CN103205416B - Extraction method of fetal DNA (deoxyribonucleic acid) in peripheral blood of pregnant woman - Google Patents
Extraction method of fetal DNA (deoxyribonucleic acid) in peripheral blood of pregnant woman Download PDFInfo
- Publication number
- CN103205416B CN103205416B CN201310133487.6A CN201310133487A CN103205416B CN 103205416 B CN103205416 B CN 103205416B CN 201310133487 A CN201310133487 A CN 201310133487A CN 103205416 B CN103205416 B CN 103205416B
- Authority
- CN
- China
- Prior art keywords
- dna
- add
- maternal blood
- concentration
- centrifugal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an extraction method of fetal DNA (deoxyribonucleic acid) in the peripheral blood of a pregnant woman and belongs to the technical field of DNA separation and extraction. The extraction method comprises the following steps of: (1) acquiring the peripheral blood of a pregnant woman, adding EDTA (Ethylene Diamine Tetraacetic Acid) for anticoagulation, and centrifuging; (2) adding a protease complexing agent and centrifuging; (3) adding Tris saturated phenol to uniformly mix, and centrifuging; (4) adding chloroform/isoamylol mixed liquor to sufficiently and uniformly mix, and centrifuging; (5) adding ethanol to sufficiently and uniformly mix, and carrying out high-speed centrifugation to obtain DNA precipitate; (6) preparing to obtain rough fetal DNA recovery liquid; and (7) obtaining a DNA extracting solution. According to the extraction method, protein is degraded into small peptide or amino acid by adopting the protease complexing agent, and therefore, high-efficiency separation between DNA and protein is achieved; the fetal DNA in the extraction solution accounts for more than 90 percent of the total DNA and can meet the requirement of prenatal screening on fetal genetic diseases; and necessary technical supports are provided for developing the prenatal screening on non-invasive fetal genetic diseases.
Description
Technical field
The invention belongs to separation and Extraction DNA technique field.
Background technology
Various the abnormal of the aspects such as body structure, intelligence or metabolism that comprise that just exist when inborn defect refers to baby due.China is one of neonatal country occurred frequently of defect in the world, and 2,000 ten thousand newborn infants of the annual birth in the whole nation have 5% to have inborn defect, within every 30 seconds, just has a defect youngster birth.Inborn defect can cause by inherited genetic factorss such as chromosome aberration, transgenations, also can be by due to these two kinds of factor interactions or other unknown causes.Existing scientific payoffs demonstration transgenation is brought into play importance in inborn defect occurs, so be the important means that reduces inborn defect to the Prenatal Screening of foetal DNA variation.
The material that foetal DNA variation detects must be from fetus self, traditional antenatal diagnosis is to be based upon the cytogenetic diagnosis carrying out on the invasive bases such as amniocentesis, villus aspiration, although diagnosis accurately, but because its operation has traumatic, easily cause intrauterine infection, miscarriage, even fetus had a certain impact, belong to invasive inspection.And the extraction of fetal DNA in maternal plasma dissociative DNA only need to gather pregnant woman blood plasma sample, without puncture amniotic cavity and insertion chorionic villi, have fetus without any wound, can belong to Noninvasive fetus inherited disease Prenatal Screening in advantages such as First Trimester diagnosis, safety and be easy to promote.
Because the total concn of dissociative DNA in common human plasma is 1-100 ng/mL, foetal DNA only accounts for 5%～7% of total DNA in pregnant woman blood plasma, and ratio is lower.A large amount of source of parents DNA backgrounds have increased the difficulty detecting for fetus dissociative DNA undoubtedly, and the foetal DNA being free in female blood is that short-movie is disconnected, are generally less than 200bp, are difficult for catching.Therefore, from maternal blood, be enriched to high density and highly purified foetal DNA is the key of carrying out the antenatal Fetal screening of Noninvasive, need to develop special technology.
