CN110106230A - A kind of enrichment method of maternal blood foetal DNA - Google Patents
A kind of enrichment method of maternal blood foetal DNA Download PDFInfo
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Abstract
The present invention provides a kind of enrichment method of maternal blood foetal DNA, the present invention captures fetus excretion body, to extract DNA from fetus excretion body mainly by the magnetic microsphere of fixed specific antibody in conjunction with the excretion body in pregnant woman blood plasma.The method of the present invention can from pregnant woman blood plasma/serum isolating fetal excretion body, and therefrom extract foetal DNA, very efficient to obtain extremely micro foetal DNA, method is simple, practical, efficient, is also applied for processing while multiple samples.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of enrichment method of maternal blood foetal DNA.
Background technique
In China, annual Newborn Birth-defects up to 800,000-120 ten thousand carry out Prenatal Screening as early as possible and diagnosis are optimal
First counter-measure, however diagnostic method conventional at present is all to have traumatic method, has certain danger to fetus and pregnant woman
It is dangerous, therefore the method for developing new Non-invasive Prenatal Diagnosis has important clinical meaning.Lo in 1997 etc. has found in pregnant woman
There are a small amount of free fetal dnas with regard to middle for peripheral blood, to make it possible Non-invasive Prenatal Diagnosis.
However the foetal DNA content dissociated in maternal blood is rare, and is mingled in mother body D NA a large amount of in blood plasma
Together, this has caused great difficulties to the detection of fetus dissociative DNA with separating, how from maternal blood efficiently concentrating is swum
From foetal DNA become the focus of currently associated research.
Currently, the method for separating free fetal dna from Maternal plasma has very much, including electrophoresis enrichment and Beads enrichment method
Deng.But since foetal DNA content is low, and the clip size of foetal DNA and the clip size difference of maternal circulation DNA are unknown
Aobvious, part is that fragment length is identical, therefore is actually difficult with separation of the electrophoresis such methods by different size of segment
Realize the efficiently concentrating of foetal DNA, this method be easy to cause pollution there is also a large amount of damages；And currently based on magnetic bead
Separation method specificity is not high, and the rate of recovery is low, and higher cost, is also not easy to obtain the fetus dissociative DNA of efficiently concentrating.
With the development of noninvasive pre-natal diagnosis technology, clinical research and diagnostic application are no longer satisfied in utilization parent blood
The risk profile of the carry out related disease of foetal DNA in slurry, and the diagnostic requirements of some hereditary diseases are also increasingly urgent to, if from
In Maternal plasma by micro foetal DNA carry out efficient physical separation be also carry out fetus prenatal genetic disease diagnosis premise it
One.
Therefore there is an urgent need to develop a kind of new efficient, accurate, method for can be realized foetal DNA physical separation.
Summary of the invention
Goal of the invention: technical problem to be solved by the invention is to provide a kind of enrichment sides of maternal blood foetal DNA
Method.
A kind of technical solution: in order to solve the above technical problems, the technical solution adopted by the present invention are as follows: maternal blood fetus
The enrichment method of DNA, the enrichment method the following steps are included:
1) maternal blood is collected, is stored using anticoagulant tube, upper plasma is centrifugated；
2) separating step 1) blood plasma in excretion body；
3) by washed antibody magnetic bead in (cleaned label being had to the magnetic bead (secondary antibody magnetic bead) of secondary antibody in centrifuge tube
It is placed in clean centrifuge tube), fetal specific antibody is added, mildly tilts and rotates to be incubated for and obtain the anti-of special sex modification
Body magnetic bead；
4) antibody magnetic bead for being coated with special sex modification is added in the blood plasma excretion body suspension of step 2) separation, mixes,
Rotation is incubated for 30-60min and obtains reaction solution；
5) reaction solution in step 4) adsorbs magnetic bead with magnetic frame, inhales and abandon reaction solution, then uses PBS buffer solution
Cleaning is primary, in the state of magnetic frame absorption, inhales and abandons supernatant；
6) eluent is added on step 5) treated magnetic bead, is detached from magnetic frame, mixes, is placed at room temperature for, collects elution
Liquid is fetus excretion liquid solution；
7) extract fetus excretion liquid solution in DNA to obtain the final product.
