CN108330187B - Method and kit for quantitatively detecting X chromosome in cell - Google Patents
Method and kit for quantitatively detecting X chromosome in cell Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and relates to a method and a kit for quantifying an X chromosome in a cell. The method of the invention comprises the following steps: 1) designing amplification primers by using regions (XIDMRs) with a plurality of X chromosome inactivation specific DNA differential methylation sites (XIDMSs) on a plurality of X chromosomes as probes; 2) establishing copy number reference value ranges of all probes and all X chromosomes in normal male and female samples; and 3) detecting and analyzing the number and structural information of the X chromosome in the unknown karyotype cells. The kit of the present invention comprises: 1) a reagent for sulfite conversion of the DNA; 2) primers and reagents for PCR amplification of the probe region; and 3) primers and reagents for methylation detection of PCR amplification products. The invention can effectively find out whether the number and the structure of the X chromosome are abnormal and the abnormal type exists in the cell.
Description
Technical Field
The invention belongs to the field of biological medicine and reagent detection, and relates to an X chromosome quantitative detection method and a kit. In particular to a method for quantitatively detecting X chromosome in cells and a kit based on the method. In particular to a method and a kit for quantitatively detecting the X chromosome in cells based on the inactivation specific DNA differential methylation region of the X chromosome.
Background
The prior art discloses that human somatic cells contain 23 pairs of chromosomes, among which 22 pairs of autosomes, 1 pair of sex chromosomes. Sex chromosomes include the X chromosome and the Y chromosome, and can determine sex: the sex chromosomes of a normal male are XY and contain 1X chromosome, while the sex chromosomes of a normal female are XX and contain 2X chromosomes. The number and structural abnormality of X chromosome is related to multiple sex chromosome diseases, including Klinefelter syndrome (Klinefelter syndrome), Turner syndrome (Turner syndrome), Trisomy X syndrome (Trisomy X syndrome), etc. Quantitative detection of X chromosome in cells to obtain the quantity and structure information of X chromosome in cells can provide important intermediate results for diagnosis of multiple sex chromosome diseases.
In the prior art, karyotype analysis can accurately acquire the quantity and structural information of X chromosomes in cells, and the method is widely applied to clinic and scientific research, but the method needs cell culture and has a long period; and needs experienced detection personnel to observe and identify, and is difficult to develop on a large scale; high throughput genetic testing techniques, such as comparative genomic hybridization chips, single nucleotide polymorphism typing chips, and whole genome sequencing techniques, are cost prohibitive; conventional molecular genetics detection methods, such as fluorescent quantitative PCR, capillary electrophoresis and the like, are difficult to detect high-incidence chimera and structural abnormality in diseases.
Studies have revealed that the number of X chromosomes in somatic cells varies between males and females, and that it is necessary to maintain the same or close transcriptional levels of most X-linked genes between sexes by a dose compensation mechanism. In humans and mammals, this dose compensation is accomplished by X chromosome inactivation (X chromosome inactivation), i.e., 1X chromosome is randomly in an inactivated state (Xi) in female somatic cells, and about 80% of the genes on the inactivated X chromosome lose transcriptional activity; studies have shown that X chromosome inactivation occurs during embryonic development, is very stable, can be maintained for life, and its molecular mechanism is accomplished by epigenetic modifications, including changes in DNA methylation, non-coding RNA, and histone modifications. Inactivation of the X chromosome follows the n-1 principle, i.e.no matter how many X chromosomes are present in the cell, the other X chromosomes are inactivated except that 1X chromosome retains transcriptional activity (Xa).
The study also discloses that DNA methylation is involved in the X chromosome inactivation process. In humans and mammals, DNA methylation is mainly the replacement of the hydrogen atom (H) at carbon atom number 5 of the cytosine (C) base in CpG dinucleotide sequences by a methyl group (-CH)3) Substitution; CpG dinucleotides are often clustered in a gene promoter region to form a CpG island, the DNA methylation of the CpG island in the promoter region is related to the transcription activity of the gene, the hypomethylation generally indicates that the gene is in a transcription activation state, and the hypermethylation generally indicates that the gene is in a transcription inhibition state; on the inactivated X chromosome, the promoter region of the inactivated target gene is in a hypermethylated state, while on the activated X chromosomeThe promoter region of the response gene is in a hypomethylated state.
