to Fetal genome, but detecting all is higher
It is carried out under mother body D NA background, easily causes false positive or false negative result.In order to eliminate as much as mother body D NA background interference,
The accuracy rate for improving diagnosis in addition to plasma DNA usually also needs that the leucocyte DNA of pregnant woman is extracted and divided simultaneously
Analysis, to exclude false positive and false negative result.For the burden for reducing pregnant woman, blood is carried out using dissociative DNA heparin tube at present and is adopted
Collection, upper plasma detect dissociative DNA, and the leucocyte of lower layer is detected for mother body D NA.
Currently, mainly comprising the steps that cracking is red thin for the extracts kit of leucocyte genomic DNA on the market
Born of the same parents obtain more pure leucocyte；Leucocyte is cracked, the dissociation of protein and DNA is promoted；Purifying obtains pure genome
DNA；Dissolving DNA, elution obtain Genomic DNA solution.
However it is found after gained white blood cell detection, the chromosome results abnormity of normal person.This Novel presentation is a plurality of
There is a large amount of nonspecific chromosome segment copy number and increases and lack in chromosome, and dosage is higher and relatively low, with clinic reality
Border result is not inconsistent, and influences effective judgement to testing result.
As shown in Figure 1, when -3 < Z value < 3 (the Z value of chromosome be used to describe chromosome value and normal person's average value it
Between distance, be measure chromosome whether a Yi Chang standard), i.e., in a dotted box in the range of when, chromosome is numerical value
Be it is normal, super go beyond the scope thinks chromosome abnormality.It will be seen from figure 1 that the sample of this Healthy People only has two dyes
In the normal range, and more serious exception occurs the Z value of colour solid for remaining chromosome.Fig. 2 is another displaying side of Fig. 1
Formula, abscissa indicate the position of each chromosome, and ordinate represents each site in sequencing data and compares successful reads
Number.A more uniform straight line should be presented in every chromosome of normal sample, rather than waveform shown in Fig. 2 frame is (see Fig. 3
In to the amplification effect figure of part in Fig. 2 frame).
In summary, the extracting method of existing Whole Blood Genomic DNA is difficult to solve to occur in testing result above-mentioned a plurality of
The problem of false positive that a large amount of segment copy numbers of chromosome increase or decrease.
Summary of the invention
The main purpose of the present invention is to provide the methods and kit of genomic DNA in a kind of extraction whole blood, to reduce
The false positive issue occurred in testing result.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of extract genomic DNA in whole blood
Method, this method comprises: isolating leucocyte from whole blood；Leucocyte is interrupted, the leucocyte after being interrupted；Air exercise
The leucocyte having no progeny carries out cell cracking and the crosslinking of DNA- protein solution, obtains genomic DNA.
Further, it from including: that erythrocyte cracked liquid is added into whole blood the step of isolating leucocyte in whole blood, obtains
Crack slurries；Cracking slurries are filtered, leucocyte is obtained.
Further, it carries out ultrasound to leucocyte to interrupt, the leucocyte after being interrupted；Preferably, the item that ultrasound interrupts
Part are as follows: power is 10~50%, and ultrasound 5~40 seconds suspends 5~30 seconds, and total time is 0.5~1 hour.
Further, the leucocyte having no progeny of fighting each other carries out cell cracking and the crosslinking of DNA- protein solution, obtains genomic DNA
The step of include: that cell cracking and DNA- protein solution are carried out using salt ion solution and the leucocyte had no progeny of Proteinase K air exercise
Crosslinking obtains solution crosslinking mixed liquor；DNA in solution crosslinking mixed liquor is precipitated and purified, genomic DNA is obtained.
Further, in the step of precipitating and purify, using the mixture of isopropanol and magnetic bead in solution crosslinking mixed liquor
DNA precipitated and purified；Preferably, the volumetric mixture ratio of isopropanol and magnetic bead is 500:10~100:10.