Wherein, fetal specific antibody is to include but be limited to PLAP, HLA-G, CK-7 etc. in the step 3).
Wherein, the fetal specific antibody in the step 3) and the mass volume ratio of antibody magnetic bead are (0.4~40): 50
μg/μL。
Wherein, the incubation time in the step 3) is 30~60min.
Wherein, in the step 4) blood plasma excretion body suspension be coated with special sex modification antibody magnetic bead volume ratio
40∶1。
Wherein, 1~2h of adsorption time in the step 5).
Wherein, in the step 6) eluent and magnetic bead volume ratio 1: 1.
The utility model has the advantages that compared with prior art, the present invention have the advantages that following characteristic and:
1, the method for the present invention can from pregnant woman blood plasma/serum isolating fetal excretion body, and therefrom extract foetal DNA, it is non-
Often efficient to obtain extremely micro foetal DNA, method is simple, practical, efficient, is also applied for processing while multiple samples.
Compared with previous fetus enrichment method, the biggest advantage is to can be by foetal DNA and mother body D NA physical separation for this method
It comes, other than the detection for the chromosome times type difference that can carry out routinely having carried out at present, common list can also be carried out
The diagnosis of gene genetic disease, if b- thalassemia, colour blindness, hemophilia, albinism etc.；Furthermore which meets Mendelian inheritance
The gene mutation bring disease of rule can also carry out the detection of early stage by foetal DNA, as familial multiple colon ceases
Meat, hereditary hearing impairment etc..The fetal DNA purity that the method for the present invention obtains is high, can directly carry out PCR amplification, the height in downstream
Library etc. is built in flux sequencing, is had a good application prospect.
2, pregnant woman blood plasma sample can be the fresh blood plasma for obtaining and being also possible to freeze after separation, and sample initial amount is 500 μ
L-5mL, as long as last 10 μ L-100 μ L of elution volume.The method of the present invention obtain DNA fragmentation length 75bp-500bp it
Between, it is between 100bp-300bp mostly absolutely, this conforms exactly to the fragment length regularity of distribution of fetus dissociative DNA.
3, the method for the present invention is without harmful reagents such as phenol chloroforms, it is only necessary to which conventional excretion body separation and minim DNA extract
Reagent kit.It is special using classical antigen-antibody because this characteristic of fetus specific antigen is contained in excretion body surface face
The principle that the opposite sex combines can separate foetal DNA.The separation of excretion body is not only able to enriches fetal information, Er Qie
The inhibitor that the subsequent experimentals such as high valence ion can be completely removed during processing washing is conducive to efficiently opening for subsequent experimental
Exhibition.
Detailed description of the invention
Fig. 1 is flow chart of the present invention；
Fig. 2 is the DNA banking process schematic diagram of embodiment 1；
Fig. 3 is the fluorescent quantitation amplification curve of sample in embodiment 2 as a result, wherein 1-1 to 1-8 is male fetus sample
This；
Fig. 4 is the fluorescent quantitation amplification curve of sample in embodiment 3 as a result, wherein 4-1 to 4-5 is determining Tang Shi comprehensive
Fetus sample is levied, 4-6 to 4-8 is normal fetus sample.
Specific embodiment
With reference to the accompanying drawings and examples, technical solution of the present invention is described in detail.