The extraction that in human plasma or serum, the technology of purifying concentrated dissociative DNA and RNA and test kit are mainly used in total DNA in blood plasma, can not meet the needs of the also concentrated foetal DNA of purifying from pregnant woman blood plasma or serum, be difficult to carry out fetus inherited disease Prenatal Screening, and the reagent of import and test kit expensive.Chinese patent 200610013153.5 discloses a kind of method of enrichment fetal DNA in maternal plasma dissociative DNA, can effectively improve the extraction yield of fetal DNA in maternal plasma DNA, but its extraction yield for foetal DNA is too low, be difficult to guarantee the accuracy of Prenatal Screening, can not meet the requirement of fetus inherited disease Prenatal Screening.
Summary of the invention
The invention provides the extracting method of foetal DNA in a kind of maternal blood, adopting proteolytic enzyme recombiner is little peptide or amino acid by protein degradation, thereby realize DNA and protein high efficiency separation, in extracting solution, foetal DNA accounts for the more than 90% of total DNA, can meet the needs of fetus inherited disease Prenatal Screening, for carrying out the guarantee that provides the necessary technical of Noninvasive fetus inherited disease Prenatal Screening.
The technical solution used in the present invention is:
In maternal blood, an extracting method for foetal DNA, comprises the steps:
(1) gather maternal blood 5-10mL, add EDTA anti-freezing, the centrifugal 5-10 minute of 1200-1700g at 4 ℃, draws supernatant liquor;
(2) in step (1) gained supernatant liquor, add proteolytic enzyme recombiner, the centrifugal 5-10 minute of 1200-1700g at 4 ℃, draws upper strata water;
(3) in the water of step (2) gained upper strata, add the saturated phenol of Tris to mix, the centrifugal 25-35 minute of 4000-4600g at 4 ℃, draws supernatant liquid;
(4) in step (3) gained supernatant liquid, add chloroform/primary isoamyl alcohol mixed solution fully to mix, 4000-4600g at 4 ℃, centrifugal 5-10 minute, draws upper water solution;
(5) in step (4) gained upper water solution, adding volumetric concentration is that the ethanol of 95 % fully mixes, and 7000-9000g high speed centrifugation 5-10 minute at 4 ℃, obtains DNA precipitation;
(6) add 20-100mL TE dissolving DNA precipitation, the DNA solution that TE dissolves carries out polyacrylamide gel electrophoresis, and cma staining is selected to cut object band and fixed, and obtains foetal DNA and slightly reclaims liquid;
(7) foetal DNA slightly reclaims 100-200mL dehydrated alcohol precipitation DNA for liquid, and 20-100mL TE dissolving DNA precipitation, obtains DNA extraction liquid.
In proteolytic enzyme recombiner, the concentration of Proteinase K is that the concentration of 5-20mg/mL, Tryptase is 1000-5000pg/mL, and the concentration of papoid is that 100-400ng/mL and pepsic concentration are 5-10ng/mL.
In the mixed solution of chloroform/primary isoamyl alcohol, the volume ratio of chloroform/primary isoamyl alcohol is 24:1.
The proteolytic enzyme recombiner that adds supernatant liquor cumulative volume 8-14% in step (2).
In step (3), add the saturated phenol of Tris with upper water equal volume.
In step (4), add and the isopyknic chloroform/primary isoamyl alcohol of supernatant liquid mixed solution.
In step (5), volumetric concentration is that the add-on of the ethanol of 95 % is 1.5-3.0 times of supernatant water liquor capacity.
Described in step (5), the temperature of ethanol is 4 ℃.
