CN103215350B - A kind of assay method of the fetal DNA in maternal plasma DNA content based on mononucleotide polymorphism site - Google Patents
A kind of assay method of the fetal DNA in maternal plasma DNA content based on mononucleotide polymorphism site Download PDFInfo
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Abstract
The invention provides the assay method of a kind of fetal DNA in maternal plasma DNA content based on mononucleotide polymorphism site, including the screening of mononucleotide polymorphism site, the extraction of site DNA in blood plasma, pcr amplification reaction, the step such as calculating of foetal DNA amount.The method method of operating is simple, uses the amplification based on mononucleotide polymorphism site, it is to avoid use the Y chromosome genetic locus mark to be difficult to measure the content of the fetal DNA in maternal plasma dissociative DNA of pregnant female's tire, be widely used, the degree of accuracy high.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of fetal DNA in maternal plasma based on mononucleotide polymorphism site
The assay method of DNA content, further relates to the application of this assay method, such as antenatal detection.
Background technology
Currently, antenatal detection is increasingly paid close attention to by people, and antenatal detection is the effective measures reducing Fetal malformation
One of.But mother and fetus can be caused certain risk by traditional invasive diagnosis technology, thus limit clinically
It uses.
In recent years, non-invasive pre-natal diagnosis receives more and more attention, and fetal DNA in maternal plasma dissociative DNA send out
Now open new approach for non-invasive pre-natal diagnosis undoubtedly.Therefore, around pregnant woman blood plasma dissociative DNA research increasingly
Many, have also been developed many potential clinical practices.Such as, the exception of foetal DNA content and many gestation phases in Maternal plasma
The disease closed is relevant, including pre-eclampsia, premature labor, ante partum hemorrhage, placenta accreta formation, fetal Down syndrome and other tires
Youngster's chromosomal aneuploidy.Therefore, the fetal DNA analysis of mother's blood plasma can as monitoring foetus health potential mark it
One.
But, it owing to the content of fetus dissociative DNA in mother's blood plasma only accounts for the 5%-30% of sum, is in a kind of high parent
Under the background of DNA so that we usually can be too low because of the content of fetus dissociative DNA during carrying out noninvasive pre-natal diagnosis
And present false-negative result.In order to the dissociative DNA in same mother source is distinguished, choose fetus dissociative DNA label at present main
From Y chromosome genetic locus, this makes the detection of fetus dissociative DNA content be confined to cherish in the middle of the pregnant woman of male fetus.Cause
This, develop a kind of new technology being capable of wide variety of detection fetal DNA in maternal plasma DNA content, be a far-reaching work
Make.
Content of the invention
Goal of the invention: it is an object of the invention to provide a kind of simple to operate, the degree of accuracy is high, in widely used pregnant woman blood plasma
The assay method of foetal DNA content.
Technical scheme: a kind of fetal DNA in maternal plasma DNA based on mononucleotide polymorphism site that the present invention provides contains
The assay method of amount, comprises the following steps:
Step one, the screening of mononucleotide polymorphism site: from the list at US National Biotechnology Information center (NCBI)
In nucleotide polymorphic site storehouse (dbSNP), Preliminary screening goes out the mononucleotide polymorphism site meeting following condition:
(1) little gene frequency (MAF) is between 0.4-0.5；
(2) a length of 50-70bp of DNA fragmentation of this pleomorphism site is comprised；
(3) any mononucleotide polymorphism site is not comprised between site upstream and downstream distance site 1-60bp；
(4) DNA fragmentation comprising this pleomorphism site is not located at copy number range of variation in genome mutation database
Within；
(5) fragment residing for this pleomorphism site is special in the range of human genome, it is ensured that this fragment is the mankind
It is unique on genome；
(6) it is compatible for comprising between the DNA fragmentation of this pleomorphism site, i.e. the DNA fragmentation of this pleomorphism site
Between, be not above the complementary series of 8bp, simultaneously the G/C content of the DNA fragmentation of each pleomorphism site should 45-55% it
Between.