1, reagent of the present invention:
Serum/plasma dissociative DNA extracts kit, producer: Tiangeng, article No.: DP339
Blood plasma excretion body extracts kit, producer: invitrogen, article No.: 4484451
It is coated with the magnetic bead of antibody, producer: invitrogen, article No.: 11201D
Fetal specific antibody HLA-G, producer: BD Biosciences, article No.: 557557
HieffNGSTM Fast Tagment DNA Library Prep Kit forProducer: assist sage is raw
Object, article No.: 12206ES08
Nuclease free water, producer: Ambion, article No.: AM9932
PBS solution, producer: Ambion, article No.: AM9624
BSA, producer: NEB, article No.: B9001
Quantitative fluorescent PCR reagent TB Green PremixEx Taq, producer: TaKaRa, article No.: RR420Q
Gender related gene primer, producer: the raw work in Shanghai
AMELX:forward primer:5 '-cagcttcccagtttaacttctg-3 '
Reverse primer:5 '-ctctcctataccacttagtcag-3 '
AMELY:forward primer:5 '-cagcttcccagtttaacttctg-3 '
Reverse primer:5 '-tgcccaaagttagtaattttacct-3 '
Down syndrome related gene primer, producer: the raw work in Shanghai
DSCR:forward primer:5 '-cagaaatcccagttcatgttgct-3 '
Reverse primer:5 '-cattcccgggtgccatgaacagt-3 '
GAPDH:forward primer:5 '-ctccctctttctttgcagcaa-3 '
Reverse primer:5 '-cagctctcataccatgagtcct-3 '
2, laboratory apparatus of the present invention:
Applied Biosystems 7500Real Time PCR System PCR instrument
Eppendorf high speed freezing centrifuge
Magnetic frame
1 isolating fetal excretion body of embodiment extracts DNA
It selects from four gestational ages of Suzhou City Hospital of Traditional Chinese Medicine to be 14-18 weeks pregnant woman, number 1-4, wherein No. 1-3 is male
Tire, sample 4 are female's tire.The venous blood of 3mL pregnant woman is extracted respectively, and EDTA is anticoagulant, and then 4 DEG C, 1600g is centrifuged 10min, in absorption
Clearly in the cone die bed centrifuge tube of 15mL, 4 DEG C, 16000g is centrifuged 10min, and the careful supernatant that shifts is to new clean preservation pipe
In, the separation of foetal DNA is carried out as follows.
1, excretion body separates:
According to blood plasma excretion body extracts kit specification, pregnant woman blood plasma interior excretion body is extracted, the specific steps are as follows:
1.1 take 200 μ L of plasma sample in 1.5mL centrifuge tube respectively, and the excretion body separating liquid in 40 μ L kits is added；
1.2 vortex oscillators mix 10s or more, have cotton-shaped muddy appearance in solution at this time, are then placed into 4 DEG C of refrigerators
30min。
1.3 take out blood plasma and the mixing liquid of excretion body separating liquid out of refrigerator, and 10000g is centrifuged under room temperature
10min, careful inhale abandon supernatant, retain precipitating.
Precipitating is resuspended in the PBS solution of 1.4 40 μ L of addition, and the liquid after resuspension is the excretion body in blood plasma.
2, it is coated with the preparation of the magnetic bead of special sex modification antibody
For fetus excretion body, special sex modification is carried out to the antibody on magnetic bead, the specific steps are as follows:
2.1 illustrate according to magnetic bead kit, clean antibody magnetic bead；
2.2 to take 50 cleaned μ L to be coated with the magnetic bead of antibody special in 0.4~40 μ g fetus in 1.5mL centrifuge tube, is added
Heterogenetic antibody HLA-G is mildly tilted and is rotated 30~60min of incubation；
Centrifuge tube is placed on magnetic frame suction after 2min by 2.3 abandons supernatant；
2.4 take down centrifuge tube from magnetic frame, and the PBS solution that 1ml contains BSA are added, magnetic bead is resuspended；
2.5 repeat step 2.3 and 2.4 twice；
Centrifuge tube is placed on magnetic frame suction abandoning supernatant after 2min by 2.6 obtains the magnetic bead for being coated with special sex modification antibody；
3, the capture of fetus excretion body
3.1 take the blood plasma excretion body of step 1 that being coated in the magnetic bead of special sex modification antibody in step 2 is added, and rotate
Mix 1h；
Centrifuge tube is placed into 2min on magnetic frame by 3.2, and inhales abandoning supernatant；
3.3 take down centrifuge tube from magnetic frame, and the PBS solution that 1ml contains BSA are added, magnetic bead is resuspended；
3.4 repeat step 3.2 and 3.3 twice；
3.5 take down centrifuge tube from magnetic frame, and 40 μ L PBS solutions are added, magnetic bead is resuspended；
Centrifuge tube is placed on magnetic frame and draws supernatant after 2min by 3.6, and fetus excretion body is contained in this supernatant.
4, the DNA in the DNA and fetus excretion body in pregnant woman blood plasma is directly extracted using conventional method.
This experiment is tested using the serum/plasma dissociative DNA extracts kit of Tiangeng biotech firm.