Research on this, quantitative information on the X chromosome in cells can be obtained by comparing the methylation levels of CpG sites on the X chromosome in male and female samples, which are unmethylated in the activated X chromosome but highly methylated only in the inactivated chromosome, and which are mainly located in the promoter region of the inactivation target gene and are present in clusters to form a DNA differentially methylated region (XIDMR) specific to inactivation of the X chromosome, based on which quantitative information on the X chromosome in cells can be obtained by quantitative analysis of the DNA methylation state of the relevant region.
Based on the current research situation of the prior art, the inventors of the present application propose to provide a method for quantifying the X chromosome in human cells, and a kit for quantifying the X chromosome in human cells.
Disclosure of Invention
The invention aims to provide a method for quantitatively detecting an X chromosome in a human cell and a kit based on the method based on the current research situation of the prior art, in particular to a method for quantitatively detecting an X chromosome in a cell based on a DNA differential methylation region specific to inactivation of the X chromosome and a kit.
The invention provides a method for quantitatively detecting an X chromosome in a human cell based on the principle that the methylation level of a plurality of differential methylation sites (XIDMS) in a DNA differential methylation region (XIDMR) with specific inactivation of the X chromosome is positively correlated with the quantity of the X chromosome in a cell, the methylation level of a plurality of XIDMS in a plurality of XIDMRs is quantified, and the quantity and structural information of the X chromosome in the cell can be acquired.
The principle of quantifying the X chromosome in the present invention is shown in FIG. 1;
the method is realized according to the following technical scheme:
screening and verifying X chromosome inactivation specific DNA differential methylation sites and regions (namely XIDMS and XIDMR), and selecting XIDMR containing a plurality of XIDMS as a marker; detecting the methylation level of each XIDMSs in cells with known karyotypes, and calculating the normal reference value range of the copy number of each XIDMR and the whole X chromosome; and obtaining the number and structure information of the X chromosome in the unknown karyotype cells according to the ratio of methylated fragments to unmethylated fragments in each XIDMS in the XIDMR.
Specifically, the method for quantitatively detecting the X chromosome in the human cells comprises the following steps:
1) designing corresponding methylation detection primers by using regions (XIDMR) with multiple DNA differential methylation sites (XIDMS) specific to inactivation of X chromosome on X chromosome as markers
Selecting male and female cells with normal karyotype, extracting DNA, and performing whole genome methylation detection by using Infinium management 450 BeadChips (450 k chip) of Iluutina to obtain methylation level information of CpG sites on X chromosome;
the XiDMS chosen should correspond to being unmethylated on the activated X chromosome (Xa) and highly methylated on the inactivated X chromosome (Xi); in male cells, methylation levels were close to 0 with only 1 Xa, while in female cells, methylation levels were close to 0.5 with 1 each of Xa and Xi;
the specific screening conditions in the method of the invention are as follows:
in male cells, the CpG sites are methylated to a degree of less than 0.05 and the standard deviation is less than 0.02; in female cells, the methylation degree of CpG sites is between 0.4 and 0.6, the standard deviation is less than 0.05, and the total 108 CpG sites meet the screening standard, and the sites are mostly positioned near the 5' end promoter region of the gene: 101 CpG sites are located within 2kb upstream and downstream of a Transcription Start Site (TSS);
performing salt sulfate PCR (polymerase chain reaction) and then Sanger sequencing (BSP) and pyrosequencing verification on candidate sites meeting the screening condition, screening the candidate sites and a gene region of which a plurality of nearby CpG sites all meet the XIDMS standard to serve as XIDMR and serve as a detection marker, and designing an amplification primer;
2) establishing a range of copy number references for each XIDMR and the whole X chromosome in a normal karyotype cell sample