In step one, the screening of mononucleotide polymorphism site may utilize software and completes, and comprises the following steps:
(following steps 1 obtain with reference to the accompanying drawings, but, this step and accompanying drawing 1 do not show and filter out this pleomorphism site
The a length of 50-70bp of DNA fragmentation；Additionally, two ends 50-60bp does not comprise SNP site please according to the power 1 changed before on accompanying drawing 1
Modification)
(1) depend on from the mononucleotide polymorphism site storehouse (dbSNP) at US National Biotechnology Information center (NCBI)
Filter according to little gene frequency numerical value, extract SNP position between 0.4-0.5 for the little gene frequency
Point, the fragment length extracting is 50-70bp；
(2) mononucleotide polymorphism site that step (1) obtains is compared with human genome, extract at human gene
It is unique mononucleotide polymorphism site in group；
(3) mononucleotide polymorphism site that filtration step (2) obtains, extracts site upstream and downstream distance site 1-60bp
Between do not comprise the mononucleotide polymorphism site of any mononucleotide polymorphism site；
(4) mononucleotide polymorphism site that step (3) is obtained and US National Biotechnology Information center (NCBI)
Genome mutation database (DGV) in copy number variation (CNV) information compare, filter out comprise copy number variation
(CNV) mononucleotide polymorphism site of information；
(5) mononucleotide polymorphism site that step (4) obtains is compared with BWA index data base, and filter, make residue
Mononucleotide polymorphism site DNA fragmentation between be compatible, i.e. between the DNA fragmentation of this pleomorphism site, do not have
Having more than the complementary series of 8bp, the G/C content of the DNA fragmentation of each pleomorphism site should be between 45-55% simultaneously.
Step 2, the extraction of site DNA in blood plasma: extract dissociative DNA in test plasma, and isolate and include that step one obtains
The dissociative DNA fragment of the site sequence obtaining: first according to the site design probe filtering out；Probe is tied to biomagnetic beads
On；Sample DNA is joined in the biomagnetic beads with probe, make DNA hybridize with probe；Wash away unnecessary DNA profiling；To tie up
Fixed sample DNA elutes from biomagnetic beads.
Step 3, pcr amplification reaction: to the mononucleotide polymorphism site sequences Design PCR primer obtaining in step one,
And utilize the dissociative DNA fragment that polymerase chain reaction (PCR) amplification step two separates, obtain pcr amplification product；
Step 4, the calculating of foetal DNA amount: obtain pcr amplification product order-checking, and each site of statistical analysis to step 3
Order-checking fragment amount, according to the ratio between not iso-allele, calculate the amount of foetal DNA.
Wherein, in step 2, described test plasma is obtained by following methods: extraction 5-10ml maternal blood, centrifugal point
From acquisition blood plasma.
Wherein, in step 2, including the separation method of the dissociative DNA fragment of the site sequence of step one acquisition, including with
Lower step:
(1) the site sequence design probe obtaining according to step one；
(2) it is tied to probe in biomagnetic beads；
(3) dissociative DNA in the test plasma of extraction is joined in the biomagnetic beads with probe, make DNA miscellaneous with probe
Hand over；
(4) unnecessary DNA is washed away with the cleaning fluid in commercialization magnetic bead kit；
(5) with the eluent in commercialization magnetic bead kit, the sample DNA of binding is eluted from biomagnetic beads.
Beneficial effect: the assay method of the fetal DNA in maternal plasma DNA content that the present invention provides, method of operating is simple, adopts
With the amplification based on mononucleotide polymorphism site, it is to avoid use Y chromosome genetic locus mark to be difficult to measure pregnant female's tire
The content of fetal DNA in maternal plasma dissociative DNA, be widely used, the degree of accuracy high.
The method obtains special single nucleotide polymorphism site by screening, is capable of the content of Accurate Determining foetal DNA, keeps away
There is false-negative result in the dissociative DNA impact exempting from mother source, and accuracy is high.Meanwhile, the present invention can use second generation high pass
Measuring sequence platform, the degree of accuracy is high, data volume is big, greatly reduce cost.
Figure of description
Fig. 1 is the flow chart of the screening technique of mononucleotide polymorphism site of the present invention.
Fig. 2 is the flow chart of the assay method of fetal DNA in maternal plasma DNA content of the present invention.
Fig. 3 is the measurement result of fetal DNA in maternal plasma DNA content of the present invention.
Detailed description of the invention
According to following embodiment, the present invention may be better understood.But, as it will be easily appreciated by one skilled in the art that reality
Execute the concrete material proportion described by example, process conditions and result thereof and be merely to illustrate the present invention, and should also will not limit
The present invention described in detail in claims processed.
