CN113005194A - Primer composition, product and method for detecting abnormal haplotype of chromosome small segment - Google Patents
Primer composition, product and method for detecting abnormal haplotype of chromosome small segment Download PDFInfo
- Publication number
- CN113005194A CN113005194A CN202110460592.5A CN202110460592A CN113005194A CN 113005194 A CN113005194 A CN 113005194A CN 202110460592 A CN202110460592 A CN 202110460592A CN 113005194 A CN113005194 A CN 113005194A
- Authority
- CN
- China
- Prior art keywords
- chromosome
- primer composition
- product
- abnormal
- primer
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a primer composition, a product and a method for detecting abnormal haplotype of a chromosome small segment, wherein the primer composition comprises a primer for specifically amplifying an abnormal region of the chromosome small segment Xq22.2 and specifically amplifying SNP sites in 2M at the upstream and downstream of the Xq22.2, the primer composition can be used for analyzing the abnormal chromosome small segment from a parent source, and the risk chromosome of the chromosome abnormal segment is determined according to the family relation and the haplotype analysis principle according to the male, female and offspring or the male, female and the parent carrying the abnormal segment, so that the genetic condition of the abnormal chromosome small segment of an embryo is judged by analyzing the haplotype of the embryo.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of embryo transplantation detection, in particular to a primer composition, a product and a method for detecting abnormal haplotypes of a small chromosome segment.
[ background of the invention ]
More than 200 chromosomal diseases are known in humans. Most chromosomal disorders are caused by an abnormal number of chromosomes, with down's syndrome being the most prevalent. Another part of the disease is caused by deletion or duplication of chromosome fragments (CNVs), which are collectively called chromosome microdeletion/microreplication syndrome. Chromosomal microdeletions/microreplications refer primarily to abnormalities in chromosomal segments of less than 5Mb in size, and chromosomal copy number variations play an important role in the diagnosis of clinical symptoms and in the identification of fetal chromosomal disorders. The chromosome diseases have various clinical phenotypes, and are manifested by multiple congenital malformations, limb disabilities, developmental delay, intellectual impairment, epilepsy, autism, learning disorder and the like (J Mol Diagn 2014,16:519e 526; http:// dx. doi. org/10.1016/J. joldx. 2014.05.002).
The chromosomal microdeletion/microreplication results in a mutation of genetic material in the reproductive cells and fertilized eggs of the previous generation. The method for preventing the birth of the child with the genetic disease is still the most effective means at present, and the birth of the child is prevented by carrying out selective abortion at 16-20 weeks of gestation through prenatal diagnosis in the past. Pre-implantation genetic diagnosis (PGD) is a new diagnosis technology developed by combining an assisted reproduction technology and a molecular biology technology, 5-10 blastocyst stage cells are obtained through an embryo biopsy technology to carry out genetic diagnosis analysis (consensus of genetics diagnosis/screening experts before embryo implantation, journal of Chinese medicine 2018,35(2):151-155), and normal and flawless healthy embryos are selected to be implanted into the uterus to continue pregnancy, so that the purpose of good birth is achieved, complications such as bleeding and infection caused by the traditional prenatal diagnosis technology and psychosomatic pain caused by abortion and induction to pregnant women can be avoided, and ethical disputes caused by the complications can be avoided.
The current methods for detecting chromosome copy number before embryo implantation mainly include Fluorescence In Situ Hybridization (FISH), microarray comparative genomic hybridization (aCGH), single nucleotide polymorphism microarray technology (SNP array), and high throughput Sequencing (NGS). The FISH is adopted to carry out chromosome copy number analysis, the karyotype of the chromosome in the metaphase of the peripheral blood of the couple of a patient needs to be verified in advance, and the fluorescence intensity and the specificity of an analysis probe are detected on interphase nucleus. Microarray technology is technically difficult, lack of expertise and high cost. The resolution of the current PGT-A and PGT-SR technology for detecting the copy number abnormality of the embryo chromosomes is about 4-10Mb, and the requirement of detecting the microdeletion/microreplication of the embryo chromosomes cannot be met.
Therefore, there is a need to develop a primer composition, product and method for detecting abnormal haplotypes of chromosomal small segments to overcome the deficiencies of the prior art and to solve or alleviate one or more of the above problems.
