WO2013163935A1

WO2013163935A1 - Method for isolating and obtaining pure and complete fetus genome dna

Info

Publication number: WO2013163935A1
Application number: PCT/CN2013/074618
Authority: WO
Inventors: 蔡勇平
Original assignee: 上海绿宇生物科技有限公司
Priority date: 2012-05-04
Filing date: 2013-04-24
Publication date: 2013-11-07
Also published as: CN103382469A

Abstract

Provided is a method for isolating and obtaining pure and complete fetus genome DNA, comprising the following steps: isolating a nucleated erythrocyte from the maternal (pregnant woman) peripheral blood, identifying the individual hereditary feature subtype of the mother and/or father of the fetus; conducting high fidelity whole genome DNA amplification on the signal cell isolated from the nucleated erythrocyte; identifying the individual hereditary feature subtype of the fetus according to the laws of hereditary, and determining a fetus-originating genome DNA. The fetus-originating pure whole genome DNA provided by the present invention lays a material foundation for prenatal heredity diagnosis.

Description

A pure fetal complete genomic DNA isolation and acquisition method

The invention belongs to the field of medicine, in particular to a method for separating and obtaining intact genomic DNA of a pure fetus. Background technique

Prenatal genetic diagnosis is based on genetic counseling, mainly through genetic testing and imaging examination, to make a clear diagnosis of high-risk fetuses, so that people can make appropriate choices or treatments during pregnancy to reduce birth. Defect rate, improve eugenic quality and population quality.

Prenatal genetic diagnosis includes both invasive and non-invasive types. The former mainly includes amniocentesis, villus sampling, cord blood sampling, fetal mirror and embryo biopsy, etc. The latter includes ultrasound examination, maternal peripheral serum marker determination, non-invasive fetal gene and cytogenetic analysis.

At present, the prenatal genetic diagnosis is still mainly based on traumatic methods. Amniocentesis and villus sampling are the most commonly used. However, there are the following risks when the fetus is invasive: 1. Fetal transient bradycardia; 0.1%~0.9% of the subjects had premature birth or intrauterine death; 3. Umbilical cord placental oozing after umbilical cord blood collection; 4. Amniotic cavity infection; 5. Amniotic membrane rupture; 6. Fetal malformation.

In the noninvasive method, ultrasound examination can only distinguish morphologically some fetuses with obvious congenital malformations, and it is impossible to identify fetuses with normal morphology and other genetic defects. The detection of maternal peripheral serum markers is indirect detection from maternal serum markers, so the items examined are limited. The current screening is mainly Down's screening, which is only one of the trisomy syndromes, the vast majority of genetic diseases. No similar non-invasive methods can be used for testing. Summary of the invention

In order to overcome the deficiencies in the prior art, the inventors of the present application provide a method for isolating and identifying intact genomic DNA of a fetus from the peripheral blood of a pregnant woman, which is capable of non-invasively obtaining pure fetal complete genomic DNA, thereby being comprehensive The application of accurate prenatal genetic diagnosis provides a material basis. Accordingly, it is an object of the present invention to provide a method for the isolation and acquisition of pure fetal complete genomic DNA.

The method for obtaining the purified fetal complete genomic DNA of the present invention comprises the following steps:

A) separating nucleated red blood cells from the maternal (pregnant) peripheral blood;

B) extracting genomic DNA from the fetal mother and/or father's somatic cells or separating single cells for high-fidelity whole-genome DNA amplification to identify individual genetic traits of the fetus mother and/or father;

C) Separating single cells from nucleated red blood cells for high-fidelity whole-genome DNA amplification;

D) using the individual genetic characteristics of the fetal mother and/or father as a control, identifying the nucleated red blood cell single-cell whole-genome DNA amplification products according to the genetic law and determining the individual genetic characteristics of the fetus, thereby determining the fetal source. Genomic DNA.

According to the present invention, the maternal (pregnant) peripheral blood is the peripheral venous blood of the mother (pregnant woman) at 9 to 38 weeks of gestation.

According to the present invention, the individual genetic trait typing includes genotyping identification using a SNP combination or a STR combination, etc., preferably a SNP combination.

According to the present invention, the method for separating nucleated red blood cells comprises: immunomagnetic bead sorting or magnetic activated cell sorting, flow cell sorting or fluorescence activated cell sorting, charge flow separation, Culture screening enrichment or density gradient centrifugation.

According to the present invention, the somatic cells of the mother and/or father are derived from: dandruff, oral epithelium or peripheral blood of a pregnant mother.

According to the present invention, the method for identifying an individual's genetic traits includes: gene chip method, PCR sequencing method, MGB fluorescent PCR method, restriction fragment length polymorphism method, single strand conformation polymorphism method, denaturing gradient gel Electrophoresis, flight mass spectrometry or denaturing high performance liquid chromatography. The invention has the following advantages:

1. Non-invasive, taking prenatal blood from pregnant women, no trauma to the fetus, and minimal impact on pregnant women.

2. Advance, genetic testing at the earliest 9 weeks of pregnancy, early detection, early prevention, early diagnosis, early treatment of genetic diseases.