Reagent used in the present invention and software are respectively as follows:
Reagent:
QiAamp Blood Mini Kit (Qiagen) extracts DNA kit
Taq archaeal dna polymerase is purchased from MBI company
DNTPs is purchased from TOYOBO company
Probe, primer are synthesized by Shanghai bio-engineering corporation
TruSeq DNA HT Sample Prep Kit Support (illumina) sequencing kit
The common redistilled water of ddH2O (making by oneself)
DNTP TOYOBO company
Buffer Takara company
20 μM of forward primer Shanghai bio-engineering corporations
20 μM of reverse primer Shanghai bio-engineering corporations
Archaeal dna polymerase MBI company
The maternal plasma DNA software that 10ngDNA extracts:
Self-editing perl script, BWA
Embodiment 1
Based on the assay method of the fetal DNA in maternal plasma DNA content of mononucleotide polymorphism site, comprise the following steps:
Step one, the screening of mononucleotide polymorphism site: from the list at US National Biotechnology Information center (NCBI)
In nucleotide polymorphic site storehouse (dbSNP), Preliminary screening goes out the mononucleotide polymorphism site meeting following condition:
(1) little gene frequency (MAF) is between 0.4-0.5；
(2) a length of 50-70bp of DNA fragmentation of this pleomorphism site is comprised；
(3) any mononucleotide polymorphism site is not comprised between site upstream and downstream distance site 1-60bp；
(4) DNA fragmentation of the 50-70bp comprising this site is not located at copy number variation model in genome mutation database
Within enclosing；
(5) fragment residing for this site is special in the range of human genome, it is ensured that this fragment is at human genome
On be unique；
(6) it is compatible between the DNA fragmentation of the 50-70bp comprising this pleomorphism site, namely this 50-70bp
DNA fragmentation between, be not above the complementary series of 8bp, the G/C content of each fragment should be between 45-55% simultaneously.
The screening of mononucleotide polymorphism site may utilize software and completes, and comprises the following steps:
(following steps 1 obtain with reference to the accompanying drawings, but, this step and accompanying drawing 1 do not show and filter out this pleomorphism site
The a length of 50-70bp of DNA fragmentation；Additionally, two ends 50-60bp does not comprise SNP site please according to the power 1 changed before on accompanying drawing 1
Modification)
(1) depend on from the mononucleotide polymorphism site storehouse (dbSNP) at US National Biotechnology Information center (NCBI)
Filter according to little gene frequency numerical value, extract SNP position between 0.4-0.5 for the little gene frequency
Point, the fragment length extracting is 50-70bp
(2) mononucleotide polymorphism site that step (1) obtains is compared with human genome, extract at human gene
It is unique mononucleotide polymorphism site in group；
(3) mononucleotide polymorphism site that filtration step (2) obtains, extracts site upstream and downstream distance site 1-60bp
Between do not comprise the mononucleotide polymorphism site of any mononucleotide polymorphism site；
(4) mononucleotide polymorphism site that step (3) is obtained and US National Biotechnology Information center (NCBI)
Genome mutation database (DGV) in copy number variation (CNV) information compare, filter out comprise copy number variation
(CNV) mononucleotide polymorphism site of information；
(5) mononucleotide polymorphism site that step (4) obtains is compared with BWA index data base, and filter, make residue
Mononucleotide polymorphism site DNA fragmentation between be compatible, i.e. between the DNA fragmentation of this pleomorphism site, do not have
Having more than the complementary series of 8bp, the G/C content of the DNA fragmentation of each pleomorphism site should be between 45-55% simultaneously.
Step 2, the extraction of site DNA in blood plasma: extract pregnant more than 12 weeks maternal blood 5-10ml, enter by centrifugal process
Promoting circulation of blood slurry separates, and isolated blood plasma QiAamp Blood Mini Kit kit extracts DNA free in blood plasma；Take
Following steps: the site sequence design probe obtaining according to step one；It is tied to probe in biomagnetic beads；To be measured by extract
In blood plasma, dissociative DNA joins in the biomagnetic beads with probe, makes DNA hybridize with probe；With in commercialization magnetic bead kit
Cleaning fluid wash away unnecessary DNA；Under the sample DNA bound being eluted from biomagnetic beads with the eluent in kit again
Come,.
Step 3, pcr amplification reaction: to the mononucleotide polymorphism site sequences Design PCR primer obtaining in step one,
And utilize the dissociative DNA fragment that polymerase chain reaction (PCR) amplification step two separates, obtain pcr amplification product；
Polymerase chain reaction condition:
Wherein, PCR reaction system is 15 μ l, comprising:
ddH2O 10.25μl
dNTP 1.5μl
Buffer 1.5 μ l(magnesium ion containing 20mM buffer solution)
20 μM of forward primer 0.3 μ l
20 μM of reverse primer 0.3 μ l
Archaeal dna polymerase 0.15 μ l
10ngDNA 1.0μl
PCR reaction condition: (1) 94 DEG C of for4min, 1cycle, (2) 94 DEG C of for30s, 53 DEG C of for50s, 70 DEG C
For1min, 32cycle, (3) 72 DEG C of for7min, 1cycle；Carry out in PE9600 type PCR instrument.
