阅读说明：本技术 一种产前亲权关系鉴定的方法 (Antenatal paternity relationship identification method ) 是由 陈洪亮 于 2019-11-12 设计创作，主要内容包括：一种产前亲权关系鉴定的方法,利用环化扩增放大仅仅只需提供母亲外周10ul-1.2ml血浆,在母亲外周血浆中提取的游离DNA已经包含了胎儿的游离DNA,所以母亲和胎儿只需一份样品即可。由于只需抽取孕妇的静脉血,操作简便,且血量少,因此不会对孕妇和胎儿造成创伤,且孕10周后即可鉴定。(A method for prenatal paternity testing utilizes cyclic amplification to provide only 10ul-1.2ml of plasma from the mother's periphery, where the extracted free DNA from the mother's periphery already contains the fetal free DNA, so that only one sample is needed for the mother and the fetus. Because only the venous blood of the pregnant woman needs to be extracted, the operation is simple and convenient, and the blood volume is small, the wound to the pregnant woman and the fetus can not be caused, and the identification can be carried out after 10 weeks of pregnancy.)

1. A method for prenatal paternity testing, comprising: which comprises the following steps:

designing a site and a primer;

extracting sample DNA of a pregnant woman sample and a pending male sample;

step three, preparing a sample DNA fragment;

step four, processing the sample DNA fragments before sequencing;

step five, sequencing;

sixthly, carrying out post-processing on the sequencing data, and screening homozygous SNP (single nucleotide polymorphism) sites of the pregnant woman sample and the undetermined male sample;

step seven, comparing the pregnant woman sample with the male sample to be determined at each same homozygous SNP locus, defining the locus with the same highest base type as a positive locus, defining the locus as a negative locus if the highest base types are different, and counting the number of the positive loci and the number of the negative loci;

and step eight, carrying out paternity judgment according to the positive locus or the negative locus obtained in the step seven.

2. The method of prenatal paternity testing as claimed in claim 1, wherein: the third step specifically comprises: circularizing the sample DNA, digesting the circularized product, amplifying the digested circulating nucleic acid, and disrupting the amplified circulating nucleic acid.

3. The method of prenatal paternity testing as claimed in claim 1, wherein: and in the step five, the sequencing is high-throughput sequencing.

4. A method of prenatal paternity testing as claimed in claim 3, wherein: the fourth step comprises: amplifying a fragment containing the SNP locus, purifying the amplified PCR product, repairing the end of the PCR product, screening to obtain a flat-end DNA fragment, connecting joints to obtain a DNA fragment with joints, amplifying and purifying the PCR product to obtain a small fragment library, and detecting the library concentration and the fragment size of the small fragment library.

5. The method of prenatal paternity testing as claimed in claim 1, wherein: and in the fifth step, the sequencing is Nanopore sequencing.

6. The method of prenatal paternity testing as claimed in claim 4, wherein: the fifth step comprises the following steps:

opening a starting port of the Nanopore sequencer, and checking whether small bubbles exist under a valve cover;

preparing and loading the sample DNA fragment obtained in the fourth step into a SpotoN flow cell: preparing a priming mix: uniformly mixing RBF and nucleic-free water, sucking 800ul of RBF and nucleic-free water, injecting the RBF and nucleic-free water into a primary port, and standing at room temperature for 5 min; library preparation: RBF, LLB, nucleic-free water and DNA library are mixed evenly, 75ul of solution is absorbed and dripped into a sample inlet hole, a cover is covered, and sequencing is prepared:

running a program according to MinKNOW instructions, and sequencing;

obtaining a file of the fastq sequencing results from the nanopore sequencer;

and aligning a plurality of short sequences obtained from each long sequence in the previous step with each other by adopting a multiple alignment algorithm, and calibrating the bases.

7. The method of prenatal paternity testing as claimed in claim 5, wherein: the fourth step comprises: repairing, screening and purifying the tail ends of the sample DNA fragments to obtain cohesive tail ends, connecting joints to obtain joint-added DNA fragments, and screening and purifying the joint-added DNA fragments to obtain DNA to be sequenced.

8. The method of prenatal paternity testing as claimed in claim 1, wherein: and in the second step, the pregnant woman sample is pregnant woman peripheral blood or a pregnant woman leucocyte sample.

9. The method of prenatal paternity testing as claimed in claim 7, wherein: and in the second step, when the pregnant woman sample is the peripheral blood of the pregnant woman, 10ul-1.2ml of blood plasma needs to be collected, and then the free DNA in the pregnant woman blood plasma sample is extracted by using a magnetic bead method.

Technical Field

The invention relates to the field of paternity test, in particular to a method for effectively identifying prenatal paternity relationship by using a small amount of blood plasma.

Background

The paternity test is to analyze genetic characteristics from the morphological structure or physiological function similar characteristics of offspring and parents according to the theory and practice of human genetics, judge suspicious father-son relationship or mother-son relationship and make positive or negative conclusions. The earliest approach to determining paternity for the detection of genetic polymorphisms was restriction fragment length polymorphism analysis using restriction enzymes on a Variable Number of Tandem Repeats (VNTRs) in the human genome. With the advancement of technology, DNA Polymerase Chain Reaction (PCR), enabling shorter nucleic acid fragments to be used for analysis, has focused the analysis targets on shorter Short Tandem Repeats (STRs) in VNTR, coupled with the use of multiplex PCR technology, rapidly enabled typing detection of STR loci for forensic and criminal investigation. STRs are also currently the most commonly used genetic markers.

