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A detection method and application of human y chromosome label site sy1291

A technology of Y chromosome and detection method, applied in the field of detection of human Y chromosome tag site sY1291, can solve problems such as deletion of AZFc region, and achieve the effects of expanding detection range, rapid detection, and high deletion consistency rate

Active Publication Date: 2021-04-23
上海五色石医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] However, in follow-up studies, using the sY1291 detection method recommended by EAA / EMQN, it was found that there were some false positives. In the samples with AZFc region deletion, more than 99% of sY1291 was missing, while AZFc in normal samples Regions can also be deleted, that is, a certain population polymorphism is present in normal samples (Krausz et al; 2009)

Method used

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  • A detection method and application of human y chromosome label site sy1291
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  • A detection method and application of human y chromosome label site sy1291

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Embodiment 1

[0031] Example 1: The present invention combines PCR product agarose electrophoresis for sY1291 site detection

[0032] Using the PCR primers shown in SEQ ID No.1 and SEQ ID No.2 (wherein SEQ ID No.2 does not need to be labeled with fluorescein), the male genomic DNA was carried out according to the PCR reaction system shown in Table 2 and the PCR reaction conditions shown in Table 3 Amplification, PCR amplification products were electrophoresed with 2.0% agarose.

[0033] A total of 6 male samples from No. 1 to No. 6 were divided into two types according to the detection results and phenotype of the classic deletion sites in the AZFc region (sY255 and sY254). Samples No. 3 and No. 4 were male infertility samples with classic deletion sites in the AZFc region , samples No. 1, No. 2, No. 5 and No. 6 are normal male samples with normal semen parameters. The detection method of the classic deletion sites (sY255 and sY254) in the AZFc region adopts the detection method recommende...

Embodiment 2

[0039] Example 2: The present invention combines PCR product capillary electrophoresis fluorescent fragment analysis to detect sY1291 site

[0040] Using PCR primers shown in SEQ ID No.1 and SEQ ID No.2 (wherein SEQ ID No.2 is labeled with FAM fluorescein), according to the PCR reaction system shown in Table 4 and the PCR reaction conditions shown in Table 5, to 1169 cases Male DNA samples were amplified by PCR, and the PCR products were subjected to capillary electrophoresis using a 3500Dx genetic analyzer.

[0041] The 1169 male samples included 232 male samples with normal semen parameters and 937 male infertile samples. All samples were identified by the kit approved by the China Food and Drug Administration for the detection of AZF microdeletions (human chromosome AZF region microdeletion nucleic acid kit (PCR-fluorescent probe method, National Machinery Note 20153400764) for the canonical mapping of the AZFc region. Points (sY255 and sY254) were detected, and the sY1291...

Embodiment 3

[0054] Example 3: The present invention combines the real-time fluorescent probe method to detect the sY1291 site

[0055] Using the PCR primers shown in SEQ ID No.1 and SEQ ID No.2 (SEQ ID No.2 does not label fluorescein), the fluorescent probe shown in SEQ ID NO.3, according to the PCR reaction system shown in Table 8 and Table 9 The PCR reaction conditions were shown for real-time fluorescence detection. The internal reference gene (sex-determining gene, SRY) recommended by EAA / EMQN for AZF microdeletion detection was detected in this system at the same time. When the fluorescent signal of the internal reference gene exists, the sample with sY1291 site amplification curve is the sample without sY1291 site deletion ( image 3 ), and those without sY1291 locus amplification curve are samples with sY1291 locus deletion ( Figure 4 ).

[0056] Table 8 PCR reaction system of the present invention combined with real-time fluorescence detection of sY1291 site

[0057] .

[...

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Abstract

The invention belongs to the technical field of in vitro nucleic acid detection, and specifically relates to a detection method of a human Y chromosome tag site sY1291 and an application thereof. The present invention provides a new sY1291 site detection strategy, that is, redesign specific PCR amplification primers in the 38bp large fragment repeat region upstream of the sY1291 site detection region recommended by EAA / EMQN, and the new PCR amplification primers detect region I The 5' end of the site is 38 bp away from the 3' end of the sY1291 site detection region recommended by EAA / EMQN, and the new PCR amplification primers can simultaneously detect four regions I, II, III, and IV. The detection strategy of the present invention combines agarose electrophoresis, fluorescent label primer capillary electrophoresis or fluorescent probe method to form different detection methods of sY1291 site. The method for detecting the tag site sY1291 of the human Y chromosome provided by the present invention has strong specificity and a wide detection area of ​​the Y chromosome, and is more suitable for the expanded detection of the AZF microdeletion of the human Y chromosome.

Description

technical field [0001] The invention belongs to the technical field of human in vitro nucleic acid detection, and in particular relates to a detection method and application of a human Y chromosome tag site sY1291. Background technique [0002] The human Y chromosome is unique to males. In terms of genetic law, the Y chromosome can only be inherited from father to son, that is, the Y chromosome shows the characteristics of paternal inheritance. At the same time, there are sex-determining genes such as SRY on the Y chromosome, genes or key regions related to male sperm production, such as Azoospermia Factor (AZF). However, due to the existence of a large number of highly homologous sequences in the Y chromosome itself, large deletions in the Y chromosome can occur during the genetic process, the most common being the microdeletion of the AZF region. [0003] Due to the paternal inheritance of the Y chromosome itself, polymorphic genetic markers on the Y chromosome are often...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q2565/125C12Q2563/107
Inventor 赵书民彭朝敏周巍金云舟
Owner 上海五色石医学科技有限公司
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