Further, salt ion solution includes: the NaCl of 0.01~0.5mol/L, the Na2HPO4 of 2~6mmol/L, 0.1
The SDS of the EDTA of the KH2PO4 of~3mmol/L, 0.1~2mmol/L and 0.5~5%.
According to the second aspect of the application, a kind of kit for extracting genomic DNA in whole blood, kit packet are provided
Include erythrocyte cracked liquid, leucocyte is interrupted with re-suspension liquid, write cell lysis buffer and DNA- protein solution crosslinked fluid.
Further, write cell lysis buffer includes the NaCl of 0.01~0.5mol/L, the Na2HPO4 of 2~6mmol/L, 0.1
The SDS of the EDTA of the KH2PO4 of~3mmol/L, 0.1~2mmol/L and 0.5~5%.
Further, DNA- protein solution crosslinked fluid is Proteinase K.
Further, kit further includes DNA precipitated liquid and DNA purifying magnetic bead, and preferably DNA precipitated liquid is isopropanol.
Apply the technical scheme of the present invention, the method that leucocyte processing is interrupted by creative use, by by DNA and
Protein, which interrupts, is easy to be cleaved reconciliation crosslinking, to make institute to make the site exposure of DNA- protein cross for small fragment
The DNA of extraction is relatively more comprehensively complete.And obtained DNA fragmentation meet after the needs of continuing library, do not need to re-start again and beat
It is disconnected.It is different to leucocyte chromosome Z value caused after fixed to targetedly solve the preservation liquid of dissociative DNA heparin tube
Often, the phenomenon that there are a large amount of fragment deletions and repeating.
Detailed description of the invention
The accompanying drawings constituting a part of this application is used to provide further understanding of the present invention, and of the invention shows
Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.In the accompanying drawings:
Fig. 1 shows the Z value situation display diagram of chromosome detected by method according to prior art；
Fig. 2 shows the displayings of the another way of the Z value situation of chromosome detected by method according to prior art
Figure；And
Fig. 3 shows the amplification effect figure of the Z value situation of chromosome shown in frame according to fig. 2.
Fig. 4 shows the Z value situation display diagram of chromosome detected according to the method for the present invention；
Fig. 5 shows the display diagram of the another way of the Z value situation of chromosome detected according to the method for the present invention；
And
Fig. 6 shows the amplification effect figure of the Z value situation of the chromosome according to Fig. 5 frame.
Fig. 7 and Fig. 8 is examined after showing the library sequencing constructed according to the application and the extracted genomic DNA of existing method
The autosomal Z value situation display diagram of each item measured；Wherein, Fig. 7 shows the Z value situation of the 1st to No. 11 chromosome, figure
8 show the Z value situation of the 12nd to the 22nd article of chromosome；Fig. 7 and Fig. 8 orbicular spot represents the application method, and square represents existing
There is method；
Fig. 9 is shown No. 1 sample of A group in the embodiment of the present invention and is passed through using the application method extraction leucocyte genomic DNA
After constructing library sequencing, using bioinformatics to the analyses and comparison result of sequencing data；And
Figure 10 shows No. 1 sample of B group in the embodiment of the present invention and existing method is used to extract leucocyte genomic DNA through structure
After building library sequencing, using bioinformatics to the analyses and comparison result of sequencing data.
Specific embodiment
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.Below in conjunction with embodiment, the present invention will be described in detail.
As background technique is previously mentioned, but the chromosome result of white blood cell detection normal person as the result is shown obtained by existing method
It is abnormal.This Novel presentation is a large amount of nonspecific chromosome segment copy number increase occur in a plurality of chromosome and lack
It loses, dosage is higher and relatively low, is not inconsistent with clinical practice result, influences effective judgement to testing result.
To solve the above-mentioned problems, present inventor has made intensive studies above-mentioned phenomenon, finds after analysis, trip
From having some ingredients, such as formaldehyde in the preservative agent that DNA saves pipe (heparin tube), there is fixed function to leucocyte, although avoiding
Pollution to dissociative DNA, but cause the exception of leucocyte sequencing result.Therefore speculate, leucocyte is by heparin tube
Free aldehyde caused by cell fixed function, cause DNA and protein that excessively crosslinking occurs, so cause to extract obtain it is white
Cell DNA is imperfect, and final sequencing result occurs abnormal.