Blood plasma and serum are to detect the topmost Specimen origin of free nucleic acid that is released into blood, and human peripheral blood dissociative DNA molecule is usually to exist with the form of protein bound, and chromatinic basic structure is combined and is formed with histone by DNA exactly.In blood plasma, contain abundant albumen simultaneously, comprise albumin and scleroproein etc., be easy to same DNA combination, therefore removing protein is the key of extracting peripheral blood dissociative DNA.The principle of preparation DNA is should DNA is separated with protein, lipid and carbohydrate etc., keeps again the integrity of DNA molecule.At present, adopt Proteinase K (proteinase K) to be hydrolyzed to protein, in order to improve hydrolysis efficiency, proteolytic enzyme recombiner of the present invention not only contains Proteinase K, also increased the Tryptase (fibrinolysin) of lower concentration, Mu melon Dan Bai Mei ﹙ Papain ﹚ and stomach en-.Wherein, Tryptase can play katalysis in fibrinolysis process, stomach en-, as a kind of digestible protein enzyme, can be decomposed into protein little peptide fragment, and papoid is a kind of proteolytic enzyme that all can decomposing protein under acid, neutral, alkaline condition.Therefore, proteolytic enzyme recombiner is solubilising protein efficiently, and secondary protein structure is disappeared, and the degradation of proteins into little peptide or amino acid, thereby DNA and protein is separated.Nuclease in the rapid lysing cell of proteolytic enzyme recombiner energy and as killed cells, to guarantee that DNA molecular is not degraded, can make maternal blood dissociative DNA concentration effect improve on the original basis 20% simultaneously.
In fetus dissociative DNA leaching process, the use of antithrombotics, centrifugal speed etc. all can affect extracts the amount of DNA and the accuracy of experimental result, reliability.Centrifugation technique is used for the removing of peripheral blood cells, the objects such as precipitation of the removing of plasma proteins and 200bp DNA fragmentation, and centrifugal speed, time can affect the removing of peripheral blood cells and plasma proteins, and the purity of DNA and yield.Too low temperature makes the insufficient and degraded of impact to protein of enzyme reaction, thereby fetus dissociative DNA is polluted, and too high temperature degradable DNA molecular affects the quality and quantity of centrifugal final acquisition DNA, by Temperature Setting, is therefore 4 ℃.
In order to guarantee to extract the amount of DNA and the accuracy of experimental result, reliability, the application all adjusts and optimizes parameters such as anticoagulant centrifugal speed, time, temperature etc., to improve the reliability of extracting sample.
Adopt respectively Proteinase K and proteolytic enzyme recombiner to carry out DNA extraction, extract and the results are shown in following table:
The beneficial effect that adopts technique scheme to produce is:
It is little peptide or amino acid by protein degradation that the application adopts proteolytic enzyme recombiner, thereby realize DNA and protein high efficiency separation, in extracting solution, foetal DNA accounts for the more than 90% of total DNA, can meet the needs of fetus inherited disease Prenatal Screening, for carrying out the guarantee that provides the necessary technical of Noninvasive fetus inherited disease Prenatal Screening.
Embodiment
Embodiment 1
(1) gather maternal blood 8mL, add EDTA anti-freezing, 1700g is centrifugal 8 minutes at 4 ℃, draws supernatant liquor;
(2) in step (1) gained supernatant liquor, add the proteolytic enzyme recombiner of supernatant liquor volume 14%, 1700g is centrifugal 5 minutes at 4 ℃, draws upper strata water;
(3) in the water of step (2) gained upper strata, add the saturated phenol of isopyknic Tris to mix, 4300g is centrifugal 28 minutes at 4 ℃, draws supernatant liquid;
(4) in step (3) gained supernatant liquid, add isopyknic chloroform/primary isoamyl alcohol mixed solution fully to mix, 4000g at 4 ℃, centrifugal 10 minutes, draws upper water solution; In the mixed solution of chloroform/primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24:1.
(5) in step (4) gained upper water solution, add 2.5 times of volumes ethanol (95 %, v/v) fully mix, 9000g high speed centrifugation is 5 minutes at 4 ℃, obtains DNA precipitation; The temperature of ethanol is 4 ℃.
(6) add 50mL TE dissolving DNA precipitation, the DNA solution that TE dissolves carries out polyacrylamide gel electrophoresis, and cma staining is selected to cut object band and fixed, and obtains foetal DNA and slightly reclaims liquid;
(7) foetal DNA slightly reclaims 180mL dehydrated alcohol precipitation DNA for liquid, and 100mL TE dissolving DNA precipitation, obtains DNA extraction liquid.