Step 4, the calculating of foetal DNA amount:
Obtaining pcr amplification product order-checking to step 3, can be selected for existing second generation high throughput sequencing technologies, the present invention adopts
With illumina order-checking platform.Concrete operations are carried out according to illumina order-checking flow process: be first prepared by library；Including end
Modifying, adding A to 3 ' end, two ends adjunction head, select the DNA plus joint, amplification reaches suitable applied sample amount；Then template is miscellaneous
Send on flow cell, carry out bridge amplification formation bunch；Finally order-checking is performed to template sequence.
Add up according to the allelic reading of mononucleotide polymorphism site, calculate the dissociative DNA of mother and fetus
Ratio.When calculating foetal DNA content, the site information of required consideration is as follows: maternal gene type is for isozygotying, and fetus genotype is miscellaneous
Close.
The computational methods of foetal DNA content are as follows:
Calculate all qualified sites (formula 1), obtain foetal DNA content list, take the intermediate value (formula of this list
2)；
Formula 1:Zi=Xi/(Xi+Yi)
I: qualified a certain site；
Xi: foetal allele quantity on i site；
Yi: mother's allele quantity on i site；
Zi: fetus content on i site.
Formula 2: μ=Median (Z)
The foetal DNA content of μ: a certain sample, takes the intermediate value of Z list.
Above formula can be calculated Median corresponding foetal DNA content；
Analyzing 25 male fetus samples by above-mentioned techniqueflow, its foetal DNA content has passed through Y chromosome mark
Note calculates, and compares our test result therewith, and result is shown in Fig. 3, Y-axis be the inventive method test result with pass through
Y chromosome marks the difference being calculated between mean value, it can be seen that, difference range is interval in [-0.004,0.004], says
Bright assay method of the present invention is accurate.
Claims (3)
1. the mensuration side of the fetal DNA in maternal plasma amount of DNA based on mononucleotide polymorphism site of a non-detection disease purpose
Method, it is characterised in that: comprise the following steps:
Step one, the screening of mononucleotide polymorphism site: from mononucleotide polymorphism site storehouse Preliminary screening go out to meet with
The mononucleotide polymorphism site of lower condition:
(1) little gene frequency is between 0.4-0.5；
(2) a length of 50-70bp of DNA fragmentation of this pleomorphism site is comprised；
(3) any mononucleotide polymorphism site is not comprised between site upstream and downstream distance site 1-60bp；
(4) comprise the DNA fragmentation of this pleomorphism site and be not located in genome mutation database within copy number range of variation；
(5) fragment residing for this pleomorphism site is special in the range of human genome；
(6) it is compatible for comprising between the DNA fragmentation of this pleomorphism site, i.e. between the DNA fragmentation of this pleomorphism site,
Being not above the complementary series of 8bp, the G/C content of the DNA fragmentation of each pleomorphism site should be between 45-55% simultaneously；
Wherein, the screening of mononucleotide polymorphism site may utilize software and completes, and comprises the following steps:
A) according to little gene frequency number from the mononucleotide polymorphism site storehouse at US National Biotechnology Information center
Value filters, and extracts mononucleotide polymorphism site between 0.4-0.5 for the little gene frequency, the fragment length extracting
For 50-70bp；
B) mononucleotide polymorphism site obtaining step (a) compares with human genome, extracts on human genome
It is unique mononucleotide polymorphism site；
C) mononucleotide polymorphism site that filtration step (b) obtains, extracts between the upstream and downstream distance site 1-60bp of site
Do not comprise the mononucleotide polymorphism site of any mononucleotide polymorphism site；
D) genome mutation at the mononucleotide polymorphism site that step (c) is obtained and US National Biotechnology Information center
In database, copy number variation information compares, and filters out the mononucleotide polymorphism site comprising copy number variation information；
E) mononucleotide polymorphism site obtaining step (d) compares with BWA index data base, and filters, and makes remaining list
It is compatible between the DNA fragmentation of nucleotide polymorphic site, i.e. between the DNA fragmentation of this pleomorphism site, not super
Crossing the complementary series of 8bp, the G/C content of the DNA fragmentation of each pleomorphism site should be between 45-55% simultaneously；
Step 2, comprises the extraction of site sequence dissociative DNA fragment: extract dissociative DNA in test plasma, and isolate in blood plasma
Including the dissociative DNA fragment of the site sequence of step one acquisition；
Step 3, pcr amplification reaction: to the mononucleotide polymorphism site sequences Design PCR primer obtaining in step one, and profit
The dissociative DNA fragment separating by PCR amplification step two, obtains pcr amplification product；
Step 4, the calculating of foetal DNA amount: obtain pcr amplification product order-checking, and the piece in each site of statistical analysis to step 3
Hop count amount, according to the ratio between not iso-allele, calculates the amount of foetal DNA, and the computational methods of foetal DNA content are as follows:
Calculate all qualified sites with formula 1, obtain foetal DNA content list, take the intermediate value of this list with formula 2:
Formula 1:Zi=Xi/(Xi+Yi)
I: qualified a certain site；
Xi: foetal allele quantity on i site；
Yi: mother's allele quantity on i site；
Zi: fetus content on i site；
Formula 2: μ=Median (Z)
The foetal DNA content of μ: a certain sample, takes the intermediate value of Z list.