5, the building of micro foetal DNA sequencing library
According to HieffNGSTMFast Tagment DNA Library Prep Kit specification provide banking process into
The building of row sequencing library, basic principle are as follows: it is based on swivel base enzyme process, forHigh-flux sequence platform development carries out
The building of DNA library.
6, experimental result
6.1 are added various concentration fetal specific antibody and different incubation time comparisons
Table 1
Through the results show, the fetal specific antibody of addition is more, and incubation time is more long, and obtained DNA concentration is got over
It is high.
6.2 sequencing result
Sequencing result is referring to table 2, from table 2 it can be seen that the concentration of either fetal concentrations or different chromosomes, first divides
The result that isolated human fetal excretion body extracts DNA again is all got well than directly extracting the result that pregnant woman blood plasma DNA is obtained, it was demonstrated that this method
Feasibility and reliability.
Table 2
2 sex identification of embodiment
1, samples sources
The pregnant woman that 100 parts of plasma samples are 14-18 weeks from the gestational age of Suzhou City Hospital of Traditional Chinese Medicine, every part of about 1mL.Through excessively high
Flux sequencing result is it is known that wherein 60 entitled male tire, 40 entitled female's tires, this sequencing result also with pregnant woman's postpartum born child gender
Unanimously.
2, the sample in step 1 is extracted to the DNA in fetus excretion body according to the method for embodiment 1；Using conventional method
Directly extract the DNA in pregnant woman blood plasma.
3, sex identification
3.1 design sex relevant gene primers:
For the gene of X chromosome are as follows: AMELX, for the gene of Y chromosome are as follows: AMELY.If male fetus, then it is
It can detecte X chromosome, may also detect that Y chromosome；If female child, then X chromosome may only be detected.Primer
Sequence such as the following table 3:
Table 3
3.2 quantitative fluorescent PCR
The DNA obtained using fluorescence quantifying PCR method to step 1 carries out the amplification of specific gene, amplification system and anti-
Answer condition such as the following table 4:
Table 4
Fluorescence quantitative PCR instrument is run according to following table 5 program
Table 5
3.3 experimental result
Table 6
From the data of table 5 it is found that by means of the present invention, isolating fetal excretion body extracts foetal DNA progress again first
Sex identification can go out 60 male fetus and 40 female childs in 100 samples with precise Identification, and use and directly extract
Pregnant woman blood plasma carries out sex identification, and accuracy rate only has 80% and 90%, uses this method as a result, demonstrating there are false negative
The fetus information extracted is more accurate, no parent interference.
The noninvasive pre-natal diagnosis of 3 pregnant woman of embodiment
1, samples sources
The pregnant woman that 100 parts of plasma samples are 14-18 weeks from the gestational age of Suzhou City Hospital of Traditional Chinese Medicine, every part of about 1mL.Through excessively high
Flux sequencing result is it is known that wherein there is 5 fetal diagnosis with Down syndrome.
2, the sample in step 1 is extracted to the DNA in fetus excretion body according to the method for embodiment 1；Using conventional method
Directly extract the DNA in pregnant woman blood plasma.
3, the identification of Down syndrome
3.1 design Down syndrome related gene primers
For exception of the Down syndrome disease on No. 21 chromosomes, DSCR specific primer and reference gene are designed
GAPDH specific primer determines the fetus with Down syndrome according to the difference of the GAPDH and DSCR ratio that amplify.
Primer sequence such as the following table 7:
Table 7
3.2 quantitative fluorescent PCR
The DNA obtained using fluorescence quantifying PCR method to step 1 carries out the amplification of specific gene, amplification system and anti-
Answer condition such as the following table 8:
Table 8
Fluorescence quantitative PCR instrument is run according to following table 9 program
Table 9
3.3 experimental result
Table 10
By this illustration method, isolating fetal excretion body first extracts foetal DNA again and carries out disorder in screening, can accurately reflect
The fetus with Down syndrome is made, and uses and directly extracts pregnant woman blood plasma progress sex identification, accuracy rate only has
80%, there are false negatives as a result, to demonstrate the fetus information that is extracted with this method more accurate, for noninvasive pre-natal diagnosis
Market has huge application value.