In the present embodiment, in proteolytic enzyme recombiner, the concentration of Proteinase K is that the concentration of 5mg/mL, Tryptase is 2000pg/mL, and the concentration of papoid is that 100ng/mL and pepsic concentration are 6ng/mL.
After testing, in the extracting solution obtaining in the present embodiment, to account for the massfraction of total DNA be 91% to foetal DNA.
Embodiment 2
(1) gather maternal blood 10mL, add EDTA anti-freezing, 1500g is centrifugal 10 minutes at 4 ℃, draws supernatant liquor;
(2) in step (1) gained supernatant liquor, add the proteolytic enzyme recombiner of supernatant liquor volume 12%, 1600g is centrifugal 6 minutes at 4 ℃, draws upper strata water;
(3) in the water of step (2) gained upper strata, add the saturated phenol of isopyknic Tris to mix, 4000g is centrifugal 35 minutes at 4 ℃, draws supernatant liquid;
(4) in step (3) gained supernatant liquid, add isopyknic chloroform/primary isoamyl alcohol mixed solution fully to mix, 4600g is centrifugal 5 minutes at 4 ℃, draws upper water solution; In the mixed solution of chloroform/primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24:1.
(5) in step (4) gained upper water solution, add 2.0 times of volumes ethanol (95 %, v/v) fully mix, 7000g high speed centrifugation is 10 minutes at 4 ℃, obtains DNA precipitation; The temperature of ethanol is 4 ℃.
(6) add 20mL TE dissolving DNA precipitation, the DNA solution that TE dissolves carries out polyacrylamide gel electrophoresis, and cma staining is selected to cut object band and fixed, and obtains foetal DNA and slightly reclaims liquid;
(7) foetal DNA slightly reclaims 150mL dehydrated alcohol precipitation DNA for liquid, and 80mL TE dissolving DNA precipitation, obtains DNA extraction liquid.
In the present embodiment, in proteolytic enzyme recombiner, the concentration of Proteinase K is that the concentration of 10mg/mL, Tryptase is 1000pg/mL, and the concentration of papoid is that 300ng/mL and pepsic concentration are 8ng/mL.
After testing, in the extracting solution obtaining in the present embodiment, to account for the massfraction of total DNA be 90% to foetal DNA.
Embodiment 3
(1) gather maternal blood 5mL, add EDTA anti-freezing, 1300g is centrifugal 6 minutes at 4 ℃, draws supernatant liquor;
(2) in step (1) gained supernatant liquor, add the proteolytic enzyme recombiner of supernatant liquor volume 10%, 1200g is centrifugal 10 minutes at 4 ℃, draws upper strata water;
(3) in the water of step (2) gained upper strata, add the saturated phenol of isopyknic Tris to mix, 4600g is centrifugal 25 minutes at 4 ℃, draws supernatant liquid;
(4) in step (3) gained supernatant liquid, add isopyknic chloroform/primary isoamyl alcohol mixed solution fully to mix, 4200g at 4 ℃, centrifugal 6 minutes, draws upper water solution; In the mixed solution of chloroform/primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24:1.
(5) in step (4) gained upper water solution, add 3.0 times of volumes ethanol (95 %, v/v) fully mix, 8000g high speed centrifugation is 8 minutes at 4 ℃, obtains DNA precipitation; The temperature of ethanol is 4 ℃.
(6) add 100mL TE dissolving DNA precipitation, the DNA solution that TE dissolves carries out polyacrylamide gel electrophoresis, and cma staining is selected to cut object band and fixed, and obtains foetal DNA and slightly reclaims liquid;
(7) foetal DNA slightly reclaims 100mL dehydrated alcohol precipitation DNA for liquid, and 20mL TE dissolving DNA precipitation, obtains DNA extraction liquid.
