CN107502668B - Detection method of human Y chromosome tag site sY1291 and application thereof - Google Patents
Detection method of human Y chromosome tag site sY1291 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of in vitro nucleic acid detection, and particularly relates to a detection method of a human Y chromosome tag site sY1291 and application thereof. The invention provides a novel sY1291 site detection strategy, namely, a specific PCR amplification primer is redesigned in a large segment repeat region of 38bp upstream of an sY1291 site detection region recommended by EAA/EMQN, the 5 'end of a novel PCR amplification primer detection region I is 38bp away from the 3' end of the sY1291 site detection region recommended by EAA/EMQN, and the novel PCR amplification primer can simultaneously detect four regions I, II, III and IV. The detection strategy of the invention combines different detection methods of forming the sY1291 locus such as agarose electrophoresis, fluorescence labeling primer capillary electrophoresis or fluorescence probe method. The detection method of the human Y chromosome tag locus sY1291 provided by the invention has strong specificity and wide detection Y chromosome region, and is more suitable for the amplification detection of human Y chromosome AZF microdeletion.
Description
Technical Field
The invention belongs to the technical field of human in-vitro nucleic acid detection, and particularly relates to a detection method and application of a human Y chromosome tag locus sY 1291.
Background
The human Y chromosome is unique to males. In the genetic rule, the Y chromosome can only be inherited by father to son, namely the Y chromosome presents the characteristics of paternal inheritance. Also, sex-determining genes such as SRY, genes or key regions associated with male spermatogenesis, such as Azoospermia Factor (AZF), are located on the Y chromosome. However, due to the large number of highly homologous sequences present on the Y chromosome itself, deletion of large chromosomal regions, most commonly microdeletions of the AZF region, may occur on the Y chromosome during the genetic process.
Because of the characteristic of paternal inheritance of the Y chromosome, the polymorphic genetic markers on the Y chromosome are often used for identifying the same paternal line among different individuals in forensic DNA analysis, and as two boys with different parents share the Y chromosome of the parents, the two boys theoretically have the same haplotype of the polymorphic genetic markers on the Y chromosome. However, this rule of judgment is disabled when one of the boys has a microdeletion of the Y chromosome and the other has not. Therefore, in the case of performing the same paternal test using the Y chromosome polymorphic genetic marker, the detection of microdeletion of the Y chromosome is also necessary, and in particular, the detection of microdeletion of the Y chromosome is more important when the conclusion of exclusion of the same paternal line is given. For individuals considering the presence of a microdeletion of the Y chromosome, it is also necessary to know how large the deletion range is, whether the polymorphic sites of the Y chromosome used for paternal testing in the vicinity thereof are covered.
Also of concern with Y chromosome microdeletion abnormalities is the field of male infertility. AZF is a critical area associated with male spermatogenesis. AZF was first proposed by Tiepolo and Zuffardi in 1976, who thought AZF to be a gene site present in the long arm of the Y chromosome that controls spermatogenesis. Numerous studies have demonstrated that at least 3 non-overlapping regions are present in the 5 th and 6 th spacers of the long arm of the Y chromosome, which are involved in spermatogenesis, namely AZFa, AZFb and AZFc. Dysspermia caused by the deletion is an important cause of male infertility and is manifested by primary Azoospermia (Azoospermia) or Oligospermia (Oligospermia), wherein the most common deletion region is the AZFc region. In view of this, in the best practice guidelines for Y chromosome microdeletion molecular diagnosis of european male association (EAA) and european molecular genetic experiment quality control collaboration network (EMQN) in 2013 edition, detection primers and methods for AZFa regions, AZFb regions, classical tag sites of AZFc regions, corresponding tag sites, and detection pathways for AZF microdeletion are recommended.