In conclusion by means of the invention it is also possible to being combined with conventional detection technological perfectionism, such as high-flux sequence skill
Art, fluorescent quantitative PCR technique etc. are available fine in fetus acquisition of information, sex identification and noninvasive pre-natal diagnosis
Application, it is as a result more accurate compared with conventional isolating fetal DNA technique, reliably.
Obviously, the above embodiment is merely an example for clearly illustrating the present invention, and is not to of the invention
The restriction of embodiment.For those of ordinary skill in the art, it can also be made on the basis of the above description
Its various forms of variation or variation, there is no necessity and possibility to exhaust all the enbodiments, these changes extended out
Change or change and is also among protection scope of the present invention.
Sequence table
<110>Southeast China University
<120>a kind of enrichment method of maternal blood foetal DNA
<141> 2019-04-29
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> AMELX: forward primer
<400> 1
cagcttccca gtttaacttc tg 22
<210> 2
<211> 22
<212> DNA
<213> AMELX:reverse primer
<400> 2
ctctcctata ccacttagtc ag 22
<210> 3
<211> 22
<212> DNA
<213> AMELY: forward primer
<400> 3
cagcttccca gtttaacttc tg 22
<210> 4
<211> 24
<212> DNA
<213> AMELY:reverse primer
<400> 4
tgcccaaagt tagtaatttt acct 24
<210> 6
<211> 23
<212> DNA
<213> DSCR: forward primer
<400> 6
cagaaatccc agttcatgtt gct 23
<210> 6
<211> 23
<212> DNA
<213> DSCR:reverse primer
<400> 6
cattcccggg tgccatgaac agt 23
<210> 7
<211> 21
<212> DNA
<213> GAPDH: forward primer
<400> 7
ctccctcttt ctttgcagca a 21
<210> 8
<211> 22
<212> DNA
<213> GAPDH: reverse primer
<400> 8
cagctctcat accatgagtc ct 22
Claims (8)
1. a kind of enrichment method of maternal blood foetal DNA, which is characterized in that the enrichment method the following steps are included:
1) maternal blood is collected, is stored using anticoagulant tube, upper plasma is centrifugated；
2) separating step 1) blood plasma in excretion body；
3) in centrifuge tube fetal specific antibody is added, mildly tilting and rotating incubation is had in washed antibody magnetic bead
The antibody magnetic bead of special sex modification；
4) antibody magnetic bead that step 3) is coated with special sex modification is added in the blood plasma excretion body suspension of step 2 separation, mixes
Even, rotation is incubated for 30-60min and obtains reaction solution；
5) reaction solution in step 4) adsorbs magnetic bead with magnetic frame, inhales and abandon reaction solution, is then cleaned with PBS buffer solution
Once, it in the state that magnetic frame adsorbs, inhales and abandons supernatant；
6) eluent is added on step 5) treated magnetic bead, is detached from magnetic frame, mixes, is placed at room temperature for, collecting eluent is
For fetus excretion liquid solution；
7) extract fetus excretion liquid solution in DNA to obtain the final product.
2. the enrichment method of maternal blood foetal DNA according to claim 1, which is characterized in that in the step 3)
Antibody magnetic bead be the magnetic bead for being marked with general secondary antibody.
3. the enrichment method of maternal blood foetal DNA according to claim 1, which is characterized in that in the step 3)
Fetal specific antibody is one of PLAP, HLA-G or CK-7.
4. the enrichment method of maternal blood foetal DNA according to claim 1, which is characterized in that in the step 3)
Fetal specific antibody and antibody magnetic bead mass volume ratio be 0.4 ~ 40:50 μ g/ μ L.
5. the enrichment method of maternal blood foetal DNA according to claim 1, which is characterized in that in the step 3)
Incubation time be 30 ~ 60min.
6. the enrichment method of maternal blood foetal DNA according to claim 1, which is characterized in that in the step 4)
The volume ratio 40:1 of blood plasma excretion body suspension and the antibody magnetic bead for being coated with special sex modification.
7. the enrichment method of maternal blood foetal DNA according to claim 1, which is characterized in that in the step 5)
1 ~ 2h of adsorption time.
8. the enrichment method of maternal blood foetal DNA according to claim 1, which is characterized in that in the step 6)
The volume ratio 1:1 of eluent and magnetic bead.
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