In the present embodiment, in proteolytic enzyme recombiner, the concentration of Proteinase K is that the concentration of 20mg/mL, Tryptase is 3000pg/mL, and the concentration of papoid is that 400ng/mL and pepsic concentration are 5ng/mL.
After testing, in the extracting solution obtaining in the present embodiment, to account for the massfraction of total DNA be 91% to foetal DNA.
Embodiment 4
(1) gather maternal blood 7mL, add EDTA anti-freezing, 1200g is centrifugal 5 minutes at 4 ℃, draws supernatant liquor;
(2) in step (1) gained supernatant liquor, add the proteolytic enzyme recombiner of supernatant liquor volume 8%, 1400g is centrifugal 8 minutes at 4 ℃, draws upper strata water;
(3) in the water of step (2) gained upper strata, add the saturated phenol of isopyknic Tris to mix, 4500g is centrifugal 32 minutes at 4 ℃, draws supernatant liquid;
(4) in step (3) gained supernatant liquid, add isopyknic chloroform/primary isoamyl alcohol mixed solution fully to mix, 4300g at 4 ℃, centrifugal 8 minutes, draws upper water solution; In the mixed solution of chloroform/primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24:1.
(5) in step (4) gained upper water solution, add 1.5 times of volumes ethanol (95 %, v/v) fully mix, 7500g high speed centrifugation is 9 minutes at 4 ℃, obtains DNA precipitation; The temperature of ethanol is 4 ℃.
(6) add 80mL TE dissolving DNA precipitation, the DNA solution that TE dissolves carries out polyacrylamide gel electrophoresis, and cma staining is selected to cut object band and fixed, and obtains foetal DNA and slightly reclaims liquid;
(7) foetal DNA slightly reclaims 200mL dehydrated alcohol precipitation DNA for liquid, and 80mL TE dissolving DNA precipitation, obtains DNA extraction liquid.
In the present embodiment, in proteolytic enzyme recombiner, the concentration of Proteinase K is that the concentration of 13mg/mL, Tryptase is 5000pg/mL, and the concentration of papoid is that 200ng/mL and pepsic concentration are 10ng/mL.
After testing, in the extracting solution obtaining in the present embodiment, to account for the massfraction of total DNA be 92% to foetal DNA.
Claims (7)
1. an extracting method for foetal DNA in maternal blood, is characterized in that it comprises the steps:
(1) gather maternal blood 5-10mL, add EDTA anti-freezing, the centrifugal 5-10 minute of 1200-1700g at 4 ℃, draws supernatant liquor;
(2) in step (1) gained supernatant liquor, add proteolytic enzyme recombiner, the centrifugal 5-10 minute of 1200-1700g at 4 ℃, draws upper strata water; In described proteolytic enzyme recombiner, the concentration of Proteinase K is that the concentration of 5-20mg/mL, Tryptase is 1000-5000pg/mL, and the concentration of papoid is that 100-400ng/mL and pepsic concentration are 5-10ng/mL;
(3) in the water of step (2) gained upper strata, add the saturated phenol of Tris to mix, the centrifugal 25-35 minute of 4000-4600g at 4 ℃, draws supernatant liquid;
(4) in step (3) gained supernatant liquid, add chloroform/primary isoamyl alcohol mixed solution fully to mix, 4000-4600g at 4 ℃, centrifugal 5-10 minute, draws upper water solution;
(5) in step (4) gained upper water solution, adding volumetric concentration is that the ethanol of 95 % fully mixes, and 7000-9000g high speed centrifugation 5-10 minute at 4 ℃, obtains DNA precipitation;
(6) add 20-100mL TE dissolving DNA precipitation, the DNA solution that TE dissolves carries out polyacrylamide gel electrophoresis, and cma staining is selected to cut object band and fixed, and obtains foetal DNA and slightly reclaims liquid;
(7) foetal DNA slightly reclaims 100-200mL dehydrated alcohol precipitation DNA for liquid, and 20-100mL TE dissolving DNA precipitation, obtains DNA extraction liquid.