[ summary of the invention ]
In view of the above, the present invention provides a primer composition, a product and a method for detecting abnormal haplotype of chromosome small segment, which can be used for analyzing abnormal chromosome small segment from parents, determining risk chromosome of chromosome abnormal segment according to family relationship and haplotype analysis principle according to male, female and offspring or male, female and parents carrying abnormal segment, and further judging the genetic condition of abnormal chromosome small segment of embryo by analyzing embryo haplotype.
In one aspect, the invention provides a primer composition for detecting abnormal haplotype of chromosome small segment, which comprises a primer for specifically amplifying an abnormal region of the chromosome small segment Xq22.2 and specifically amplifying SNP sites in upstream and downstream 2M of Xq22.2.
The above aspects and any possible implementations further provide an implementation that the specifically amplified xq22.2 chromosomal small fragment is abnormal for a human specifically amplified xq22.2 chromosomal small fragment.
The above aspects and any possible implementations further provide an implementation where the sequence of the specific amplification xq22.2 chromosome small fragment abnormal region is seq id NO: 151-181 primer pair.
The above aspects and any possible implementations further provide an implementation where the primer sequence for specifically amplifying the SNP sites in 2M upstream and downstream of xq22.2 is seq id NO: 1-150 and 182-289 primer pairs.
The aspect as defined above and any possible implementation form further provides an implementation form wherein the primer composition comprises seq id NO: 1-289.
The above aspects and any possible implementation manners further provide a detection product before embryo implantation, wherein the detection product is prepared from the primer composition.
The above aspects and any possible implementations further provide an implementation, and the detection product is a detection kit.
The above aspects and any possible implementations further provide an implementation, and the detection kit includes:
a reagent for library construction comprising: specific binding primers, universal PCR primers, multiplex PCR polymerase, DNA ligase, terminal repair enzyme, dNTPs and reaction buffer solution;
the reagent for purifying the reaction product of the PCR comprises a product purification magnetic bead and a buffer solution.
The above aspects and any possible implementations further provide an implementation, and the detection kit further includes a negative quality control material and a positive quality control material.
The above aspects and any possible implementation manners further provide a detection method before embryo implantation, which is implemented by the detection product, and the detection method comprises the following steps:
s1: isolating sample genomic DNA;
s2: performing multiplex PCR amplification on the enriched target region DNA by using the primer composition;
s3: purifying PCR products and sequencing;
s4: comparing and counting;
s5: and (6) analyzing the data.
Compared with the prior art, the invention can obtain the following technical effects:
1) universality: the method of the invention analyzes 289 SNPs in the dup Xq22.2(102740001_103300000) chromosome small fragment abnormal region, and can be used for gene diagnosis and embryo pre-transplantation diagnosis of different partners;
2) dup xq22.2(102740001_103300000) chromosomal small-fragment abnormal region and multisite SNP sequencing: based on the second generation sequencing technology, the method can analyze a plurality of SNPs near the dup Xq22.2(102740001_103300000) chromosome small fragment abnormal region and the dup Xq22.2(102740001_103300000) chromosome small fragment abnormal region without depending on known probes and designed probes;
3) high flux: based on a high-throughput sequencing technology, the method can detect dup Xq22.2 (102740001-103300000) chromosome small fragment abnormality at high throughput, and can analyze a large number of samples at one time by adding different tag sequences to each sample;
4) the cost is low: with the continuous development of sequencing technology and the continuous reduction of sequencing cost, the cost of analyzing the dup Xq22.2(102740001_103300000) chromosome small segment abnormity is also continuously reduced;
5) high sensitivity: can be used for analyzing 3-5 cells. Therefore, the method is suitable for detection before embryo transplantation in the test-tube infant technology;
6) specificity: the invention selects high-frequency polymorphic sites with CHB (northern Han) and CHS (southern Han) minimum allele frequencies larger than 0.2 in the range of 2Mb upstream and downstream of a dup Xq22.2(102740001_103300000) chromosome small fragment in thousand human genome planning data (http:// www.ncbi.nlm.nih.gov/variation/tools/1000 genes /), removes polynucleotide (polyN) and polymorphic sites with GC content of more than 70% in a 50bp sequence upstream and downstream of the sites, selects a region of 258 SNP sites aligned to hg19 of a human genome and a dup Xq22.2(102740001_103300000) chromosome small fragment as a target region, and designs 289 pairs of primers which have high specificity.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
[ detailed description ] embodiments
In order to better understand the technical scheme of the invention, the following detailed description of the embodiment of the invention.