Oxidation of unmethylated cytosine (C) in the DNA sample to be tested to uracil (U) by sulfite OxidationAmplifying each XIDMR by using the DNA sample to be detected converted by the sulfite as a template, analyzing the methylation detection result of each XIDMS in each XIDMR in male and female cells with normal karyotype, and respectively calculating the mean value of the methylation of each site in the male sample
And mean value in female samples
And the standard deviation;
in the present invention, XIDMS for quantitative analysis of X chromosome is selected as follows:
<0.05, standard deviation< 0.02；0.37 < 
<0.55, standard deviation<0.05; according to the methylation level M of each XIDMS in the sampleXRespectively calculating the copy number of the sites, and obtaining the XIDMR copy number N according to the average value of the copy numbersRThereby reducing the effect of random bias;
NRthe calculation formula is as follows:
due to the fact that
Close to 0, so a simplified formula can also be used:
in formula (1), n is the selected XIDMS number in each XIDMR, i is each XIDMS sequence number, and is the methylation level of each sample in the corresponding XiDMS;
separately counting in normal karyotype male samples
Mean value of
Standard deviation of
(ii) a In normal karyotype female samples
Mean value of
Standard deviation of
(ii) a To be provided with
For XIDMR corresponding to the reference value range of X chromosome 1 copy, to
A range of reference values for the corresponding X chromosome 2 copy;
reference value N of total X chromosome copy number of karyotype normal sampleXCalculated for the selected plurality of XIDMR, the formula is:
p in equation (3) is the selected number of XIDMR,
the number of copies calculated for each XIDMR; separately counting in normal karyotype male samples
Mean value, standard deviation of
(ii) a In normal karyotype female samples
Mean value of
Standard deviation of
(ii) a To be provided with
For XIDMR corresponding to the reference value range of X chromosome 1 copy, to
A range of reference values for the corresponding X chromosome 2 copy;
3) detecting X chromosome quantity and structure information in unknown karyotype cells
Detecting the methylation level of each XIDMS in a plurality of XIDMRs for unknown karyotype samples, and calculating the copy number of each XIDMR according to formula (1)
Calculating the total X chromosome copy number according to the formula (3)
(ii) a If it is not
And
judging that the number of X chromosomes in the cell is 1 or 2 if the number of X chromosomes is within the reference value range of 1 copy or 2 copies of the X chromosomes; if it is not
Deviation from the reference value of 1 copy or 2 copies, based on
Obtaining the number of X chromosomes in cells; if part
And if the deviation is 1 copy or 2 copies of the reference value, judging that the X chromosome in the cell has structural abnormality, and giving the abnormal karyotype of the X chromosome according to the copy number.
Meanwhile, the invention also provides an X chromosome quantitative detection kit based on the XIDMR marker.
Said XIDMR is respectively positioned atSAT1，UXTAndUTP14Agene promoter regions comprising 6, 6 and 9 XIDMS, respectively; the above-mentionedSAT1The gene is located in the X chromosome short arm (Xp), and is positioned in Xp22.1, and the XIDMR sequence is shown in SEQ ID NO: as shown in figure 1, the first and second main bodies, UXTthe gene is located in the X chromosome short arm (Xp), and is positioned in Xp11.23-p11.22, and the sequence of XIDMR is shown in SEQ ID NO: as shown in figure 2, the first and second,UTP14Athe gene is located in the long arm of the X chromosome (Xq), is positioned at Xq26.1, and has a sequence shown in SEQ ID NO: 3 is shown in the specification;
the kit comprises a pretreatment reagent for DNA methylation detection, wherein the DNA pretreatment adopts a sulfite oxidation method to oxidize unmethylated cytosine (C) into uracil (U) and convert the unmethylated cytosine (C) into thymine (T) in subsequent PCR reaction;
the kit contains the sulfite after oxidation treatmentSAT1，UXT1AndUTP14Areagents and primers for amplifying DNA sequences of the gene promoter regions; the amplification uses the Polymerase Chain Reaction (PCR), wherein the amplificationSAT1The primer pair sequence of the gene XIDMR is SEQ ID NO: 4 and SEQ ID NO: 5; amplification ofUXTThe primer pair sequence of the gene XIDMR is SEQ ID NO: 6 and SEQ ID NO: 7; amplification ofUTP14AThe primer pair sequence of the gene XIDMR is SEQ ID NO: 8 and SEQ ID NO: 9, wherein SEQ ID NO: 5. SEQ ID NO: 7 and SEQ ID NO: 9, labeling the 5' end of the sequence primer by using biotin;