The extracting method of whole blood DNA in the prior art is had studied, as much as possible further with the " paramagnetic particle method of Ying Ruicheng
Extracts kit is measured in poba gene group DNA " for, which only utilizes cracking in leucocyte cracking, protein isolate matter
The digestion promotion protein dissociation of salt ion and Proteinase K in liquid, but salt ionic concentration and Proteinase K in this method
Digestion the protein in sample with DNA crosslinking cannot all be completely removed, thus cause the exception for building library, this exception passes through
After the amplification of two generation high-flux sequences, what normal chromosomal all had that a large amount of segment copy numbers of a plurality of chromosome increase or decrease shows
As, the interpretation to sequencing result is directly affected, it is final to generate abnormal false positive results.
To sum up, the phenomenon that leading to chromosome abnormality in testing result for dissociative DNA heparin tube, present inventor's base
In the situation for promoting protein dissociation that can not still solve the above problems using the digestion of salt ion and Proteinase K merely, further
It proposes and interrupt on the one hand exposure DNA- protein cross site to leucocyte, on the other hand can also carry out piece to DNA
Duan Hua saves the DNA fragmentation in subsequent Library development flow, then again by salting liquid and Proteinase K to the solution of DNA and protein
Crosslinked action, so that solution crosslinking is more thorough, to keep the DNA extracted relatively more complete, yield is higher.
In addition, having two methods of centrifugation column purification and magnetic beads for purifying in the purifying and absorption of genomic DNA.Centrifugal column
Type absorption makes DNA solution pass through adsorbent thin film by high speed centrifugation, and DNA is trapped on film.This method lacks of both having
Point: the adsorption efficiency of one side DNA is lower, and another aspect elution efficiency is lower, and the yield for eventually leading to DNA is lower.Magnetic bead is inhaled
Attached rule is specifically bind by the charge and DNA of magnetic bead surfaces realizing absorption and purifying to DNA.Use magnetic bead
When method carries out nucleic acid extraction, yield height, high-quality DNA can be not only obtained, additionally it is possible to which the automation of implementation process eliminates multiple
Miscellaneous artificial extraction procedure is adapted to requirement of the market for nucleic acid extraction efficiency.Therefore, the improvement project of the application is purifying
Shi Caiyong magnetic beads for purifying not only makes the yield of DNA higher, but also purity is also higher.So that using the present processes
As shown in Fig. 4, Fig. 5 and Fig. 6, Z value is respectively positioned in the range in dotted line frame the Z value situation of detected chromosome, shows to contaminate
Colour solid numerical value is normal, and every chromosome is linear rather than waveform.
Based on the studies above as a result, applicant proposed the technical solutions of the application.In a kind of typical implementation of the application
In example, a kind of method for extracting genomic DNA in whole blood is provided, this method comprises: isolating leucocyte from whole blood；Dialogue
Cell is interrupted, the leucocyte after being interrupted；The leucocyte having no progeny of fighting each other carries out cell cracking and DNA- protein solution is handed over
Connection, obtains genomic DNA.
This method is by improving existing Whole Blood Genomic DNA extracting method, after sub-argument goes out leucocyte, first dialogue
Cell carries out interrupting processing, and the effect for interrupting processing is the macromoleculars such as DNA breakage and protein, on the one hand makes DNA break Cheng Shi
When the small molecule of length, on the other hand the cross-linking part of DNA- protein can be exposed.Then by being split to leucocyte
Solution release intracellular matter, is then crosslinked DNA- protein solution, so that DNA is sufficiently released, and then obtains relatively completeer
Whole genomic DNA.