It should be understood that the described embodiments are only some embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the examples of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The invention provides a primer composition, a product and a method for detecting abnormal haplotype of a chromosome small segment, which can be used for analyzing the abnormal chromosome small segment from a parent source, determining the risk chromosome of the chromosome abnormal segment according to a family relation and a haplotype analysis principle according to a male prescription, a female prescription and offspring or a male prescription, a female prescription and a parent carrying the abnormal segment, and further judging the genetic condition of the abnormal chromosome small segment of an embryo by analyzing the haplotype of the embryo.
The primer composition comprises a primer for specifically amplifying an abnormal region of a small chromosome segment Xq22.2 and specifically amplifying SNP sites in upstream and downstream 2M of Xq22.2. The specific amplification Xq22.2 chromosome small fragment is abnormal for human specific amplification Xq22.2 chromosome small fragments. The sequence of the specific amplification Xq22.2 chromosome small fragment abnormal region is SEQID NO: 151-181 primer pair. The primer sequence of the SNP locus in the upstream and downstream 2M of the specific amplification Xq22.2 is SEQ ID NO: 1-150 and 182-289 primer pairs. The primer composition comprises SEQ ID NO: 1-289.
The invention also provides a detection product before embryo implantation, and the detection product is prepared from the primer composition. The detection product is a detection kit. The detection kit comprises:
a reagent for library construction comprising: specific binding primers, universal PCR primers, multiplex PCR polymerase, DNA ligase, terminal repair enzyme, dNTPs and reaction buffer solution;
the reagent for purifying the reaction product of the PCR comprises product purification magnetic beads and a buffer solution;
negative quality control product and positive quality control product.
The invention also provides a detection method before embryo implantation, which is realized by the detection product, and the detection method comprises the following steps:
s1: isolating sample genomic DNA;
s2: performing multiplex PCR amplification on the enriched target region DNA by using the primer composition;
s3: purifying PCR products and sequencing;
s4: comparing and counting;
s5: and (6) analyzing the data.
The invention comprises an analysis method for analyzing chromosome small segment abnormality by using single nucleotide polymorphism sites, which can carry out linkage analysis on all chromosome small segment abnormalities, and has quicker detection method and more accurate detection result.
First definition
"reads" refers to the sequence fragments obtained by sequencing.
"Single Nucleotide Polymorphism (SNP)" refers to a DNA sequence polymorphism caused by a variation of a single nucleotide at the genome level.
"Haplotype" (Haplotype) refers to a group of related single nucleotide polymorphisms located in a specific region of a chromosome and tending to be inherited as a whole to offspring, and is also called a Haplotype or Haplotype.
"effective sites (information SNPs)" can provide polymorphic sites of genetic information while avoiding the selection of SNP sites having high homology, high GC content in adjacent sequences or polynucleotide sequences.
(2) Primer composition
The invention provides a primer group design method for detecting chromosome small fragment abnormality, which consists of a specific amplification Xq22.2 (102740001-103300000) chromosome small fragment abnormal region and Single Nucleotide Polymorphism (SNP) sites which are closely linked in the range of 2Mb at the upstream and downstream of the region.
In the present invention, using xq22.2(102740001_103300000) as an example, 289 pair of sequence-specific primers (as shown in table 1) were designed for human xq22.2. Wherein the primer for specifically amplifying the Xq22.2 comprises a primer pair with the number 151-181; the primers for specifically amplifying SNP sites which are closely linked within the range of 2Mb upstream and downstream of Xq22.2 comprise primer pairs with the numbers of 1-150 and 182-289.
These primers are characterized in that: the sequence is unique on a target chromosome; the position-specific oligonucleotides have the same annealing temperature; 289 pairs of primers were mixed into 4 PCR reaction tubes, and 289-fold reactions were carried out in four PCR reaction tubes.
TABLE 1 chromosomal Small segment anomaly Xq22.2(102740001_103300000) detection primers
In a most preferred embodiment, the primer composition of the present invention comprises all primer pairs numbered 1-289 in Table 1.