Single Nucleotide Polymorphisms (SNPs) are third generation genetic markers that result from the occurrence of two bases at a particular nucleotide position due to a single base mutation, the frequency of the least one of which in the population is not less than 1%. The characteristics of the STR are distinct from those of the first generation RFLP and the second generation STR in that the difference of the lengths is taken as a genetic marker. The distribution of SNPs is dense, if calculated with the frequency of 1%, more than 300 ten thousand SNP genetic markers exist in the human genome, which can reach the limit of the number of polymorphic sites of the human genome, and therefore, the SNP genetic markers are considered as the genetic markers with the best application prospect. Has wide application in medical genetics, population genetics and pharmacogenomics. In forensic physical evidence testing, high importance is also paid to the abundant content and genetic stability of SNP.

Prenatal paternity testing, also known as embryonic paternity testing and fetal paternity testing, refers to the identification of the father of a fetus in the biological sense by genetic technology. The prior prenatal paternity test technology is to extract DNA substances from fetal villi or amniotic fluid of pregnant women, and to identify and detect STR of the fetus and compare the detected STR with DNA of a suspected father so as to confirm the paternity.

Disclosure of Invention

In order to solve the above problems, the present invention provides a technical solution of a method for antenatal paternity test, which comprises the following steps:

designing a site and a primer;

extracting sample DNA of a pregnant woman sample and a pending male sample;

step three, preparing a sample DNA fragment;

step four, processing the sample DNA fragments before sequencing;

step five, sequencing;

sixthly, carrying out post-processing on the sequencing data, and screening homozygous SNP (single nucleotide polymorphism) sites of the pregnant woman sample and the undetermined male sample;

step seven, comparing the pregnant woman sample with the male sample to be determined at each same homozygous SNP locus, defining the locus with the same highest base type as a positive locus, defining the locus as a negative locus if the highest base types are different, and counting the number of the positive loci and the number of the negative loci;

and step eight, carrying out paternity judgment according to the positive locus or the negative locus obtained in the step seven.

Further preferred is: the third step specifically comprises: circularizing the sample DNA, digesting the circularized product, amplifying the digested circulating nucleic acid, and disrupting the amplified circulating nucleic acid.

Further preferred is: and in the step five, the sequencing is high-throughput sequencing.

Further preferred is: the fourth step comprises: amplifying a fragment containing the SNP locus, purifying the amplified PCR product, repairing the end of the PCR product, screening to obtain a flat-end DNA fragment, connecting joints to obtain a DNA fragment with joints, amplifying and purifying by PCR to obtain a small fragment library, and detecting the library concentration and the fragment size of the small fragment library.

Further preferred is: and in the fifth step, the sequencing is Nanopore sequencing.

Further preferred is: the fifth step comprises the following steps:

1) opening a starting port of the Nanopore sequencer, and checking whether small bubbles exist under a valve cover;

2) preparing and loading the sample DNA fragment obtained in the fourth step into a SpotoN flow cell: preparation of primingmix: uniformly mixing RBF and nucleic-free water, sucking 800ul of RBF and nucleic-free water, injecting the RBF and nucleic-free water into a primary port, and standing at room temperature for 5 min; library preparation: uniformly mixing RBF, LLB, nucleic-free water and DNA library, sucking 75ul of the mixture, dripping the mixture into a sample inlet, and covering a cover to prepare sequencing;

3) running a program according to MinKNOW instructions, and sequencing;

4) obtaining a file of the fastq sequencing results from the nanopore sequencer;

5) and aligning a plurality of short sequences obtained from each long sequence in the previous step with each other by adopting a multiple alignment algorithm, and calibrating the bases.

Further preferred is: the fourth step comprises: repairing, screening and purifying the tail ends of the sample DNA fragments to obtain cohesive tail ends, connecting joints to obtain joint-added DNA fragments, and screening and purifying the joint-added DNA fragments to obtain DNA to be sequenced.

Further preferred is: and in the second step, the pregnant woman sample is pregnant woman peripheral blood or a pregnant woman leucocyte sample.

Further preferred is: and in the second step, when the pregnant woman sample is the peripheral blood of the pregnant woman, 10ul-1.2ml of blood plasma needs to be collected, and then the free DNA in the pregnant woman blood plasma sample is extracted by using a magnetic bead method.

Compared with the prior art, the invention has the following advantages:

the method of the invention utilizes a different mode of DNA amplification: the method reduces the requirement on the blood sample amount, adopts the cyclic amplification, selects more samples in a sequencing mode, can select second-generation sequencing and third-generation sequencing, and has different meanings of more choices on time and cost;

in addition, the invention uses the third generation sequencing nanopore to circularly amplify the free DNA, thereby solving the disadvantages of the application of the third generation sequencer in the aspect of plasma free DNA, simultaneously, the steps of experiment, sequencing and data analysis SNP typing are different, the experiment time of the embodiment is fast, the requirement on the experiment environment is low, and the requirement on blood transportation is further reduced;

in conclusion, the invention solves the technical problem that the client is inconvenient to collect the sample, realizes the purpose of collecting a small amount of plasma and achieving the paternity test accuracy and the actual operability, meets the requirements of most clients, popularizes the sample collection method and improves the practicability and the actual operability of the whole prenatal paternity relationship test.

Detailed Description

The following describes in detail embodiments of the present invention.