the kit is provided with a pairSAT1，UXTAndUTP14Aquantification of methylation of XIDMS in Gene promoter region XIDMR quantification of intracellular X chromosomeSeparating out; the quantitative method employed pyrosequencing, in which,SAT1the pyrosequencing primer of the gene XIDMR is SEQ ID NO: 10;UXTthe pyrosequencing primer of the gene XIDMR is SEQ ID NO: 11;UTP14Athe pyrosequencing primer of the gene XIDMR is SEQ ID NO: 12; analyzing the number of X chromosomes in the cells by using a formula (1) after obtaining methylation data;
in the present invention, the analysis comprises the steps of:
1. extracting DNA in a sample;
2. oxidizing unmethylated cytosine bases in the DNA sample to uracil using sulfite;
3. using corresponding primer pairsSAT1，UXT1AndUTP14Acarrying out PCR amplification on the gene promoter region, and replacing uracil with thymine;
4. pyrosequencing the PCR product, and obtaining a methylation value according to the relative signal intensity of cytosine and thymine in corresponding XINDMS;
5. and calculating the number and structure information of the X chromosome in the cell by combining the reference values obtained by the known karyotype samples.
In the present invention, the sample is peripheral blood ex vivo, most commonly.
The experimental data of the present invention prove that, AT1，UXT1andUTP14Athe methylation level of the promoter region XIDMS of the gene can be used for quantitative analysis of the X chromosome in the cell.
The beneficial effects and the demonstration with practicability of the invention are as follows:
1) the invention uses XIDMS as a probe for detecting the quantity and the structural information of the X chromosome, carries out relative quantification on the quantity of the X chromosome, can accurately acquire the copy number information of each probe region, and finds the quantity and the structural abnormality of the X chromosome.
2) Based on the conventional operation method of a molecular genetic laboratory, compared with the current common karyotype analysis in clinic, the invention has the advantages of no need of cell culture, simple and convenient operation and convenient automation and parallel detection. Therefore, the detection time is shortened to be less than 6 hours compared with more than 3 days of karyotype analysis, and a larger amount of samples can be detected.
3) The detection kit provided by the invention is based on methylation detection of a small number of regions, and compared with high-throughput methods such as comparative genome chips, second-generation sequencing and the like which are widely applied in scientific research in recent years, the cost is less than 1/10, the data analysis is simple and convenient, and special biological information analysts are not needed.
4) The detection kit provided by the invention has the advantages that each XIDMS fragment detected in the detection kit contains a plurality of XIDMS, a plurality of sites are simultaneously detected and averaged, and compared with common molecular genetic detection methods such as fluorescence quantitative PCR (polymerase chain reaction), the detection kit has more accurate quantification, can not only discover the abnormality of the quantity and structure of X chromosomes, but also provide important intermediate results for the diagnosis of common chromosome diseases.
Drawings
FIG. 1 quantitative determination of X chromosome inactivation specific DNA differential methylation sites (XIDMS),
wherein N is the number of X chromosomes in the cell, a, 1X chromosome in the cell is an activated X chromosome, the other X chromosomes are inactivated, CpG sites on the activated X chromosome are in a hypomethylated state (white dots) in a promoter region of an inactivated target gene of the X chromosome, and CpG sites on the inactivated X chromosome are in a hypermethylated state (black dots); b. in the case of different numbers of X chromosomes in the cells, the methylation levels of XIDMS are significantly different, and mCpG in Xa: activating methylated CpG fragments on chromosome X, mCpG in Xi: methylated CpG fragment on inactive X chromosome, umCpG in Xa: unmethylated CpG fragments on the activated X chromosome, umCpG in Xi: inactivating unmethylated CpG fragments on the X chromosome; c. in the case of different numbers of X chromosomes in cells, there is a significant difference in the ratio of unmethylated to methylated fragments in XIDMS.