(3) Test product
The invention provides a detection product for detecting abnormal Xq22.2(102740001_103300000) of a chromosome small fragment, which comprises the primer composition disclosed by the invention.
In a particular embodiment, the test product is a test kit comprising:
the reaction reagent for library construction includes: specific binding primers, universal primers, multiple PCR polymerase, DNA ligase, terminal repair enzyme, dNTPs and reaction buffer;
② a reagent for purifying PCR reaction products.
The specific binding primer is the primer composition of the invention.
Preferably, the reagents for PCR product purification include product purification magnetic beads and buffers. The kit can also comprise a negative quality control product and a positive quality control product.
(4) Detection method
The invention provides a detection method for in vitro detection of chromosome small segment abnormality Xq22.2(102740001_103300000), which comprises the following steps:
firstly, separating the genomic DNA of a sample;
secondly, the primer composition is utilized to carry out multiplex PCR amplification to enrich the DNA of the target area;
purifying PCR product and sequencing;
comparing and counting;
data analysis.
Preferably, the sample is from an embryo or whole blood.
The acquisition of the embryo genome DNA is that when the embryo develops to the blastocyst stage, 3-5 peripheral trophoblast cells are taken out, and the genome DNA in the cells is enriched by using a whole genome amplification method. Optionally, the amplification product is purified using magnetic beads.
The target region DNA molecule enrichment adopts a multiplex PCR amplification method. Specific principles and methods DNA molecules are enriched into fragments of a certain size in a comparative set, see the instructions provided by the manufacturer. In one embodiment of the invention, the DNA fragment has a size of 125-275 bp.
Preferably, the library is constructed according to the standard library construction process of NEXTflex Rapid DNA-Seq Kit in the second step. In the process, the target region is amplified into DNA molecules concentrated in 125-275 bp by multiplex PCR, linkers for sequencing are added at two ends, and different tag sequences (barcode) are added to each sample, so that the data of a plurality of samples can be distinguished in the data obtained by one-time sequencing.
Preferably, the sequencing method employed is a high throughput sequencing method. In a particular embodiment, the sequencing platform is the Illumina Miseq sequencing platform. More preferably, the sequencing depth is 300X to 3000X, i.e., each specific PCR amplification product is sequenced 300 to 3000 times, e.g., in an embodiment of the invention, the sequencing depth is 1000X, i.e., the strip of specific PCR amplification product is sequenced 1000 times.
Preferably, when the DNA molecules to be tested are from multiple test samples, each sample is tagged with a different tag sequence (barcode) for sample discrimination during sequencing (Micah Hamady, Jeffrey J Walker, JKirk Harris et al, error-correcting coded primers for sequencing Methods,2008,5(3)), thereby allowing for simultaneous sequencing of multiple samples.
In the present invention, the genomic reference sequence may be from a public database. For example, the human genomic sequence may be a human genomic reference sequence in the NCBI or UCSC databases.
In the present invention, in data analysis, firstly removing bases with sequence quality value less than 30 and the following sequences, selecting sequences with length greater than 30bp, aligning the sequences to human Genome HG19(Genome Build 37.3) by any sequence Alignment program, such as BWA (BWA-Wheeler Alignment Tool, v0.7.15) software, which is available to those skilled in the art, to obtain the position of the read on the reference Genome, and then filtering out the sequences with Alignment quality value of 0 or Alignment length less than 95% of the total length. Finally, VarScan (v2.3.6) software is used for detecting the SNP of the corresponding chromosome target region in the data, the lowest sequencing depth of the detected SNP is 30X, and finally the haplotype SNP coverage factor and the genotype are analyzed.
The method can be used for diagnosing the chromosome small segment abnormal dup Xq22.2(102740001_103300000) embryo before transplantation for applicable people, and is favorable for providing genetic counseling and clinical decision basis. dup Xq22.2(102740001_103300000) gene diagnosis, embryo pre-transplantation diagnosis and prenatal diagnosis can effectively prevent the birth of dup Xq22.2(102740001_103300000) children. The population to which the invention is applicable can be dup Xq22.2(102740001_103300000) carriers and patients.
The above examples of suitable persons are only used for the present invention, and should not be construed as limiting the scope of the present description.