In each region of AZF, EAA/EMQN recommended classical sites for detection of microdeletions in the corresponding region, such as sY84 and sY86 in the AZFa region, sY134 and sY127 in the AZFb region, and sY255 and sY254 in the AZFc region. Among AZFa, AZFb and AZFc regions, the most common deletion region is the AZFc region, accounting for over 79% of the total deletion. In addition to the above classical deletion sites, EAA/EMQN also recommends extended detection sites and detection methods for each region, which extended detection sites, in addition to their validation function in the case of suspected detection of classical sites, also confirm the microdeletion range of the Y chromosome. Among them, sY1291 is the most important among the extended detection sites of AZFc regions and has potential clinical research value (Krausz et al; 2013). This site is located in a sub-region of the AZFc region called "gr/gr", and was earlier used in the study of the evolution of Y chromosome lineage tree deletions (repling et al; 2003), and it was suggested that a "gr/gr" deletion with family genetic polymorphisms may affect the occurrence of spermatozoa by eliminating almost half of the genetic information of the AZFc region, and is an important risk factor for male infertility.
However, in the subsequent studies, the detection method of sY1291 recommended by EAA/EMQN is adopted to find that certain false positive exists, and in the sample with the deletion of AZFc region, sY1291 shows that more than 99% is deleted, while in the normal sample, the AZFc region can also show the deletion, i.e. certain human polymorphism is shown in the normal sample (Krausz et al; 2009).
Therefore, a more reliable sY1291 site detection strategy and method is needed for better analysis of male AZF microdeletions.
Disclosure of Invention
The invention aims to provide a detection method of a human Y chromosome tag locus sY1291 with strong specificity and high consistency with the deletion of an AZFc region classical deletion locus and application aiming at the defects of an sY1291 detection method recommended by EAA/EMQN and the practical requirements of sY1291 deletion detection.
The invention provides a detection method of a human Y chromosome tag locus sY1291, which relates to a strategy (detection strategy) of an amplification region of a PCR primer of the sY1291 locus, designs a new specific PCR amplification primer and detects the human Y chromosome tag locus sY 1291; the method comprises the following specific steps:
(1) and determining the amplification region of the PCR primer of the sY1291 site. The strategy is to avoid the amplification region of the PCR primer of the sY1291 locus recommended in the Y chromosome microdeletion molecular diagnosis operation manual (2013 edition) jointly published by European Male scientific Association/European molecular genetic experiment quality control cooperative network (EAA/EMQN), and design a new specific PCR amplification primer in the large-fragment repeat region near the EAA/EMQN recommended detection region. Therefore, the detection area selected by the invention comprises: region I (ChrY: 25,504,754-25,505, 032), region II (ChrY: 25,207,966-25,208, 244), region III (ChrY: 26,841,711-26,841, 989), region IV (ChrY: 27,120,413-27,120, 691). The I area and the II area are positioned at the upstream of the detection area of the EAA/EMQN recommended sY1291 site, and the III area and the IV area are positioned at the downstream of the detection area of the EAA/EMQN recommended sY1291 site; wherein, the 5 'end of the I region is 38bp away from the 3' end of the EAA/EMQN recommended sY1291 site detection region (see attached figure 1).
(2) Designing PCR amplification primers with the nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2, wherein the PCR amplification primers cover the I region (ChrY: 25,504,754, 25,505, 032), the II region (ChrY: 25,207,966-25,208, 244), the III region (ChrY: 26,841,711-26,841, 989) and the IV region (ChrY: 27,120,413-27,120, 691) of the human Yq11.223-Yq11.23 region;
the sequence homology of the I region (ChrY: 25,504,754, 25,505, 032), the II region (ChrY: 25,207,966-25,208, 244), the III region (ChrY: 26,841,711-26,841, 989) and the IV region (ChrY: 27,120,413-27,120, 691) of the Yq11.223-Yq11.23 region amplified by the PCR amplification primer (SEQ ID No.1 and the nucleotide sequence shown in SEQ ID No. 2) provided by the invention is more than 99%.
