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Kit for detecting estrogen acceptor alpha gene upstream TA repeated sequence, and its detection method

An estrogen receptor and repeat sequence technology, which is applied in the fields of life science and biology, can solve the problems of poor primer specificity, affecting sequencing results, and non-specific band interference of amplified bands, and achieves high accuracy, amplified bands, etc. With single, good sequencing results

Active Publication Date: 2013-12-25
FUZHOU ADICON CLINICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the existing primers for detecting the TA repeat sequence upstream of the estrogen receptor α gene have poor specificity, and the amplified bands have non-specific band interference, which affects the sequencing results

Method used

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  • Kit for detecting estrogen acceptor alpha gene upstream TA repeated sequence, and its detection method
  • Kit for detecting estrogen acceptor alpha gene upstream TA repeated sequence, and its detection method
  • Kit for detecting estrogen acceptor alpha gene upstream TA repeated sequence, and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A kit for detecting the TA repeat sequence upstream of the estrogen receptor alpha gene, including

[0032] (1) Red blood cell lysate 10×: (NH 4 CL 82mg / ml, NAHCO 3 8.4mg / ml, EDTA-NA 2 3.72mg / ml, DEPC-ddH 2 O make up to the prepared volume)

[0033] (2) Nucleic acid extract: (Tris 1.2114mg / ml, Chelex100 50mg / ml, 10%SDS 0.01%),

[0034] (3) PCR amplification reaction solution: including 10 μl of PCR buffer, 1 μl of dNTPs, 0.4 μl of Taq enzyme, and 0.4 μl of upstream and downstream primers TAF / TAR; among them, the sequences of upstream and downstream primers TAF / TAR are:

[0035] TAF: AACTCGATTTTGGCTTCA

[0036] TAR: GATCTTCTCGGTTCACTTG

[0037] (4) Enzyme purification solution: including EXOI (exonuclease) 0.5 μl, CIP (alkaline phosphatase) 0.1 μl, water 1.4 μl.

Embodiment 2

[0039] The using method of kit of the present invention:

[0040] (1) Nucleic acid extraction: Take 500ul of whole blood, put it into a 1.5ml centrifuge tube, and add 1ml of red blood cell lysate. Flip up and down to make it completely mixed, rotate and pulsate for 15 seconds, then put it into a centrifuge for centrifugation, the centrifugation speed is 5000rpm, 10min. Pour off the upper layer, and it can be seen that there is a bloody precipitate at the bottom of the centrifuge tube. Add 500ul red blood cell lysate, repeat this lysis step once. Centrifuge at 5000rpm for 5min, and finally suck up all the upper layer with a pipette and discard it, so that the bloody precipitate at the bottom of the centrifuge tube will no longer have lysate. Mix well, put in a metal bath at 100°C for 10 minutes, 12000rpm for 5min, take the supernatant and save it at -20°C for later use.

[0041] (2) PCR amplification: PCR amplification system: 2*Buffer 10 μl, dNTPs 1 μl, Taq enzyme 0.4 μl, u...

Embodiment 3

[0054] Adopt kit of the present invention to detect clinical specimen

[0055] 186 whole blood samples were taken, and the number of repetitions of the TA repeat sequence upstream of the estrogen receptor a gene was detected according to the method described in Example 2. The results are as follows:

[0056] Number of specimens (total 186)

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Abstract

The invention discloses a kit for detecting an estrogen acceptor alpha gene upstream TA repeated sequence, and its detection method. The kit which comprises an erythrocyte lysate, a nucleic acid extract, a PCR amplification reaction solution, and an enzyme purifying solution is characterized in that the PCR amplification reaction solution comprises a PCR buffer solution, d NTP, a Taq enzyme, an upstream primer TAF and a downstream primer TAR; the sequence of the upstream primer TAF is AACTCGATTTTGGCTTCA; and the sequence of the downstream primer TAR is GATCTTCTCGGTTCACTTG. The polymorphism of the human estrogen acceptor alpha gene upstream TA repeated sequence can be detected through adopting the kit to amplify Er alpha gene in a sample to be detected and combing with a Sanger sequencing technology, so the specificity is good and the accuracy is high.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to a kit and a detection method for detecting the TA repeat sequence upstream of the estrogen receptor alpha gene. Sanger sequencing technology is used to detect multiple TA repeat sequences upstream of the human estrogen receptor alpha gene. It can be detected with good specificity and high accuracy. Background technique [0002] Hypomenorrhea in young women is a common symptom in gynecology. Its hazard is to affect the implantation of fertile eggs in the endometrium, leading to repeated miscarriages and infertility, which are the prodromal symptoms of amenorrhea. Estrogen is the main hormone that regulates menstruation, but it is found clinically that a group of patients have normal estrogen levels and no medical, surgical or congenital diseases, which are called unexplained hypomenorrhea. At the same time, hysteroscopy was performed on a large number of patients wi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 葛婵方国伟王淑一
Owner FUZHOU ADICON CLINICAL LAB INC
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