Primer for TaqMan probe real-time fluorescence PCR (Polymerase Chain Reaction) detection on DNA SRY (Deoxyribonucleic Acid Sex-Determining Region on the Y Chromosome) gene of human genome of trace sample

A technology of SRY gene and real-time fluorescence, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., to achieve the effect of simple preparation process, strong specificity and low cost

Inactive Publication Date: 2017-09-01
谱天福信(天津)分子诊断技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of TaqMan probe real-time fluorescent PCR method to the detection of SRY gene has not been reported yet.

Method used

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  • Primer for TaqMan probe real-time fluorescence PCR (Polymerase Chain Reaction) detection on DNA SRY (Deoxyribonucleic Acid Sex-Determining Region on the Y Chromosome) gene of human genome of trace sample
  • Primer for TaqMan probe real-time fluorescence PCR (Polymerase Chain Reaction) detection on DNA SRY (Deoxyribonucleic Acid Sex-Determining Region on the Y Chromosome) gene of human genome of trace sample
  • Primer for TaqMan probe real-time fluorescence PCR (Polymerase Chain Reaction) detection on DNA SRY (Deoxyribonucleic Acid Sex-Determining Region on the Y Chromosome) gene of human genome of trace sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Preparation of primers and probes for the SRY gene TaqMan probe real-time fluorescent PCR detection kit for micro-sample human genomic DNA

[0040] The full length of the SRY gene is 887bp, without introns, and encodes a protein of 204 amino acids. According to the SRY gene (gene sequence number: NM_003140, updated on 2016.10.7) sequence published by the NCBI database, specific primers and probes were designed, as shown in Table 1. When designing primer probes for SRY gene detection, select the conserved region of the SRY gene and avoid polymorphic site regions on the SRY gene to ensure the specificity of the primer probes.

[0041] Table 1 is used to detect the specific primer probe combination of SRY gene

[0042]

[0043] Wherein SRY F is a forward primer, and SRY R is a reverse primer to amplify the SRY gene region. SRY P is a detection probe for the 5'-end labeled FAM signal and the 3'-end labeled TAMRA signal, which can bind to the amplified fragment.

[004...

Embodiment 2

[0051] Sample preparation before detection of SRY gene TaqMan probe real-time fluorescent PCR detection kit for micro-sample human genomic DNA

[0052] (1) Obtain the genomic DNA of the sample to be tested (taking human peripheral blood plasma cell-free genomic DNA as an example)

[0053] Use Tianlong's nucleic acid extraction kit (magnetic bead method) (Ex-DNA serum / plasma free) to extract the free genomic DNA from the peripheral blood plasma of pregnant women, and use it to detect the SRY gene of the fetus in the mother. The extraction is completed according to the operation steps in the instruction manual.

[0054] The extracted DNA samples were tested for concentration and DNA quality with a UV spectrophotometer. The DNA concentration was required to be >5ng / μL, and the DNA quality OD260 / 280 was between 1.8 and 2.0. After quantification, the mother liquor was stored at -20°C.

[0055] (2) Dissolve and dilute the positive quality control

[0056] The SRY gene positive qu...

Embodiment 3

[0062] Detection method of SRY gene TaqMan probe real-time fluorescent PCR detection kit for micro-sample human genomic DNA

[0063] (1) Sensitivity experiment of the SRY gene TaqMan probe real-time fluorescent PCR detection kit for micro-sample human genomic DNA

[0064] Reagent composition: PCR amplification enzyme reaction mixture; TaqMan probe primer pair mixture of SRY gene and internal standard gene conservative region of Example 1; positive quality control product working solution group and negative quality control product of Example 2. Among them, the positive quality control working solution group was used as the target sample for this test.

[0065] Reaction system: add 12.5 μl of PCR amplification enzyme reaction mixture to each reaction, 1.5 μl of TaqMan probe primer pair mixture of SRY gene and internal standard gene conserved region of Example 1, 1 μl of positive quality control product working solution, 10 μl Sterile double distilled water, the total reaction s...

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Abstract

The invention provides a primer for TaqMan probe real-time fluorescence PCR (Polymerase Chain Reaction) detection on a DNA SRY (Deoxyribonucleic Acid Sex-Determining Region on the Y Chromosome) gene of a human genome of a trace sample. The primer is characterized by comprising an SRY gene conserved region primer pair, an SRY gene probe, an endogenous reference gene conserved region primer pair and an endogenous gene probe. The primer and a kit are simple in preparation process, low in cost, good in specificity and high in sensitivity, the detection rate and the accuracy of the SRY gene in DNA of trace samples such as blood spots, hair and parent free plasma can be greatly increased, and an effective detection method is provided for fields such as medicinal diagnosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to primers for real-time fluorescent PCR detection of TaqMan probes of human genome DNA SRY gene in trace samples, and also relates to a kit including the above primers. Background technique [0002] The SRY gene is the best candidate gene located on the Y chromosome Yp11.3 testicular determinant, which determines the sex differentiation and testis formation in the early stage of embryonic development, and is the key gene of sex differentiation. In the genetic disease of abnormal sex development, the main clinical feature is that the chromosomal karyotype sex is opposite to the gonad sex, manifested as abnormal genital development, differences in body phenotype and sex, abnormal hormone levels in the body, and abnormal physiological activities. The possible reasons for the results include chromosomal translocation, insertion or deletion of SRY gene during embryonic developme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/166
Inventor 李捷吴永鹏刘开春
Owner 谱天福信(天津)分子诊断技术有限公司
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