FIG. 2. pairSAT1、UXT1AndUTP14Athe verification of the XIDMR of the gene promoter region,
wherein: a-c is BSP verification result, d-f is pyrophosphoric acid verification result; a. d:SAT1the gene XIDMR; b. e:UXT1the gene XIDMR; c. f:UTP14Athe gene XIDMR; the results of the male DNA samples are shown at the top and the results of the female DNA samples are shown at the bottom.
FIG. 3. calculation of the resulting X chromosome copy number based on the XIDMS methylation level,
of these, 46, XX healthy female samples; 46, XY: a healthy male sample; TS: turner syndrome samples, showing only Xp copy numbers for samples with 46, X, i (Xq) karyotypes, - -: the normal female reference value ranges from 1.82 to 2.22.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are only illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1 validation of X chromosome inactivation specific DNA differential methylation sites and regions (i.e., XIDMS and XIDMR)
The study was conducted according to the Declaration of Helsinki Principles (Classification of Helsinki Principles) with the consent of the ethical Committee of the university of Compound Dane. All participants were from the pediatric hospital affiliated to the university of shanghai, china, participated in the study under the premise of informed consent, and signed an informed consent. The biological sample is derived from the rest sample of peripheral blood leucocyte karyotype analysis, DNA is extracted according to the kit instruction, DNA concentration and purity are detected by an ultraviolet spectrophotometer, and a sulfite reagent is used for conversion.
In this example, a primer set was usedSAT1、UXT1AndUTP14APCR amplification is carried out on the gene promoter region fragments, PCR sequencing is carried out after the products are purified, and the results show that the three gene promoter region fragments contain a plurality of CpG sites, are unmethylated in male DNA samples, are partially methylated in female DNA samples, accord with the characteristics of XIDMS, and form XIDMR (shown in figures 2.a, b and c); in this example, quantitative methylation analysis was also performed on the three fragments by pyrosequencing, and the degree of methylation of the candidate XIDMS in the male DNA sample was 0 or close to 0, and the degree of methylation in the female DNA sample was close to 50% (as shown in fig. 2.d, e, and f), which indicates that the presence of XIDMR can detect the methylation levels of multiple XIDMS in parallel, and effectively reduce the influence of random errors on the results.
Example 2 establishing reference value ranges for each XIDMR in the normal population
18 DNA samples with known karyotypes of 46, XX and 6 of 46, XY, karyotypes were used, with primer pairsSAT1、UXT1AndUTP14APCR amplification of the XIDMR fragment of the promoter region of the gene followed by quantitative methylation analysis by pyrosequencing, whereinSAT1AndUXT1the XIDMR fragment of the gene respectively comprises 6 XIDMS,UTP14Athe XIDMR fragment of the gene comprises 8 XIDMS;
for each XIDMS, the mean methylation value in the 46, XY and 46, XX karyotype samples was calculated
And
and calculating the copy number N of each sample at each XIDMR using equation 1RCalculating the number of 46, XY and 46, XX karyotypes in samples
Mean value of
And
standard deviation of
And, with
For XIDMR corresponding to the reference value range of X chromosome 1 copy, to
A range of reference values for the corresponding X chromosome 2 copy; the total X chromosome copy number N of each sample was calculated using the formula (3)XCalculating the number of 46, XY and 46, XX karyotypes in samples
Mean value of
And
standard deviation of
And
to do so by
For XIDMR corresponding to the reference value range of X chromosome 1 copy, to
A range of reference values for the corresponding X chromosome 2 copy;SAT1、UXT1andUTP14Athe gene promoter region XIDMR and the reference value ranges for the total X chromosome copy number are shown in table 1.