In the above method, existing step can be used the step of isolated leucocyte from whole blood.For example, using red thin
Cellular lysate liquid carries out cracking removing to red blood cell.In a kind of preferred embodiment, the step of leucocyte is isolated from whole blood
Include: that erythrocyte cracked liquid is added into whole blood, obtains cracking slurries；Cracking slurries are filtered, leucocyte is obtained.Specifically
Erythrocyte cracked liquid use existing erythrocyte splitting formula of liquid, can also using existing Whole Blood Genomic DNA extract
Related reagent in related kit obtains relatively pure leucocyte.
The operating procedure interrupted in the above method to leucocyte is the inventive improvements step of the application, because existing
All be after DNA extraction in technology, DNA longer to length is interrupted when constructing sequencing library, and have no precedent it is directly right
The report that cell is interrupted.In the application, the condition of operation is specifically interrupted, it can according to the actual situation, existing to DNA
It improves and optimizates to obtain on the conditioned basic interrupted.In a kind of preferred embodiment, ultrasound is carried out to leucocyte and is interrupted, is obtained
Leucocyte to after interrupting；Preferably, the condition that ultrasound interrupts are as follows: power is 10~50%, ultrasound 5~40 seconds, pause 5~30
Second, total time is 0.5~1 hour.Under the conditions of the ultrasound interrupts, the broken and DNA- protein cross of leucocyte is not only realized
The exposure in site, and the effect to DNA fragmentation is realized, fragmentation is carried out to DNA when saving subsequent builds library
Operating procedure.
It is above-mentioned leucocyte is interrupted after, the step of cell cracking to leucocyte and DNA- protein solution are crosslinked, can
To use existing operating procedure.In a kind of preferred embodiment, the leucocyte having no progeny of fighting each other carries out cell cracking and DNA- egg
The crosslinking of white matter solution, the step of obtaining genomic DNA include: the leucocyte had no progeny using salt ion solution and Proteinase K air exercise into
Row cell cracking and the crosslinking of DNA- protein solution obtain solution crosslinking mixed liquor；To solution crosslinking mixed liquor in DNA carry out precipitating and
Purifying, obtains genomic DNA.
Existing method is to directly adopt salt ion solution and protease in the case where the operation interrupted without leucocyte
K carries out cracking reconciliation crosslinking, and on the one hand since cell encapsulation acts on, the crosslink sites of DNA- protein are blanked and are not easy by egg
White enzyme K is recognized, and on the other hand, the concentration of salt ion solution is improper, so that DNA is not easy to be solved crosslinking and release.And
When above-mentioned salt ion solution and the Proteinase K processing of the application, cell cracking and DNA- albumen can be more fully played relatively
The effect of matter solution crosslinking, so that the DNA being crosslinked be made more thoroughly to release relatively, thus obtain relatively more comprehensively, it is completeer
Whole genomic DNA.
After being released by solution crosslinking, the precipitating reagent purification process of DNA can be carried out using existing routine operation.
In order to further increase the purity and yield of DNA, in a kind of preferred embodiment, above-mentioned precipitating and purifying the step of in, adopt
The DNA in solution crosslinking mixed liquor is precipitated and purified with the mixture of isopropanol and magnetic bead；Preferably, isopropanol and magnetic bead
Volumetric mixture ratio be 500:10~100:10.
It has been observed that since magnetic beads for purifying is specifically bind realizing to DNA's by the charge and DNA on surface
Absorption and purifying compare column purification using magnetic beads for purifying, not only make the yield of DNA higher, but also purity is also higher.In addition,
Can also implementation process automation, eliminate complicated artificial extraction procedure, be adapted to market for nucleic acid extraction efficiency
It is required that.
In order to further increase the lytic effect for the leucocyte that air exercise is had no progeny, to more fully make DNA from DNA- albumen
It is released in matter cross-linking agent, in a kind of preferred embodiment, salt ion solution includes: the NaCl of 0.01~0.5mol/L, and 2
The Na of~6mmol/L2HPO4, the KH of 0.1~3mmol/L2PO4, 0.1~2mM EDTA and 0.5~5%SDS.The application institute
The salt ion solution of improved salt ion solution compared to the prior art has and cracks abundant, lysis efficiency height, promotes DNA- egg
The change of space structure occurs for the cross-linking agent of white matter, and the site made it combine sufficiently exposes, and the digestion for being more advantageous to Proteinase K is made
With the advantages of enabling DNA sufficiently to discharge, finally obtain complete DNA.