(3) The PCR amplification primer is used for detecting the human Y chromosome tag locus sY1291, and the detection is divided into 3 cases:
(a) carrying out PCR amplification by adopting DNA polymerase, detecting PCR amplification products by adopting 1.0-2.0% agarose gel electrophoresis, wherein the position of sY1291 is deleted if no PCR amplification target product exists, and the position of sY1291 is not deleted if the PCR amplification target product exists;
(b) carrying out fluorescein labeling (taking FAM as an example) on the 5 'end of the nucleotide sequence shown in SEQ ID No.1 or the 5' end of the nucleotide sequence shown in SEQ ID No.2, carrying out PCR amplification by adopting DNA polymerase, carrying out capillary electrophoresis fragment analysis on a PCR product by adopting 3500Dx or a gene sequencer of the same type, wherein the site of sY1291 is not deleted when the fluorescence peak of the PCR amplification target product appears, and the site of sY1291 is not deleted when the fluorescence peak of the PCR amplification target product does not appear;
(c) designing a fluorescence labeling probe, wherein the nucleotide sequence of the fluorescence labeling probe is shown as SEQ ID No.3, detecting by adopting a real-time fluorescence PCR method, and detecting whether an sY1291 site is not deleted when a target fragment PCR amplification curve appears or not and whether the sY1291 site is deleted when the target fragment PCR amplification curve does not appear.
Wherein, in the reaction systems of the case (a) and the case (b), the working concentration of the PCR amplification primers SEQ ID number 1 and SEQ ID number 2 is 50nmol/L to 300 nmol/L; in the reaction system of the case (c), the working concentration of the PCR amplification primers SEQ ID No.1 and SEQ ID No.2 is 300nmol/L to 500nmol/L, and the working concentration of the probe SEQ ID No.3 is 150nmol/L to 250 nmol/L.
The sY1291 deletion detection method can be used for detecting the human AZF microdeletion.
The invention has the beneficial effects that:
the sY1291 detection method provided by the invention effectively expands the detection range of the AZFc region deletion region. In the invention, a new PCR amplification primer (nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO. 2) is redesigned at the upstream 38bp of the sY1291 detection region recommended by EAA/EMQN. The 5 'end of the I region (ChrY: 25,504,754-25,505, 032) detected by the new PCR amplification primer is 38bp away from the 3' end of the EAA/EMQN recommended sY1291 detection region. In addition to region I, the novel PCR amplification primers detected region II (ChrY: 25,207,966-25,208, 244), region III (ChrY: 26,841,711-26,841, 989) and region IV (ChrY: 27,120,413-27,120, 691) at the same time. The total coverage of the four regions is Yq11.223-Yq11.23 which exceeds 1912kb, and the detection range of the AZFc deletion region is effectively expanded. And the deletion consistency rate with the classical deletion sites (sY255 and sY254) of the AZFc region of the Y chromosome reaches 100 percent, thereby overcoming the problems that the detection method of the EAA/EMQN recommended sY1291 site is inconsistent with the detection result of the classical deletion sites (sY255 and sY254) of the AZFc region and the deletion false positive problem in normal male population.
The detection method provided by the invention has strong specificity and high consistency with the deletion of the AZFc region classical deletion site, can effectively assist in determining the deletion range of the AZFc region, is quick and simple to detect, and can be popularized and applied in the Y chromosome microdeletion field.
Drawings
FIG. 1: the invention provides a detection strategy of a human Y chromosome tag locus sY 1291. The attached figure shows a structural sketch of a region of a human Yq11.223-Yq11.23 in which a tag site sY1291 is positioned. Wherein, the black vertical line shows the detection region of the sY1291 site recommended by EAA/EMQN. The white vertical rectangle frame is the region detected by the PCR amplification primer of the sY1291 site provided by the invention, wherein the I site is positioned 38bp upstream from the EAA/EMQN recommended sY1291 detection region. The physical location information of the Y chromosome where each region is located is shown in the figure in the human genome database Hg19 (http:// genome. ucsc. edu /).