(5) Pre-embryo implantation screening
The invention also provides a screening method before embryo implantation for dup Xq22.2(102740001_103300000) chromosome small segment abnormality.
In a particular embodiment, the pre-embryo implantation screening method comprises the steps of (4) the detection method. Embryos not carrying dup xq22.2(102740001_103300000) chromosomal small segment abnormalities were selected for uterine implantation.
Example 1:
2 embryos dup Xq22.2(102740001_103300000) chromosomal small segment pre-implantation assays.
Family background: children had dup xq22.2(102740001_103300000) small fragment repeats and women had dup xq22.2(102740001_103300000) heterozygous repeats. In order to avoid the pain of both men and women caused by abortion or induced labor of the child suffering from the pregnancy genetic disease, after sufficient informed consent, both men and women select haplotype detection aiming at avoiding the small segment repetition of the offspring.
First, build storehouse and sequencing
(1) Obtaining whole blood samples of 3 cases of couples and sick children, extracting genome DNA, using NonaQ micro spectrophotometer (Bo-ao crystal dictionary) to control quality, the purity OD260/280 is larger than 1.2, and separating 20ng for subsequent experiment.
(2) Obtaining 2 cases of embryo sample cells of the family, carrying out whole genome amplification, purifying a whole genome amplification product by magnetic beads, quantifying by using a Qubit (Invitrogen,1x dsDNA HS Assay Kit), and separating 20ng for subsequent experiments;
(3) the preferred primer set (all primer pairs 1-289 in Table 1) of the invention was used for multiplex PCR amplification and the library was constructed according to the standard library construction procedure of NEXTflex Rapid DNA-Seq Kit.
(4) Purifying PCR products and sequencing;
in brief, the linkers used for sequencing are added at the two ends of the DNA molecules of the multiple PCR amplification product, the nucleic acid molecules grow in clusters under certain conditions, and then sequencing is carried out on the Illumina Miseq, so as to obtain the DNA fragment sequence of the target region with the fragment length distributed in 125 bp-275 bp.
The comparison and statistics
The original data generated by Illumina Miseq sequencer was used to remove bases with sequence quality value less than 30 and sequences thereafter, select sequences with length greater than 30bp, and aligned to human Genome HG19(Genome Build 37.3) using BWA (Burrows-Wheeler Alignment Tool, v0.7.15) software.
Analysis of data
And (3) detecting the SNP of the corresponding chromosome target region in the data by using VarScan (v2.3.6) software, detecting the SNP with the minimum sequencing depth of 30X, and finally analyzing the haplotype SNP coverage factor and the genotype.
Results
The results are shown in Table 2
The haplotype is constructed according to the principle, the X chromosome is a sex chromosome, only one X chromosome is arranged in a male part and a son, two chromosomes are arranged in a female part, and the chromosome inherited to the son is a risk chromosome carrying dup Xq22.2 small segment duplication.
The results show that: embryo 1 is a small repeated carrier of Xq22.2(102740001_103300000), and small Xq22.2(102740001_103300000) of embryo 2 is not abnormal.
Table 2 the results of the family and embryo analysis based on the family relationship and haplotype analysis are shown below.
Table 2 shows the haplotype detection results. Wherein F1 represents a paternal-derived DNA strand, M1 represents a maternally-derived normal DNA strand, and M0 represents a maternally-derived DNA strand carrying a small piece repeat of xq22.2(102740001_ 103300000); NO: positions 1-41, 43-62 are valid sites within 2M upstream and downstream of female part Xq22.2(102740001_103300000) in haplotyping, and position 42 is the valid site of the female part Xq22.2(102740001_103300000) repeat region.
The primer composition, the product and the method for detecting abnormal haplotypes of chromosomal small segments provided in the embodiments of the present application are described in detail above. The above description of the embodiments is only for the purpose of helping to understand the method of the present application and its core ideas; meanwhile, for a person skilled in the art, according to the idea of the present application, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present application.
As used in the specification and claims, certain terms are used to refer to particular components. As one skilled in the art will appreciate, manufacturers may refer to a component by different names. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.
It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a commodity or system that includes the element.
It should be understood that the term "and/or" as used herein is merely one type of association that describes an associated object, meaning that three relationships may exist, e.g., a and/or B may mean: a exists alone, A and B exist simultaneously, and B exists alone. In addition, the character "/" herein generally indicates that the former and latter related objects are in an "or" relationship.
