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PCR amplification primer, kit and use thereof in identification of mammal sex

A technology for amplification primers and kits, which is applied in the field of PCR amplification primers, kits, and gender identification of mammals, can solve the problems of difficult implementation, low identification accuracy, and long time required, etc. To achieve the effect of avoiding pollution

Inactive Publication Date: 2005-09-07
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the above method has low identification accuracy and takes a long time, and it is difficult to implement the methods and procedures for sex detection of animal embryos in production practice.

Method used

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  • PCR amplification primer, kit and use thereof in identification of mammal sex
  • PCR amplification primer, kit and use thereof in identification of mammal sex
  • PCR amplification primer, kit and use thereof in identification of mammal sex

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0065] Gender detection using animal genome DNA

[0066] Extraction, purification and quality inspection of genomic DNA from animal tissues

[0067] 1. Cut 0.5 mg of fresh animal muscle tissue or liver tissue into pieces and place in 4.5 mL of DNA extraction buffer (10 mmol / LTris-HCl, 0.1mol / LEDTA, 20 μg / mL RNase, 0.5% SDS pH8.0) In the homogenization tube, the homogenization tube was placed in a beaker with ice cubes, and an electric glass homogenizer was used to homogenize at a medium speed for about 90 sec. All the homogenate was poured into two 5mL centrifuge tubes, and the centrifuge tubes were placed in a beaker with ice cubes, and crushed with an ultrasonic cell pulverizer (power 400W, ultrasonic time 3 sec, interval time 4 sec, working frequency 15 times). EDTA refers to ethylenediaminetetraacetic acid.

[0068] 2. After crushing the cells, quickly place the centrifuge tube in a constant temperature shaker in a water bath and incubate at 37°C for 1 h.

[0069] 3. Ad...

example 2

[0088] Extraction, purification and quality detection of genomic DNA from animal serum

[0089] 1. Collect about 6mL of fresh blood and add it to a centrifuge tube filled with 1mL acid citrate glucose solution B (ACD solution—0.48g citric acid, 1.32g sodium citrate, 1.47g glucose, add water to 100mL). Blood can be stored at 0°C for several days or at -70°C for long-term storage before DNA preparation.

[0090] a. Fresh blood (2mL) was centrifuged at 4500r / min for 15min, the upper layer of plasma was discarded, and the light yellow layer was carefully transferred to a new centrifuge tube with a pipette, and centrifuged again at 4500r / min for 15min. After centrifugation, resuspend the buffy layer in 1.5 mL of DNA extraction buffer, incubate at 37° C. for 1 h, and follow steps 3-11 in Example 1.

[0091] b. Refrigerated blood (2mL), thawed at room temperature, transferred to a new centrifuge tube, washed with an equal volume of phosphate-buffered saline PBS (8g NaCl, 0.2g KCl, 1...

example 3

[0098] Methods of sex determination using embryos

[0099] 1. Evaluation and size determination of embryos

[0100] Embryos detected under a solid microscope were washed with PBS for 3 times and placed in a sterile room for 0.5 hours. In the culture medium, the developmental stage, embryo age and grade were evaluated.

[0101] 2. Treatment of Embryos

[0102] Collect 1~2 cells of mammalian embryos, first rinse with 0.145mol / L NaCl twice, then add embryo treatment solution (10mmol / LTris-HCl (pH8.3), 50mmol / L KCl, 2mmol / L MgCl 2 , 0.45% NP-40, 0.45% Tween-20, 0.1μg / μL proteinase K), so that the total volume is 20μL, the embryo and its treatment solution are reacted in a 55°C water bath for 60min, and then the embryo lysate is directly used as a PCR reaction template.

[0103] 3. Amplification reaction

[0104] According to the number of samples to be amplified n (n = the number of processed embryo samples + 2), take PCR reaction solution n×29.μL and Taq DNA polymerase n×0.4 ...

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Abstract

The invention relates to a PCR augmentation primer and agent box and the application in the sex identification of mammals, which is a pair of primer augmenting the 163bp area in gene Sry, sequence of basic group is: SRY5: 5'-TGAACGCCTTCATTGTGTGGTC-3',SRY3: 5'-GCTAGTAGTCTCTGTGCCTCCT-3'. The main components of the agent box are: buffer for DNA extraction, Taq DNA polymerase, PCR reaction liquid, blank control, paraffin oil and sample buffer. The applying steps of agent box in the sex identification of mammals are: (1) sample treatment, (2) augmentation, (3) product detecting. The invention is characterized by judging the existence of Y colorant layer specific fragment by detecting the augmenting product, and the avoidance of the unnecessary pollution, and the suitness for the quick(about 1 hour) sex identifying of mammal embryo in practice, the accuracy is 100%.

Description

technical field [0001] The invention belongs to a PCR detection method, a kit and primers. Background technique [0002] Polymerase Chain Reaction (Polymerase Chain Reaction), referred to as PCR, also known as in vitro amplification technology, is a new technology for in vitro enzymatic amplification of DNA jointly created by the Human Genetics Department of Cetus Corporation, the University of California and Howghes Medical College in 1985. It has the characteristics of strong specificity, high sensitivity, simple operation, rapidity and high efficiency. After the advent of PCR technology, it immediately aroused widespread interest and attention, and soon it was widely used and developed all over the world, and quickly entered the field of genetic diagnosis of genetic diseases, detection of infectious pathogens, forensic science, archaeology and molecular biology each field. [0003] PCR technology is actually an enzymatic synthesis reaction dependent on DNA polymerase in...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12Q1/68
Inventor 陈宏权洪桂云章孝荣
Owner ANHUI AGRICULTURAL UNIVERSITY
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