According to the extracted DNA of the method for the above preferred embodiment of the application,
In second of the application typical embodiment, a kind of kit for extracting genomic DNA in whole blood is provided,
Kit includes that erythrocyte cracked liquid, leucocyte are interrupted with re-suspension liquid, write cell lysis buffer and DNA- protein solution crosslinked fluid.It should
Kit realizes the erythroclasis in the blood sample acquired to heparin tube by erythrocyte cracked liquid, further passes through centrifugal filtration
And remove, to obtain relatively pure leucocyte, laggard Break Row then is resuspended in leucocyte using leucocyte re-suspension liquid, is beaten
The leucocyte having no progeny further passes through cracking and the DNA that write cell lysis buffer and DNA- protein solution crosslinked fluid realize leucocyte
Release, to obtain genomic DNA.The genomic DNA loss extracted from whole blood using the kit is relatively fewer,
Not only yield is high by DNA, but also more comprehensively, completely.
In a kind of preferred embodiment, write cell lysis buffer includes the NaCl of 0.01~0.5mol/L, 2~6mmol/L
Na2HPO4, the KH of 0.1~3mmol/L2PO4, 0.1~2mM EDTA and 0.5~5%SDS.The kit of the application is wrapping
When containing the write cell lysis buffer, have extraction time short, easy to operate, obtained DNA integrity degree is higher, with high purity and be suitable for
The advantages of extraction of cellular genome after formaldehyde substance is fixed.
In a kind of preferred embodiment, DNA- protein solution crosslinked fluid is Proteinase K.By scinderin matter, promote
The dissociation of protein and DNA.
In a kind of preferred embodiment, kit further includes DNA precipitated liquid and DNA purifying magnetic bead, preferably DNA precipitated liquid
For isopropanol.
In the kit of the application simultaneously containing the ingredient in above preferred embodiment when, gene in whole blood can not only be made
The extraction more convenient and quicker of group DNA, and DNA yield can be made high, purity is high.
Further illustrate the beneficial effect of the application below in conjunction with specific embodiments.
Embodiment 1
The present embodiment chooses the plasma sample (number 1,2,3) of three normal persons, and each sample is divided into two groups, one group
Using the present processes (A group), one group utilizes existing method (B group, no longer list detailed step herein), respectively from whole blood
The extracting genome DNA of leucocyte is carried out, and carries out sequencing and bioinformatic analysis after routinely building library, pays close attention to two
The chromosomal abnormalities of group normal sample.Wherein, in A group, the detailed process of the application extracting method is as follows:
One, the extracting method of leucocyte genomic DNA
(1) cracking of whole blood red blood cell
Addition 0.5mL is fresh first into the centrifuge tube of 1.5mL or freezes the Buffer A of the whole blood and 1.0mL that melt
(specific formula are as follows: 10mmol/L Tris-HCL pH7.6,10mmol/L NaCl, 5mmol/L MgCl2), it is mixed by inversion 10 times
After be stored at room temperature 10min.Centrifuge tube is put into a centrifuge into 16000g centrifugation 1min, supernatant is removed later, retains leucocyte
Precipitating.1.0mL Buffer A is added into centrifuge tube again, the concussion that is vortexed extremely precipitates and centrifuge tube is put into centrifugation after block disappears
16000g is centrifuged 1min in machine.Centrifuge tube is taken out from centrifuge, centrifuge tube is buckled in clean water suction after removing solution
1min is stood on paper.Period avoids precipitating block from falling off from tube bottom.More pure leucocyte after removal red blood cell can be obtained.