TABLE 1 reference value ranges for XIDMR copy number in the normal population
| Karyotype | SAT1 | UXT1 | UTP14A | |
| 46,XY | 1.00 |
1.00 |
1.01 | 1.00 |
| 46,XX | 2.05 |
2.01 |
2.01 |
2.02 |
The result shows that the method of the invention has very accurate detection on the single copy of the X chromosome, the deviation is not more than 2% in 2.58-fold standard deviation, namely 99% confidence interval, and the X chromosome abnormality such as Klinefelter syndrome, 46XX sex reversal and the like can be very sensitively distinguished in a male sample; the method has more accurate quantification for the X chromosome 2 copy sample, the deviation in the 99% confidence interval is less than 20%, and the deviation in the 99% confidence interval can reach 10% level by combining 3 XIDMR copy numbers, which is obviously superior to the conventional molecular genetics means such as fluorescence quantitative PCR and the like.
Example 3: detecting X chromosome quantity and structure information in clinical samples
By pairsSAT1、UXT1AndUTP14Amethylation detection of the promoter region XIDMR of the gene was performed on a total of 42 clinical blood samples with abnormal karyotypes, including 36 patients with turner syndrome, 3 patients with krainian syndrome, and 3 patients with trisomy X syndrome, where the patients with turner syndrome had multiple karyotypes, including 45, X, 46, X, i (xq) and 45, X and 46, X, i (xq), 46, XX, 47, XXX, 46, X, del (xp) or chimeras of 46, XY karyotypes;
extracting DNA from the sample; converting sulfite; pyrosequencing is carried out after PCR, and the detection result shows that the quantitative result of the X chromosome of the sample shows that the copy number of at least 2 XIDMRs deviates from the normal karyotype reference value range, wherein the quantitative result of the sample of the Turner syndrome patient is shown in figure 3;
the results of this example also show that the quantification method of the present invention has high accuracy for chimeras, structural abnormalities, and high copy number of the X chromosome, and the results of typing some karyotype samples are shown in table 2; if the karyotype is 46, X, i (Xq) sample, the cells contain 1 normal X chromosome and 1X chromosome long arm isochromosome, thus having 1 short arm and 3 long arms, and the detection result shows that the XIDMR located at the X chromosome short arm is single copy, and the XIDMR located at the long arm is close to 3 copies.
Table 2 detailed quantification results for a part of karyotype samples:
| karyotype | SAT1 | UXT | UTP14A | X chromosome |
| 45,X[12]/46,XX[9] | 1.47 | 1.48 | 1.43 | 1.46 |
| 45,X[3]/46,XX[32] | 1.73 | 1.56 | 2.02 | 1.77 |
| 45,X[7]/46,XX[13] | 1.77 | 1.70 | 1.65 | 1.71 |
| 45,X[27]/47,XXX[3] | 1.51 | 1.50 | 1.66 | 1.56 |
| 45,X[16]/46,X,r(X)(p11q22)[4] | 1.00 | 1.00 | 0.99 | 1.00 |
| 45,X[6]/46,X,r(X)(p21q21)[14] | 1.00 | 1.45 | 1.07 | 1.17 |
| 45,X[10]/46,X,r(X)(p22q25)[10] | 1.23 | 1.30 | 1.02 | 1.18 |
| 45,X[7]/46,X,del(X)(p11)[13] | 1.08 | 1.62 | 1.46 | - |
| 45,X[13]/46,X,i(X)(q10)[7] | 1.00 | 1.00 | 1.47 | - |
| 45,X[8]/46,X,i(X)(q10)[12] | 1.00 | 1.00 | 1.75 | - |
| 45,X[9]/46,X,i(X)(q10)[11] | 1.00 | 1.00 | 2.14 | - |
| 45,X[4]/46,X,i(X)(q10)[16] | 1.00 | 1.00 | 2.14 | - |
| 45,X[3]/46,X,i(X)(q10)[32] | 1.02 | 1.00 | 3.40 | - |
| 45,X[4]/46,X,i(X)(q10)[56] | 1.00 | 1.00 | 3.14 | - |
| 46,X,i(X)(q10) | 1.00 | 1.00 | 3.03 | - |
| 46,X,i(X)(q10) | 1.00 | 1.00 | 3.35 | - |
| 46,X,i(X)(q10) | 1.00 | 1.00 | 3.45 | - |
| 47,XXX | 2.94 | 2.90 | 3.29 | 3.04 |
| 47,XXX | 3.19 | 2.66 | 3.35 | 3.07 |