3. Accuracy, using the genomic DNA of the mother and/or father as a control, using individual-specific inheritance Labeling identifies whole-genome DNA from fetal single-cell sources to ensure accurate fetal cell and whole-genome DNA sources. Whole-genome DNA derived from different cells of the fetus can be subjected to multiple parallel diagnostic experiments and mutually confirmed to avoid genomic amplification errors. Misdiagnosis to ensure accurate and reliable diagnosis.

4. Purity, identified as the fetal genome, is derived from a single cell and is not contaminated by maternal (pregnant) DNA, thus ensuring the accuracy of subsequent genetic diagnosis.

5. Universality, access to complete whole-genome DNA derived from the fetus, selection of different sites or use of different combinations of markers and markers to perform a wide range of genes at the individual and genome-wide levels

(DNA) analysis, diagnosis of monogenic diseases, individual drug tolerance, risk of polygenic disease, and other genetic diagnoses except cytogenetic analysis. The nucleated red blood cells are abundant in the peripheral blood of pregnant women, and the life cycle is short, which is not affected by the previous pregnancy. It is the most ideal material for non-invasive identification of fetal genotypes. The present invention combines the methods of individual genetic characterization to obtain the full source of fetal origin. Genomic DNA, non-invasive, advanced, accurate, pure and universal, lays the material foundation for prenatal genetic diagnosis. detailed description

The present invention will be further described below in conjunction with specific embodiments. It is to be understood that the following examples are merely illustrative of the invention and are not intended to limit the scope of the invention.

The methods for isolating nucleated red blood cells in the following examples include, but are not limited to: magnetic activated cell sorting (MACS) or magnetic activated cell sorting, and fluorescence activated cell sorting (fluorescence activated cell sorting, FACS) or fluorescence activated cell sorting, charge flow separation (CFS), culture screening enrichment, density gradient centrifugation, and the like.

The somatic cells of the mother and/or father in the following examples are derived from, but not limited to, dandruff, oral epithelium, peripheral blood of a pre-pregnancy mother, and the like.

Methods for identifying individual genetic traits in the following examples include, but are not limited to, SNP combinations, STR combinations, and the like.

Experimental techniques for identifying individual genetic traits in the following examples include, but are not limited to, gene chips Method, PCR sequencing, MGB fluorescent PCR, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing gradient gel electrophoresis, flight mass spectrometry, denaturing high performance liquid chromatography, and the like. Example 1. Isolation and acquisition of a pure fetal complete genome

1.1. Separation of nucleated red blood cells in maternal peripheral blood

10 ml of peripheral venous blood of the mother (pregnant woman) at 9 to 38 weeks of gestation was anticoagulated with 4 mmol/L EDTA, and then diluted 1:1 with phosphate buffer (PBS, pH 7.4). In a 15 ml Falcon centrifuge tube, add 5 ml of cell separation solution Histopaque®-1077 (Sigma), and slowly add 10 ml of the diluted pregnant blood along the wall of the centrifuge tube to cover the blood in the cell separation solution.

On Histopaque®-1077, centrifuge at room temperature for 30 min at 400 xg. Aspirate the mononuclear cell layer between plasma and Histopaque®-1077 and wash once with PBS buffer (containing 5 mmol/L EDTA and 0.5% BSA) to add approximately 8 per 10 ⁷ cells ( The ratio of ^L PBS buffer (pH 7.4) was made into a mononuclear cell suspension.

The CD71 immunomagnetic beads coated with the CD71 antibody were added to the above mononuclear cell suspension at a ratio of 4 magnetic beads per target cell, and incubated at 4 ° C for 45 minutes to place the magnetic bead-labeled mononuclear cell suspension. In the Dynal MPC magnetic column, it was allowed to stand for 2 minutes to adsorb the nucleated red blood cells marked with magnetic beads and suck up the unadsorbed cells. The magnetic bead-labeled nucleated red blood cells were washed with PBS buffer and suspended in PBS buffer at an appropriate ratio.

Take 上述μί the above nucleated red blood cell suspension into 9μί PBS buffer, count with a hemocytometer under the microscope, determine the number of cells per microliter, dispense into about 100 cells per tube, and add antifreeze liquid nitrogen cryopreservation .

1.2. Identification of individual genetic characteristics of fetal mothers and/or fathers

Take pregnant women and fetal biology fathers dandruff more than 1mg each, using QIAamp DNA

Investigator Kit (QIAGEN), follow the instructions to extract genomic DNA from the dandruff of pregnant and fetal biology fathers.

Individual genetic trait typing (gene fingerprinting) identification methods are as follows:

SNP loci with high polymorphism and no interlocking relationship in Chinese population (ensure different individuals) The mother or/and father identification was performed with a repetition probability of less than one in 10 billion, and genotyping of the SNP site was detected and recorded. The reference sequence for the SNP locus can be obtained in the NCBI database.