2. the extracting method of foetal DNA in maternal blood according to claim 1, is characterized in that the volume ratio of chloroform/primary isoamyl alcohol in the mixed solution of described chloroform/primary isoamyl alcohol is 24:1.
3. the extracting method of foetal DNA in maternal blood according to claim 1, is characterized in that adding in step (2) the proteolytic enzyme recombiner of supernatant liquor cumulative volume 8-14%.
4. the extracting method of foetal DNA in maternal blood according to claim 1 and 2, is characterized in that adding in step (3) the saturated phenol of Tris with upper water equal volume.
5. the extracting method of foetal DNA in maternal blood according to claim 1, is characterized in that adding and the isopyknic chloroform/primary isoamyl alcohol of supernatant liquid mixed solution in step (4).
6. the extracting method of foetal DNA in maternal blood according to claim 1, it is characterized in that volumetric concentration in step (5) be the add-on of the ethanol of 95 % be supernatant water liquor capacity 1.5-3.0 doubly.
7. the extracting method of foetal DNA in maternal blood according to claim 1, the temperature that it is characterized in that ethanol described in step (5) is 4 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310133487.6A CN103205416B (en) | 2013-04-17 | 2013-04-17 | Extraction method of fetal DNA (deoxyribonucleic acid) in peripheral blood of pregnant woman |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310133487.6A CN103205416B (en) | 2013-04-17 | 2013-04-17 | Extraction method of fetal DNA (deoxyribonucleic acid) in peripheral blood of pregnant woman |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103205416A CN103205416A (en) | 2013-07-17 |
| CN103205416B true CN103205416B (en) | 2014-03-26 |
Family
ID=48752822
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310133487.6A Expired - Fee Related CN103205416B (en) | 2013-04-17 | 2013-04-17 | Extraction method of fetal DNA (deoxyribonucleic acid) in peripheral blood of pregnant woman |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103205416B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104164417A (en) * | 2014-06-26 | 2014-11-26 | 北京圣谷同创科技发展有限公司 | Peripheral blood fetal free DNA extraction method |
| WO2016045022A1 (en) * | 2014-09-25 | 2016-03-31 | 深圳华大基因科技有限公司 | Preserving solution for maternal peripheral blood sample and method for preserving maternal peripheral blood sample |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016562A (en) * | 2006-02-08 | 2007-08-15 | 陈熹 | Method of concentrating fetus dissociative DNA in pregnant woman blood plasma |
| WO2010085815A1 (en) * | 2009-01-26 | 2010-07-29 | Artemis Health, Inc. | Methods and compositions for identifying a fetal cell |
| CN101812501A (en) * | 2009-02-20 | 2010-08-25 | 崔汝强 | Method for detecting aphelenchoides besseyi and diagnostic reagent kit |
| CN102127818A (en) * | 2010-12-15 | 2011-07-20 | 张康 | Method for creating fetus DNA library by utilizing peripheral blood of pregnant woman |
-
2013
- 2013-04-17 CN CN201310133487.6A patent/CN103205416B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016562A (en) * | 2006-02-08 | 2007-08-15 | 陈熹 | Method of concentrating fetus dissociative DNA in pregnant woman blood plasma |
| WO2010085815A1 (en) * | 2009-01-26 | 2010-07-29 | Artemis Health, Inc. | Methods and compositions for identifying a fetal cell |
| CN101812501A (en) * | 2009-02-20 | 2010-08-25 | 崔汝强 | Method for detecting aphelenchoides besseyi and diagnostic reagent kit |
| CN102127818A (en) * | 2010-12-15 | 2011-07-20 | 张康 | Method for creating fetus DNA library by utilizing peripheral blood of pregnant woman |