FIG. 2: the present invention combined PCR agarose electrophoresis to detect sY1291 (example 1). In the figure, lanes M are PCR product molecular weight markers and 1-6 are 6 different male samples. From left to right: 6 lanes in the first group of 1-6 are EAA/EMQN recommended method for detecting 6 samples AZFc region classical deletion sites (sY255 and sY254, the amplified fragment of sY255 is 123bp, and the sY254 is 380 bp), samples No.3 and No. 4 are AZFc region deleted male samples for infertility, and samples No.1, No.2, No. 5 and No. 64 are normal male samples; the second group of lanes 1-6 are the same 6 samples, and the EAA/EMQN recommended sY1291 site detection method (the amplification fragment is 527 bp) is adopted, samples No.1 and No.2 are not deleted, and samples No. 5 and No. 6 are deletion false positive; the third group is that the method of the invention detects sY1291 site (the amplified fragment is 279 bp), and the detection result of 6 samples is consistent with the detection result of AZFc region classical deletion site.
FIG. 3 shows that sY1291 and EAA/EMQN recommended reference gene (sex-determining gene, SRY) are detected simultaneously by combining real-time fluorescence PCR (example 3), and the results show that SRY and sY1291 sites have corresponding amplification curves, and the SRY and sY1291 sites are male sY1291 site non-deletion samples.
FIG. 4 shows that sY1291 and EAA/EMQN recommended reference gene (sex-determining gene, SRY) are simultaneously detected by combining the real-time fluorescence PCR method (example 3), and the result shows that the SRY gene amplification curve is normally detected, and no sY1291 site amplification signal is detected, so that the SRY 1291 site deletion sample is male sY1291 site deletion sample.
Detailed Description
For a better understanding of the present invention, reference will now be made to the following examples. It should be understood that the following specific examples are illustrative of the effectiveness of the invention and are not to be construed as limiting the invention. In addition, the extraction of human genomic DNA from human biological samples (such as human whole blood, throat swab, saliva, hair follicle or muscle tissue, etc.) using commercial human genomic DNA extraction kits or other chemical reagents for subsequent detection is not included in the claims of the present invention, but the human genomic DNA extraction process should not be a limitation to the application of the present invention as a conventional flow or previous step of human genomic DNA detection. Thus, subsequent embodiments begin after extraction of human genomic DNA is complete.
The nucleotide sequences of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 and the modification characteristics thereof used in the examples are shown in Table 1, wherein the 5' end of the nucleotide sequence shown in SEQ ID No.2 may be modified with or without fluorescein (e.g., FAM) (for capillary electrophoresis fragment analysis) depending on the detection method of the subsequent PCR product (examples 1 and 3).
TABLE 1 PCR amplification primers and probes for sY1291 site used in the present invention
Note: the 5' end of SEQ ID No.2 may be unlabeled with fluorescein (agarose electrophoresis) or labeled with fluorescein (capillary electrophoresis).
Example 1: the invention combines PCR product agarose electrophoresis to carry out sY1291 locus detection
The male genome DNA was amplified using the PCR primers shown in SEQ ID No.1 and SEQ ID No.2 (wherein SEQ ID No.2 did not need fluorescein labeling) according to the PCR reaction system shown in Table 2 and the PCR reaction conditions shown in Table 3, and the PCR amplification product was electrophoresed using 2.0% agarose.
No. 1-6 total 6 male samples are divided into two types according to the detection result and phenotype of the AZFc region classical deletion sites (sY255 and sY254), No.3 and No. 4 samples are male infertility samples of the AZFc region classical deletion sites, and No.1, No.2, No. 5 and No. 6 samples are normal male samples with normal semen parameters. The detection method of the classical deletion sites (sY255 and sY254) of the AZFc region adopts the detection method recommended by EAA/EMQN. The detection results of the ZFc region classical deletion sites (sY255 and sY254) of the 6 samples, the detection results of the EAA/EMQN recommended sY1291 site detection method and the detection results of the sY1291 site detection method are shown in the attached figure 2.