(http://www.ncbi.nlm.nih.gov/snp/), the site accession number is as follows: rsl2041538, rsl2041538, rs7520495, rsl 160336, rs7421375, rs 11130440, rs6549011, rs7655916, rs 1503627, rs 12516484, rs6940304 , rs6938545, rs4717331, rs2317774, rs2447542, rs2920296, rs971984, rsl414252, rs7900633, rs293325, rsl0768140, rs 12223762, rs4842610, rs 10454027, rsl333100, rsl2896399, rs 12594957, rs4781118, rs9940450, rs7251903, rs6073995, rsl 1088302, rs4823460, rs2385622 , rs9814835, rsl466329, rs3013148, rs3807337, rsl484646, rs6560626, rs293325, rsl 11234106, rs4930860, rsl885193, rsl6965880.

1.3, high-fidelity whole-genome DNA amplification of nucleated red blood cells

Take the nucleated red blood cells obtained in Example 1.1, separate the single cells under the microscope, and put them into a PCR tube to perform single-cell whole-genome DNA amplification. For the specific operation method, refer to Patent 201110378904.4, and after the amplification, the DNA purification kit ( QIAGEN) Purification.

1.4. Identification of fetal-derived genomic DNA

The genomic DNA amplified in Example 1.3 was identified according to the individual genetic traits identification method described in Example 1.2, and the genotype of each genome was determined, and the genomic DNA derived from the fetal cells was determined according to different conditions as follows. :

If the individual genetic traits of the fetal mother and father are obtained in Example 1.2, the fetal genotype should be different from the father and mother genotypes, and the genotypes of each point originate from the father or mother and follow the genetic law; if in Example 1.2 Only the individual genetic characteristics of the fetus mother are obtained, the fetal genotype should be different from the mother, and one of each locus is derived from the mother; if it does not meet the above rules, it is contaminated, and the sample is discarded.

If multiple fetal genotypes are detected, pregnant women should be advised to perform ultrasonic testing of the number of fetuses to verify experimental data. 1.5. Labeling and preservation of fetal-derived genomic DNA

The fetal-derived genomic DNA obtained in Example 1.4 was labeled and stored frozen at -20 °C. Example 2-6, application

According to the method described in Example 1, 5 pregnant women aged 12-24 weeks were selected, 20 ml of peripheral blood of pregnant women were taken, and nucleated red blood cells were isolated. Ten erythrocytes were isolated from each pregnant woman for genome-wide amplification, and then the mother's somatic cells were used as controls to identify the fetal genome in the amplified genome. Four pregnant women identified 1 - 3 fetal cells, 1 pregnant woman was not detected for the first time. A further round of genome-wide amplification and identification of 10 nucleated red blood cells was obtained from the undetected pregnant women, and two of them were found to be fetal cells. The above five volunteers only identified a fetus's SNP classification, indicating that there is only one fetus, and the fetus was born one after another, which fully proves this.

The results are shown in Table 1.

Table 1,

In summary, the method of the present invention is used to separate nucleated red blood cells from peripheral blood of pregnant women, and separate single cells from these nucleated red blood cells for high-fidelity whole genome amplification, thereby obtaining fetal genomic DNA non-invasively, simply and rapidly. And successful identification of fetal SNP genotyping. At the same time, the method can be used to determine the number of fetuses, and in the case of multiple fetuses, it is also possible to accurately obtain the pure genomic DNA of each fetus.

The technique of the present invention can ensure the purity, integrity and high fidelity of the amplified fetal genomic DNA by using high-efficiency, high-fidelity and highly sensitive single-cell whole-genome DNA amplification (see the amplification effect for details). Patent 201110378904.4). This provides a material basis for comprehensive and accurate application of prenatal genetic diagnosis. It should be understood by those skilled in the art that the above-described embodiments and operating methods, instruments, and reagents not mentioned in the Summary of the Invention should be understood to be implemented by the prior art or by those skilled in the art according to the prior art.

The specific embodiments of the present invention have been described in detail above, but by way of example only, the invention is not limited to the specific embodiments described above. Any equivalent modifications and substitutions of the present invention are also within the scope of the invention. Accordingly, equivalent changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

Claim

1. A method for separating and obtaining complete fetal genomic DNA, characterized in that it comprises the following steps:

The method according to claim 1, wherein the maternal (pregnant) peripheral blood is a maternal (pregnant) peripheral venous blood of 9 to 38 weeks of gestation.

3. The method of claim 1, wherein the individual genetic trait typing comprises genotyping identification using a SNP combination or a STR combination.

4. The method according to claim 1, wherein the method for separating nucleated red blood cells comprises: immunomagnetic bead sorting or magnetic activated cell sorting, flow cytometry or fluorescence activation Cell sorting, charge flow separation, culture screening enrichment or density gradient centrifugation.

5. The method according to claim 1, wherein the mother and/or father's somatic cells are derived from: dandruff, oral epithelium or peripheral blood of a pre-pregnancy mother.

6. The method according to claim 1, wherein the method for identifying an individual's genetic feature classification comprises: a gene chip method, a PCR sequencing method, an MGB fluorescent PCR method, a restriction fragment length polymorphism method, and a single method. Chain conformation polymorphism, denaturing gradient gel electrophoresis, flight mass spectrometry or denaturing high performance liquid chromatography.