(2) cracking of leucocyte is interrupted and is cracked
Obtained leucocyte is resuspended in 100 μ L PBS solutions, resuspension, which is placed on, interrupts the enterprising Break Row of instrument.It interrupts
When, the difference condition of 3 samples use are as follows: power 10%, 20% and 50%, ultrasound 5 seconds, 15 seconds and 40 seconds suspend 5 seconds,
10 seconds and 30 seconds, total time 0.5h, 1h and 1h.The effect interrupted is the macromoleculars such as DNA breakage and protein, is on the one hand
On the other hand DNA break the cross-linking part of DNA- protein can be exposed at the small molecule of suitable length.Divide after interrupting
20 μ L Proteinase Ks and three kinds of 200 different μ L Buffer B (B1 formulas are as follows: 0.2mol/L are not added into 3 centrifuge tubes
NaCl, the Na of 4mmol/L2HPO4, the KH of 1mmol/L2PO4, 0.5mM EDTA and 0.5%SDS；B2 formula are as follows:
The Na of the NaCl of 0.01mol/L, 2mmol/L2HPO4, the KH of 0.11mmol/L2PO4, 0.1mM EDTA and 0.5%SDS；B3
Formula are as follows: the Na of the NaCl of 0.5mol/L, 6mmol/L2HPO4, the KH of 3mmol/L2PO4, 2mM EDTA and 5%SDS), whirlpool
Centrifuge tube is put in 65 DEG C after rotation concussion 10s, concussion cracking 30min on the constant temperature blending instrument of 1600rpm.The effect of Buffer B
It is lytic cell, and Proteinase K then passes through scinderin matter, promotes the dissociation of protein and DNA.
(3) precipitating and purifying of DNA
Isopropanol that 310 μ L, 510 μ L and 110 μ L are mixed well and magnetic bead are added into the centrifuge tube of above-mentioned 3 samples
Mixture is immediately placed in 25 DEG C after the concussion 5s that is vortexed, mixes 2min on the constant temperature blending instrument of 1600rpm.This step is by different
After propyl alcohol precipitates DNA, magnetic bead carries out adsorption recovery to DNA.
(4) magnetic beads for purifying
1min is stood by having added the centrifuge tube of magnetic bead to be put on magnetic frame, is thoroughly abandoned after magnetic bead is adsorbed in centrifuge tube side wall
Remove solution.750 μ L Buffer D1 are added, and (formula is 1mol/L guanidine hydrochloride, 10mmol/L Tris-HCl pH7.6,5mmol/L
EDTA, 75% ethyl alcohol), be vortexed point shake 1min, and centrifuge tube is put on magnetic frame and stands 1min, is adsorbed in centrifuge tube side to magnetic bead
Solution is discarded after wall.750 μ L Buffer D2 (formula is 20mmol/L Tris-HCl, 75% ethyl alcohol) is added, be vortexed point shake
Centrifuge tube is put on magnetic frame and stands 1min by 1min, discards solution after Magbeads is adsorbed in centrifuge tube side wall.It is of short duration from
After the heart, pipettor removes tube bottom solution, 750 μ L Buffer E (formula is 10mmol/L Tris-HCl) is added, wait what is hanged
Magnetic bead sufficiently discards solution after being adsorbed in centrifuge tube side wall again immediately.This step is to include residual in genomic DNA of going out
The impurity such as the protein, the salt ion that stay.
(5) DNA is eluted
100-200 μ L eluent is added in containing the centrifuge tube of magnetic bead after purification.The concussion that is vortexed makes magnetic bead sufficiently suspend
56 DEG C are put in after in eluent, concussion elution 10min on the constant temperature mixed instrument of 1600rpm.Centrifuge tube is put in magnetic force
2min is stood on frame, and solution is transferred to -20 in new centrifuge tube with pipettor after magnetic bead is sufficiently adsorbed in centrifuge tube side wall
It DEG C saves backup.By the principle of absorption with high salt, less salt elution, pure, high-quality genomic DNA is obtained using magnetic bead.
(7) concentration mensuration
The DNA concentration measured with Qubit method.
Two, build library (this step starts, and two groups of operations are identical)
(1) end reparation, purifying are carried out using the DNA fragmentation that BIOO kit air exercise is had no progeny.