TABLE 2 PCR reaction System for detecting sY1291 site by agarose electrophoresis
TABLE 3 PCR reaction conditions for detecting sY1291 site by agarose electrophoresis
The result shows that in the male sample with deletion of the AZFc region classical site, the sY1291 site detected by the method provided by the invention is also deleted, but the EAA/EMQN recommended sY1291 site detection method can be deleted in the sample with deletion of the AZFc classical deletion site, and can be shown as deletion or non-deletion in the normal male sample with normal semen parameters, namely the detection result of the EAA/EMQN recommended sY1291 site has false positive.
Example 2: the invention combines the capillary electrophoresis fluorescent fragment analysis of PCR products to detect sY1291 locus
PCR amplification was performed on 1169 cases of male DNA samples using PCR primers shown in SEQ ID No.1 and SEQ ID No.2 (in which SEQ ID No.2 was labeled with FAM fluorescein) according to the PCR reaction system shown in Table 4 and the PCR reaction conditions shown in Table 5, and capillary electrophoresis was performed on the PCR products using a 3500Dx genetic analyzer.
1169 male samples including 232 male samples with normal semen parameters and 937 male samples with infertility. All samples were tested for AZFc region canonical sites (sY255 and sY254) using a kit approved by the Chinese food and drug administration for AZF microdeletion detection (human chromosome AZF region microdeletion nucleic acid kit (PCR-fluorescent probe method, national institutes of record 20153400764), and for sY1291 sites recommended by EAA/EMQN.
TABLE 4 PCR reaction system for detecting sY1291 site by combining capillary electrophoresis
TABLE 5 PCR reaction conditions for detecting sY1291 site by agarose electrophoresis
From the point of view of consistency with phenotype and with detection results of classical deletion sites of AZFc region, EAA/EMQN recommends that the sY1291 detection method has a positive predictive value of 64.92% (198/305) for the deletion of AZFc region, a negative predictive value of 100.00% [ (212+652)/864] for the deletion of AZFc region, and a false positive detection rate of 11.02% [ (20+87)/(232+739) ].
TABLE 6 EAA/EMQN recommendation sY1291 locus detection method for 1169 male samples
Note: the deletion of sY255 and sY254 is detected by using a kit of national institutes of care 20153400764.
From the viewpoint of consistency with phenotype and consistency with the detection result of the classical deletion site of the AZFc region, the sY1291 detection method of the present invention has a positive predictive value of 100.00% (198/198) for the deletion of the AZFc region, a negative predictive value of 100.00% [ (232+739)/971] for the deletion of the AZFc region, and a false positive detection rate of 0.00% [ (0+0)/(232+739) ]. Obviously, compared with the sY1291 detection method recommended by EAA/EMQN, the sY1291 detection method provided by the invention can obviously improve the positive predictive value of AZFc deletion (from 64.92% to 100.00%) and obviously reduce the false positive rate of the sY1291 deletion (from 11.02% to 0.00%).
TABLE 7 detection results of 1169 male samples by the sY1291 locus detection method of the present invention
Note: the deletion of sY255 and sY254 is detected by using a kit of national institutes of care 20153400764.
Example 3: the invention combines a real-time fluorescent probe method to detect the sY1291 locus
Real-time fluorescence detection was performed using PCR primers shown in SEQ ID No.1 and SEQ ID No.2 (No fluorescein labeled in SEQ ID No. 2) and a fluorescent probe shown in SEQ ID No.3 according to the PCR reaction system shown in Table 8 and the PCR reaction conditions shown in Table 9. The EAA/EMQN recommended AZF microdeletion assay reference gene (sex-determining gene, SRY) was also tested in this system. In the presence of a fluorescence signal of an internal reference gene, a sample without deletion of the sY1291 locus is a sample without an amplification curve of the sY1291 locus (figure 3), and a sample without an amplification curve of the sY1291 locus is a sample without deletion of the sY1291 locus (figure 4).