2. the pregnant woman blood plasma based on mononucleotide polymorphism site of a kind of non-detection disease purpose according to claim 1
The assay method of middle foetal DNA amount, it is characterised in that: in step 2, described test plasma is obtained by following methods: extraction 5-
10ml maternal blood, centrifugation obtains blood plasma.
3. the pregnant woman blood plasma based on mononucleotide polymorphism site of a kind of non-detection disease purpose according to claim 1
The assay method of middle foetal DNA amount, it is characterised in that: in step 2, including the dissociative DNA piece of the site sequence of step one acquisition
The separation method of section, comprises the following steps:
(1) the site sequence design probe obtaining according to step one；
(2) it is tied to probe in biomagnetic beads；
(3) dissociative DNA in the test plasma of extraction is joined in the biomagnetic beads with probe, make DNA hybridize with probe；
(4) unnecessary DNA is washed away；
(5) sample DNA of binding is eluted from biomagnetic beads.
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|---|---|---|---|---|
| CN106795551B (en) * | 2014-09-26 | 2020-11-20 | 深圳华大基因股份有限公司 | Single-cell chromosome CNV analysis method and detection device |
| WO2016112539A1 (en) * | 2015-01-16 | 2016-07-21 | 深圳华大基因股份有限公司 | Method and device for determining fetal nucleic acid content |
| CN104846089B (en) * | 2015-05-06 | 2017-06-16 | 厦门万基生物科技有限公司 | A kind of quantitative approach of fetal cell-free DNA in maternal plasma ratio |
| CA2993362A1 (en) | 2015-07-23 | 2017-01-26 | The Chinese University Of Hong Kong | Analysis of fragmentation patterns of cell-free dna |
| CN106939334B (en) * | 2017-01-13 | 2021-01-08 | 天昊生物医药科技（苏州）有限公司 | Method for detecting fetal DNA content in plasma of pregnant woman |
| CN110291212A (en) | 2017-01-25 | 2019-09-27 | 香港中文大学 | Diagnostic applications using nucleic acid fragments |
| CN107133491B (en) * | 2017-03-08 | 2020-05-29 | 广州市达瑞生物技术股份有限公司 | Method for obtaining concentration of free DNA of fetus |
| CN107217095B (en) * | 2017-06-15 | 2021-06-04 | 广东腾飞基因科技股份有限公司 | Multiple PCR primer set for human paternity test and detection method |
| CN107194206A (en) * | 2017-06-26 | 2017-09-22 | 思畅信息科技（上海）有限公司 | A kind of screening technique in the chromosome abnormality site based on big data |
| CN108220451B (en) * | 2017-12-08 | 2020-10-27 | 北京科迅生物技术有限公司 | Detection method and kit for concentration of fetal free nucleic acid |
| CN119943152B (en) * | 2024-12-20 | 2025-10-10 | 东莞博奥木华基因科技有限公司 | Method, system, device and medium for simulating plasma free nucleic acid data of pregnant women |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012114075A1 (en) * | 2011-02-25 | 2012-08-30 | University Of Plymouth | Method for processing maternal and fetal dna |
-
2013
- 2013-03-26 CN CN201310098730.5A patent/CN103215350B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012114075A1 (en) * | 2011-02-25 | 2012-08-30 | University Of Plymouth | Method for processing maternal and fetal dna |
Non-Patent Citations (1)
| Title |
|---|
| 孕妇血浆中胎儿DNA的数量变化的研究;任晨春等;《现代妇产科进展》;20060828;第15卷(第08期);614-616 * |
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