The foregoing description shows and describes several preferred embodiments of the present application, but as aforementioned, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the application as described herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the application, which is to be protected by the claims appended hereto.
Claims (10)
1. A primer composition for detecting abnormal haplotype of a chromosome small fragment is characterized by comprising a primer for specifically amplifying an Xq22.2 chromosome small fragment abnormal region and an SNP locus in 2M upstream and downstream of the Xq22.2 chromosome small fragment abnormal region.
2. The primer composition of claim 1, wherein the specifically amplified small xq22.2 chromosome fragment is abnormal in human specifically amplified small xq22.2 chromosome fragments.
3. The primer composition of claim 1, wherein the upstream and downstream sequences of the specific amplification Xq22.2 chromosome small fragment abnormal region are SEQ ID NO: 151-181 primer pairs.
4. The primer composition of claim 3, wherein the upstream and downstream sequences of the primer for specifically amplifying the SNP sites in 2M upstream and downstream of Xq22.2 are SEQ ID NO: 1-150 and 182-289 primer pairs.
5. The primer composition of claim 4, wherein the primer composition comprises the nucleotide sequence of SEQ ID NO: 1-289.
6. A test product before embryo implantation, which is prepared from the primer composition of any one of claims 1 to 5.
7. The assay product of claim 6, wherein the assay product is an assay kit.
8. The test product of claim 7, wherein the test kit comprises:
a reagent for library construction comprising: specific binding primers, universal PCR primers, multiplex PCR polymerase, DNA ligase, terminal repair enzyme, dNTPs and reaction buffer solution;
the reagent for purifying the reaction product of the PCR comprises a product purification magnetic bead and a buffer solution.
9. The test product of claim 8, wherein the test kit further comprises a negative quality control and a positive quality control.
10. A method for testing a pre-implantation embryo by means of a test product according to any one of claims 6 to 9, characterized in that it comprises the following steps:
s1: isolating sample genomic DNA;
s2: performing multiplex PCR amplification on the enriched target region DNA by using the primer composition;
s3: purifying PCR products and sequencing;
s4: comparing and counting;
s5: and (6) analyzing the data.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110460592.5A CN113005194A (en) | 2021-04-27 | 2021-04-27 | Primer composition, product and method for detecting abnormal haplotype of chromosome small segment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110460592.5A CN113005194A (en) | 2021-04-27 | 2021-04-27 | Primer composition, product and method for detecting abnormal haplotype of chromosome small segment |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113005194A true CN113005194A (en) | 2021-06-22 |
Family
ID=76380564
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110460592.5A Pending CN113005194A (en) | 2021-04-27 | 2021-04-27 | Primer composition, product and method for detecting abnormal haplotype of chromosome small segment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113005194A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018106782A1 (en) * | 2016-12-08 | 2018-06-14 | Case Western Reserve University | Methods and compositions for enhancing functional myelin production |
| CN110578009A (en) * | 2019-11-11 | 2019-12-17 | 广东华美众源生物科技有限公司 | Multiplex amplification detection kit containing 40 InDel genetic polymorphic sites of human X chromosome |
| CN110628891A (en) * | 2018-06-25 | 2019-12-31 | 深圳华大智造科技有限公司 | Method for screening embryo for gene abnormality |
| CN111363807A (en) * | 2020-04-30 | 2020-07-03 | 中信湘雅生殖与遗传专科医院有限公司 | EVC syndrome detection method, detection composition and detection kit |
| CN111575378A (en) * | 2020-04-26 | 2020-08-25 | 中信湘雅生殖与遗传专科医院有限公司 | Detection method, detection composition and detection kit for hereditary breast cancer and ovarian cancer syndrome |
-
2021
- 2021-04-27 CN CN202110460592.5A patent/CN113005194A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018106782A1 (en) * | 2016-12-08 | 2018-06-14 | Case Western Reserve University | Methods and compositions for enhancing functional myelin production |