TABLE 8 PCR reaction system for detecting sY1291 site by combining real-time fluorescence
TABLE 9 PCR reaction conditions for the detection of sY1291 site by real-time fluorescence
The invention provides a novel detection strategy aiming at a human Y chromosome AZFc region tag site sY129 and different from an EAA/EMQN recommendation method, and provides a PCR amplification primer which can detect 4 regions before and after the EAA/EMQN recommendation sY1291 detection range and has the total length of more than 1912 kb. The detection strategy can be combined with different subsequent PCR product analysis means to form different sY1291 site detection methods, such as PCR combined agarose electrophoresis, PCR combined capillary electrophoresis fluorescent fragment analysis, real-time fluorescence PCR and the like shown in the examples. By taking an EAA/EMQN recommended sY1291 detection method and a kit approved by China CFDA for AZFc region classical site (sY255 and sY254) deletion detection as a contrast, large-sample detection shows that compared with the EAA/EMQN recommended sY1291 site detection method, the sY1291 site detection strategy and method provided by the invention have higher positive consistency, positive predicted value, negative predicted value and extremely low false positive rate with an AZFc region classical deletion region, and are beneficial to defining an AZFc deletion range.
In this specification, the invention has been described with reference to specific embodiments thereof. However, it is clear that various modifications and variations can be made without departing from the spirit and scope of the invention, such as the use of high resolution melting curves for subsequent means of PCR product analysis, or the use of different types of fluorescein labels for the primers or probes provided, or the use of the basic design principles of PCR primers to design new PCR primers or probes within the limited range (0-200bp) upstream of sY1291 recommended by EAA/EMQN. The description is thus to be regarded as illustrative instead of limiting on the invention.
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Claims (6)
1. A detection method of a human Y chromosome tag locus sY1291 is used for non-diagnostic purposes and comprises the following specific steps:
(1) determining an amplification region of an sY1291 locus PCR primer, wherein the strategy is to avoid the amplification region of the sY1291 locus PCR primer recommended in a Y chromosome microdeletion 2013 version molecular diagnosis operation guide jointly published by the European Male scientific society/European molecular genetic experiment quality control cooperation network, and designing a new specific PCR amplification primer in a large segment repetition region near the recommended detection region of the European Male scientific society/European molecular genetic experiment quality control cooperation network; the detection region I is positioned at the upstream 38bp of the detection region of the sY1291 locus recommended by the European Male scientific Association/European molecular genetic experiment quality control cooperative network; the selected detection region includes: and (3) region I: ChrY:25,504,754-25,505,032, region II: ChrY:25,207,966-25,208,244, region III: region 26,841,711-26,841,989, IV: ChrY 27,120,413-27,120, 691; the I area and the II area are positioned at the upstream of the detection area of the recommended sY1291 site of the European Male scientific Association/European molecular genetic experiment quality control cooperative network, and the III area and the IV area are positioned at the downstream of the detection area of the recommended sY1291 site of the European Male scientific Association/European molecular genetic experiment quality control cooperative network; the distance between the 5 'end of the I region and the 3' end of the detection region of the sY1291 locus recommended by the European Male scientific society/European molecular genetic experiment quality control cooperative network is 38 bp;
(2) designing a PCR amplification primer, wherein the nucleotide sequences are shown as SEQ ID No.1 and SEQ ID No.2, and the PCR amplification primer covers a region I, a region II, a region III and a region IV of a region Yq11.223-Yq11.23;
(3) the PCR amplification primer is used for detecting the human Y chromosome tag locus sY1291, and the detection is divided into 3 cases:
(a) carrying out PCR amplification by adopting DNA polymerase, detecting PCR amplification products by adopting 1.0-2.0% agarose gel electrophoresis, wherein the position of sY1291 is deleted if no PCR amplification target product exists, and the position of sY1291 is not deleted if the PCR amplification target product exists;
(b) carrying out fluorescein labeling on the 5 'end of the nucleotide sequence shown in SEQ ID No.1 or the 5' end of the nucleotide sequence shown in SEQ ID No.2, carrying out PCR amplification by adopting DNA polymerase, carrying out capillary electrophoresis fragment analysis on a PCR product by adopting 3500Dx or a gene sequencer of the same type, wherein the site where the fluorescence peak of the PCR amplification target product appears is sY1291 site deletion, and the site where the fluorescence peak of the PCR amplification target product does not appear is sY1291 site deletion;
(c) designing a fluorescence labeling probe, wherein the nucleotide sequence of the fluorescence labeling probe is shown as SEQ ID No.3, detecting by adopting a real-time fluorescence PCR method, and detecting whether an sY1291 site is not deleted when a target fragment PCR amplification curve appears or not and whether the sY1291 site is deleted when the target fragment PCR amplification curve does not appear.