(2) the DNA fragmentation adjunction head after reparation is attached, is purified using BIOO kit.
(3) PCR amplification, purifying are carried out to the DNA fragmentation after adjunction head using BIOO kit, measures concentration.
Three, quality inspection and upper machine
Machine, the nexseq500 sequenator of upper machine platform selection illumina platform, sequencing are carried out after the inspection of library library is qualified
Strategy is PE75 (Pair-End, abbreviation PE), and each sample sequencing depth is 5M.
Four, result and analysis
(1) DNA extracting concentration and purity (being shown in Table 1)
Table 1:
As shown in Table 1, the yield of the gDNA extracted with existing method is lower than the extracted gDNA of the present processes.
When the numerical value of A260/A280 is located at 1.8-2.0, when A260/A230 > 2, the DNA that extracts without protein, carbohydrate or salt from
The interference of son, the DNA purity for showing that extraction obtains are higher.From table 1 it can also be seen that the present processes extracted gDNA
Quality is more excellent.
(2) QC Quality Control result compares
Quality Control is carried out to the library of A group and B group, has been respectively compared the base ratio for reaching Q30, the reads of exact matching
The number of sites of number and exact matching, comparison result is by table 2.
Table 2:
From Table 2, it can be seen that the library of A group is compared with B group in Q30 base ratio, the reads number of exact matching and ratio side
Face is all higher；From the point of view of G/C content, the G/C content of B group is relatively more micro- to be higher than 0.38~0.42 this range.This shows with application
The extracted DNA of method constructed by Library Quality it is relatively high.
(3) sequencing Z value result compares
Sequencing result of two groups of samples after analysis of biological information is summarized, 22 often dyeing of three samples have been respectively compared
The Z value of body., horizontal axis represents chromosome numbers, and the longitudinal axis indicates Z value size.Wherein, Fig. 7 show 1 to No. 11 it is autosomal
Z value situation, Fig. 8 show 12 to No. 22 autosomal Z value situations.
Wherein, the sequencing after dot represents library constructed by the leucocyte genomic DNA that A group is extracted through the present processes
As a result, square is represented using the sequencing result behind constructed library after prior art extraction leucocyte genomic DNA.When -3 < Z value
It is normal chromosomal when<3, when Z value<-3 or Z value>3, this chromosome abnormality.The wherein chromosome bands in dotted line frame
Normal, the chromosome Z value outside dotted line frame is abnormal.
Behind library constructed by leucocyte genomic DNA it can be seen from this result with the present processes extraction
The chromosome Z value of sequencing result is normal, and in the library sequencing result for the DNA that existing method is extracted, chromosome abnormality
Ratio is higher.
(4) chromosome comparison result
Fig. 9 and Figure 10 is analyses and comparison result of the bioinformatics to sequencing data.Each figure all represents each text
The comparison situation of 23 chromosomes in library, including 22 autosomes and 1 sex chromosome.The horizontal axis of every chromosome indicates
Position on chromosome, the longitudinal axis indicate to compare successful reads quantity.
For theoretically, genomic DNA can be compared onto chromosome after interrupting by random physical by sequencing
Probability is also consistent, be presented in figure be exactly every chromosome at an arbitrary position on reads number should be equal.Simultaneously
Due to the blood that this sample used is Healthy People, the phenomenon that should not having a large amount of fragment deletions or repeat, so
Reads on item chromosome should be that a uniform line is distributed.
As shown in figure 9, in the comparison result that No. 1 sample of A group obtains after the method in the application is extracted, reads number point
Cloth is uniform, does not occur chromosomal abnormalities (No. 2 of A group are similar thus not shown with the result of No. 3 samples).
And Figure 10 shows that No. 1 sample of B group extracts the comparison knot obtained after leucocyte genomic DNA using existing method
Fruit.Reads number on a plurality of chromosome is unevenly distributed, and wave-like is presented, this shows that chromosome is lacked with a large amount of segment
It becomes estranged repetition, (result of No. 2 of B group and No. 3 samples is similar, does not also illustrate again) is not inconsistent with the truth of sample.