Non-Patent Citations (8)
| Title |
|---|
| Dorothy J. Huang等.Use of an automated method improves the yield and quality of cell-free fetal DNA extracted from maternal plasma.《CLINICAL CHEMISTRY》.2005,第51卷(第12期),全文. |
| Use of an automated method improves the yield and quality of cell-free fetal DNA extracted from maternal plasma;Dorothy J. Huang等;《CLINICAL CHEMISTRY》;20051231;第51卷(第12期);全文 * |
| 孕妇外周血中胎儿DNA 的提取和应用;高军;《国外医学妇幼保健分册》;20031231;第14卷(第1期);全文 * |
| 孕妇血浆中胎儿DNA的提取及STR基因的检测;李维春等;《安徽医学》;20100531;第31卷(第5期);全文 * |
| 应用酚提取法纯化孕妇血浆及血清中胎儿游离DNA;陈小蔓等;《中华医学遗传学杂志》;20021031;第19卷(第5期);全文 * |
| 李维春等.孕妇血浆中胎儿DNA的提取及STR基因的检测.《安徽医学》.2010,第31卷(第5期),全文. |
| 陈小蔓等.应用酚提取法纯化孕妇血浆及血清中胎儿游离DNA.《中华医学遗传学杂志》.2002,第19卷(第5期),全文. |
| 高军.孕妇外周血中胎儿DNA 的提取和应用.《国外医学妇幼保健分册》.2003,第14卷(第1期),全文. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103205416A (en) | 2013-07-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10260063B2 (en) | Enrichment of small nucleic acids | |
| CN108088727B (en) | Pretreatment method of protein in-vitro body fluid | |
| CN103952397A (en) | Method for separating free nucleic acid from blood serum or blood plasma sample by using magnetic bead | |
| CN112410327A (en) | Kit and method for extracting RNA | |
| CN103205416B (en) | Extraction method of fetal DNA (deoxyribonucleic acid) in peripheral blood of pregnant woman | |
| KR101967878B1 (en) | A method for isolating circulating cell-free nucleic acid | |
| CN104017800A (en) | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof | |
| CN113906146A (en) | Optimized ultra-low volume liquid biopsy methods, systems, and apparatus | |
| CN103421903B (en) | Alpha-thalassemia screening kit and application thereof in prenatal screening | |
| WO2023071402A1 (en) | Saliva-specific fucosylated structure-based sugar profile, detection method therefor, and application thereof | |
| CN110191951A (en) | The method and its application of noninvasive pre-natal diagnosis are carried out based on excretion body DNA | |
| CN112322584A (en) | Simple exosome extraction method | |
| US8063199B2 (en) | Method for isolating both free and protein association DNA | |
| Li et al. | Inability to detect cell free fetal DNA in the urine of normal pregnant women nor in those affected by preeclampsia associated HELLP syndrome | |
| Watson et al. | The cilia of Tetrahymena pyriformis: Fractionation of isolated cilia | |
| CN105132572B (en) | A kind of patau syndrome kit of noninvasive Prenatal Screening 21 | |
| CN106701742A (en) | Blood collection tube for protecting and stabilizing free RNA (Ribonucleic Acid) | |
| CN105506066A (en) | Cell identification method for new generation of noninvasive prenatal diagnosis field | |
| Moore et al. | Oestrous and ovarian response of the ewe to a horse anterior pituitary extract | |
| CN109609493A (en) | Extract the method and kit of genomic DNA in whole blood | |
| CN104164416A (en) | Method for high efficiently extracting nucleic acid from fresh milk | |
| Shetty et al. | Obstetric Markers as a Diagnostic Forensic Tool | |
| CN118256489B (en) | Extraction kit of circulating nucleic acid and extraction method of circulating nucleic acid | |
| CN113234717B (en) | Separation and enrichment method of circulating tumor DNA | |
| Hirschfeld et al. | The unsaturated capacity of the thyroxine-binding proteins in pregnant women and newborn infants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wu Chaoqun Inventor after: Guan Li Inventor after: Shi Shuxia Inventor before: Wu Chaoqun Inventor before: Guan Li |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: WU CHAOQUN GUAN LI TO: WU CHAOQUN GUAN LI SHI SHUXIA |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140326 |