2. The method for detecting the human Y chromosome tag site sY1291 according to claim 1, characterized in that PCR amplification primers are adopted: the sequence homologies of the I region, the II region and the III region of the human Yq11.223-Yq11.23 region amplified by SEQ ID No.1 and SEQ ID No.2 and the IV region are more than 99 percent.
3. The method for detecting the human Y chromosome tag site sY1291 according to claim 1, wherein in the reaction system of the case (a) and the case (b) in the step (3), the working concentration of the PCR amplification primers SEQ ID No.1 and SEQ ID No.2 is 50nmol/L to 300 nmol/L; in the reaction system of the case (c), the working concentration of the PCR amplification primers SEQ ID No.1 and SEQ ID No.2 is 300nmol/L to 500nmol/L, and the working concentration of the probe SEQ ID No.3 is 150nmol/L to 250 nmol/L.
4. The PCR amplification primer for detecting the human Y chromosome tag site sY1291 is characterized in that the nucleotide sequences are shown as SEQ ID No.1 and SEQ ID No. 2.
5. The PCR amplification primers for detection of the human Y chromosome tag site sY1291 as claimed in claim 4, further comprising a probe for detection of the human Y chromosome tag site sY1291, the nucleotide sequence of which is shown in SEQ ID No. 3.
6. Use of the method of detection of the human Y chromosome tag site sY1291 according to any one of claims 1 to 3 in the detection of human AZF microdeletions for non-diagnostic purposes.
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| US5840482A (en) * | 1990-10-10 | 1998-11-24 | The Regents Of The University Of California | Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ |
| FR2824144B1 (en) * | 2001-04-30 | 2004-09-17 | Metagenex S A R L | METHOD OF PRENATAL DIAGNOSIS ON FETAL CELLS ISOLATED FROM MATERNAL BLOOD |
| KR100908691B1 (en) * | 2007-03-08 | 2009-07-22 | (주)지노첵 | 남성 Male infertility diagnostic kit for detecting chromosomal microdeletion |
| KR100926255B1 (en) * | 2008-07-04 | 2009-11-12 | (주)지노첵 | Diagnostic methods for male infertility and diagnostic kits for male infertility for detecting Y chromosomal microdeletion |
| CN102703578B (en) * | 2011-07-06 | 2013-12-04 | 郭奇伟 | Multiple real-time fluorescent PCR detection kit for Y chromosome microdeletion |
| CN102304582A (en) * | 2011-09-01 | 2012-01-04 | 云南中科胚胎工程生物技术有限公司 | Sex identification method for early embryo of saanen goat |
| CN106148484B (en) * | 2015-03-27 | 2019-10-25 | 上海透景生命科技股份有限公司 | A kind of kit that diagnosis Y chromosome is micro-deleted |
| KR101817180B1 (en) * | 2016-01-20 | 2018-01-10 | 이원다이애그노믹스(주) | Method of detecting chromosomal abnormalities |
| CN106498090A (en) * | 2017-01-16 | 2017-03-15 | 上海普洛麦格生物产品有限公司 | A kind of test kit for detecting DNA mismatch repair system and application thereof |
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