| CN110628891A (en) * | 2018-06-25 | 2019-12-31 | 深圳华大智造科技有限公司 | Method for screening embryo for gene abnormality |
| CN110578009A (en) * | 2019-11-11 | 2019-12-17 | 广东华美众源生物科技有限公司 | Multiplex amplification detection kit containing 40 InDel genetic polymorphic sites of human X chromosome |
| CN111575378A (en) * | 2020-04-26 | 2020-08-25 | 中信湘雅生殖与遗传专科医院有限公司 | Detection method, detection composition and detection kit for hereditary breast cancer and ovarian cancer syndrome |
| CN111363807A (en) * | 2020-04-30 | 2020-07-03 | 中信湘雅生殖与遗传专科医院有限公司 | EVC syndrome detection method, detection composition and detection kit |
Non-Patent Citations (3)
| Title |
|---|
| HUILI XUE等: "Prenatal diagnosis of PLP1 duplication by single nucleotide polymorphism array in a family with Pelizaeus-Merzbacher disease", 《AGING》 * |
| REBECCA L. MARGRAF等: "Novel PLP1 Mutations Identified With Next-Generation Sequencing Expand the Spectrum of PLP1-Associated Leukodystrophy Clinical Phenotypes", 《CHILD NEUROLOGY OPEN》 * |
| 章容等: "1例佩-梅氏病Xq22.2片段拷贝数变异的产前诊断", 《第十一届全国遗传病诊断与产前诊断学术交流会暨第二届海峡两岸医药卫生交流协会遗传与生殖专业委员会年会论文集》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9994902B2 (en) | Methods and genotyping panels for detecting alleles, genomes, and transcriptomes | |
| AU2004217872B2 (en) | Identification of fetal DNA and fetal cell markers in maternal plasma or serum | |
| CN108220403B (en) | Method and device for detecting specific mutation site, storage medium and processor | |
| WO2014127749A1 (en) | Application of single cell genome sequencing in preimplantation genetic diagnosis | |
| CN106029899B (en) | Method, system and computer readable medium for determining SNP information in predetermined region of chromosome | |
| CN115029451B (en) | Sheep liquid phase chip and application thereof | |
| WO2024027569A1 (en) | Haplotype construction method independent of proband | |
| CN111518917B (en) | Micro haplotype genetic marker combination and method for noninvasive prenatal paternity relationship determination | |
| CN105420233B (en) | HBB gene mutation and HLA typing detection kit | |
| CN105112541B (en) | Human embryos spinal muscular atrophy mutator detection kit | |
| EP3797418A1 (en) | Method for determining the probability of the risk of chromosomal and genetic disorders from free dna of fetal origin | |
| CN116083562B (en) | SNP marker combination and primer set related to aspirin resistance auxiliary diagnosis and application thereof | |
| CN114657265B (en) | SNP marker for identifying weaning weight of alpine merino sheep and application thereof | |
| Adam et al. | GeneReviews glossary | |
| CN113005194A (en) | Primer composition, product and method for detecting abnormal haplotype of chromosome small segment | |
| US20100206316A1 (en) | Method for determining chromosomal defects in an ivf embryo | |
| CN115273972A (en) | Method for determining noninvasive prenatal paternity relationship by using micro-haplotypes | |
| CN116083592A (en) | Molecular marker related to sheep growth traits and application thereof | |
| CN114875117A (en) | Construction method and kit of gene library for detecting female infertility | |
| CN114958967A (en) | Construction method and kit of gene library for detecting male infertility | |
| CN113564266A (en) | SNP typing genetic marker combination, detection kit and application | |
| CN111286533B (en) | PAH gene multiplex PCR primer and application thereof | |
| CN112442527B (en) | Autism diagnosis kit, gene chip, gene target screening method and application | |
| CN117004708A (en) | Screening method, detection composition and detection kit for hereditary Imerslund-Grasbeck syndrome | |
| WO2017124214A1 (en) | Method for detecting chromosome robertsonian translocation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 102629 Room 302, floor 3, building 7, courtyard 19, Tianrong street, Daxing biomedical industry base, Zhongguancun Science and Technology Park, Daxing District, Beijing Applicant after: Beijing Jiabao Renhe Medical Technology Co.,Ltd. Address before: 102629 Room 203, building 6, courtyard 19, Tianrong street, Daxing biomedical industrial base, Zhongguancun Science and Technology Park, Daxing District, Beijing Applicant before: PEKING JABREHOO TECHNOIOGY Co.,Ltd. |
|
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210622 |