| 47,XXY | 2.00 | 1.84 | 2.23 | 2.02 |
| 47,XXY | 2.17 | 2.07 | 1.93 | 2.06 |
| 49,XXXXY | 4.40 | 4.02 | 4.45 | 4.29 |
SEQUENCE LISTING
<110> subsidiary pediatric hospital of double-denier university
<120> method and kit for quantitatively detecting X chromosome in cell
<130> 2016
<160> 12
<170> PatentIn version 3.5
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Claims (5)
1. A kit for quantitative detection of X chromosome in cells, characterized by comprisingSAT1，UXTAndUTP14Athe gene promoter region sequence is used as XIDMR, and contains a pretreatment reagent for detecting DNA methylation, and is used for detecting the X chromosome detection amount in cells;
the XIDMR is used for the chemical reaction of the chemical reaction,SAT1the sequence of the gene XIDMR is shown as SEQ ID NO: as shown in figure 1, the first and second main bodies,UXTthe sequence of the gene XIDMR is shown as SEQ ID NO: as shown in figure 2, the first and second,UTP14Athe sequence of the gene XIDMR is shown as SEQ ID NO: 3, respectively.
2. The kit for quantitative determination of X chromosome in cells according to claim 1, wherein the DNA pretreatment comprises oxidation of unmethylated cytosine (C) to uracil (U) by sulfite oxidation and subsequent conversion to thymine (T) by Polymerase Chain Reaction (PCR).
3. The kit of claim 1, further comprising reagents and primers for amplifying the XIDMR; amplification is performed by a PCR reaction.
4. The kit of claim 3, wherein the amplification is performedSAT1The primer pair sequence of the gene XIDMR is SEQ ID NO: 4 and SEQ ID NO: 5; amplification ofUXTThe primer pair sequence of the gene XIDMR is SEQ ID NO: 6 and SEQ ID NO: 7;amplification ofUTP14AThe primer pair sequence of the gene XIDMR is SEQ ID NO: 8 and SEQ ID NO: 9, wherein SEQ ID NO: 5. SEQ ID NO: 7 and SEQ ID NO: 9 the 5' end of the sequence primer is marked by biotin.
5. The kit of claim 1, comprising primers for pyrosequencing of XIDMR to detect DNA methylation, wherein,SAT1the pyrosequencing primer of the gene XIDMR is SEQ ID NO: 10;UXTthe pyrosequencing primer of the gene XIDMR is SEQ ID NO: 11;UTP14Athe pyrosequencing primer of the gene XIDMR is SEQ ID NO: 12.
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| CN1882700A (en) * | 2003-09-22 | 2006-12-20 | 特莱索根生物科技有限合伙公司 | Methods and kits useful for detecting an alteration in a locus copy number |
| CN101962680A (en) * | 2010-10-13 | 2011-02-02 | 南方医科大学 | Double PCR molecular diagnosis kit for detecting inactivation of X chromosome |
| CN104498607A (en) * | 2014-12-17 | 2015-04-08 | 绍兴华因生物科技有限公司 | Quantitative comparison method for detecting fragile X syndrome based on methylation level and primer used in quantitative comparison method |
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| US20070141577A1 (en) * | 2003-09-11 | 2007-06-21 | Moore Thomas F | Method |
| US9732390B2 (en) * | 2012-09-20 | 2017-08-15 | The Chinese University Of Hong Kong | Non-invasive determination of methylome of fetus or tumor from plasma |
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| CN1882700A (en) * | 2003-09-22 | 2006-12-20 | 特莱索根生物科技有限合伙公司 | Methods and kits useful for detecting an alteration in a locus copy number |
| CN101962680A (en) * | 2010-10-13 | 2011-02-02 | 南方医科大学 | Double PCR molecular diagnosis kit for detecting inactivation of X chromosome |
| CN104498607A (en) * | 2014-12-17 | 2015-04-08 | 绍兴华因生物科技有限公司 | Quantitative comparison method for detecting fragile X syndrome based on methylation level and primer used in quantitative comparison method |
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