In summary result it is found that the present processes in leucocyte extracting genome DNA yield, DNA quality, library
Quality Control and sequencing result etc. are superior to existing method.
It can be seen from the above description that the above embodiments of the present invention realized the following chievements:
1. the method that the use of the invention interrupts processing, by interrupting DNA and protein for small fragment, thus
The site exposure for making DNA- protein cross, is easy to carry out solution crosslinking by Proteinase K and lysate, to make extracted DNA
It is comprehensively complete.And obtained DNA fragmentation meet after the needs of continuing library, do not need to re-start again and interrupt.To targetedly
Solve dissociative DNA heparin tube saves the liquid chromosome Z value exception caused after fixed to leucocyte, a large amount of segments occurs and lacks
Become estranged repetition the phenomenon that.
2. the present invention adjusts the cracking formula of liquid in solution cross-linking process, leucocyte is interrupted combining, exposure
Under the premise of DNA- protein cross site, so that genomic DNA more fully discharges reconciliation and is crosslinked out, so that DNA be made to obtain
Rate is more.
3. the present invention is on the basis of interrupting processing and improved write cell lysis buffer is formulated, pure further combined with magnetic bead
Change, so that purity is higher than existing methods for extracted genomic DNA, impurity is less.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method for extracting genomic DNA in whole blood, which comprises
Leucocyte is isolated from whole blood；
The leucocyte is interrupted, the leucocyte after being interrupted；
Cell cracking and the crosslinking of DNA- protein solution are carried out to the leucocyte after described interrupt, obtain the genomic DNA.
2. according to the method described in claim 1, the step of isolating leucocyte from whole blood includes:
Erythrocyte cracked liquid is added into the whole blood, obtains cracking slurries；
The cracking slurries are filtered, the leucocyte is obtained.
3. it is interrupted according to the method described in claim 1, carrying out ultrasound to the leucocyte, it is white thin after obtaining described interrupt
Born of the same parents；
Preferably, the condition that the ultrasound interrupts are as follows: power is 10~50%, and ultrasound 5~40 seconds suspends 5~30 seconds, total time
It is 0.5~1 hour.
4. method according to claim 1 or 3 carries out cell cracking and DNA- protein to the leucocyte after described interrupt
Solution crosslinking, the step of obtaining the genomic DNA include:
Using salt ion solution and Proteinase K carries out cell cracking to the leucocyte after described interrupt and DNA- protein solution is handed over
Connection obtains solution crosslinking mixed liquor；
DNA in the solution crosslinking mixed liquor is precipitated and purified, the genomic DNA is obtained.
5. according to the method described in claim 4, the precipitating and purifying the step of in, using the mixture of isopropanol and magnetic bead
DNA in the solution crosslinking mixed liquor is precipitated and purified；Preferably, the volume mixture of the isopropanol and the magnetic bead
Than for 500:10~100:10.
6. according to the method described in claim 4, the salt ion solution includes: the NaCl of 0.01~0.5mol/L, 2~
The Na of 6mmol/L2HPO4, the KH of 0.1~3mmol/L2PO4, 0.1~2mM EDTA and 0.5~5%SDS.
7. a kind of kit for extracting genomic DNA in whole blood, the kit includes that erythrocyte cracked liquid, leucocyte interrupt use
Re-suspension liquid, write cell lysis buffer and DNA- protein solution crosslinked fluid.
8. kit according to claim 7, the write cell lysis buffer includes the NaCl of 0.01~0.5mol/L, 2~
The Na of 6mmol/L2HPO4, the KH of 0.1~3mmol/L2PO4, 0.1~2mM EDTA and 0.5~5%SDS.
9. kit according to claim 8, the DNA- protein solution crosslinked fluid is Proteinase K.
10. kit according to any one of claims 7 to 9, the kit further includes DNA precipitated liquid and DNA pure
Change magnetic bead, the preferably described DNA precipitated liquid is isopropanol.
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Application publication date: 20190412 |