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Primer group and kit

A primer set and kit technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problem of high probe cost, high difficulty of multiplex fluorescent PCR, and inability to achieve 6 STS amplification. Increase the number of problems, to achieve the effect of shortening time-consuming and easy operation

Inactive Publication Date: 2018-05-22
HAINAN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of this method is that it is fast, easy to operate, and the interpretation of the results is very easy, but the disadvantages are that the cost of the probe is high, it is difficult to set up multiple fluorescent PCR, the generation of possible by-products cannot be monitored, and it is impossible to realize six PCR assays. STS are simultaneously amplified in one multiplex PCR reaction, etc.

Method used

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  • Primer group and kit
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  • Primer group and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1 solution composition, configuration

[0051] (1) According to the primer information published in "EAA EMQN best practical guidelines for molecular diagnosis of Ychromosomal microdeletions state of the art 2013", the primers were synthesized by the primer synthesis factory. The forward primer in the primer pair adds a fluorophore at the 5' end. The fluorescent color and primer information are shown in Table 1.

[0052] Table 1: STS primer sequences and fluorescent modifications

[0053]

[0054]

[0055] (2) After obtaining the primer powder synthesized by the primer synthesis manufacturer, centrifuge at 12000rpm for 5min, and use nucleic acid-free ddH 2 O dissolves the primers. According to the primer unit on the primer information sheet, dilute to a primer stock solution with a molar concentration of 100 pmol / μL.

[0056] Vortex and mix well, then centrifuge at low speed to collect the solution at the bottom of the tube.

[0057] (3) Prepare PC...

Embodiment 2

[0063] Embodiment 2PCR operation

[0064] (1) Thaw the PCR reaction solution, enzyme solution, positive quality control, negative quality control, female quality control and blank quality control solution prepared in Example 1, vortex and mix for 5 seconds, and centrifuge at low speed for 10 seconds before use. According to the form of "specimen number + 5" required for the experiment, draw the PCR reaction solution based on 18.8 μL, and put it into a 1.5mL EP tube.

[0065] (2) Pipette 0.2uL of enzyme solution into the above-mentioned EP tube for mixing. Vortex for 5s, centrifuge at low speed for 10s and set aside

[0066](3) Pipette 19 μL of the mixture into a 0.2mL PCR tube, or 0.2mL 8-strip tube. At the same time, add 1 μL of the sample to be tested (be sure to dilute the sample DNA so that the concentration is 5-10ng / μL), and cover the tube cap. Add 1 μL of quality control DNA solution in the same way. Vortex for 5 s and centrifuge at low speed for 10 s.

[0067] (4)...

Embodiment 3ABI3500

[0070] Example 3 ABI3500 computer operation

[0071] (1) Vortex and mix the PCR product of Example 2 for 5 s, centrifuge at low speed for 10 s, and set aside.

[0072] (2) With the ratio of 9.5 μL HiDi fomamide and 0.5 μL Genescan-500 (LIZ) SIZE STD KIT, mix the solution according to the form of “PCR product number + 1”. Vortex for 5 s and centrifuge at low speed for 10 s.

[0073] (3) Pipette 10 μL of the above solution into a new 0.2mL PCR tube or 0.2mL 8-strip tube, add 1 μL of the product to it at the same time, and cover the tube cap. Vortex for 5 s and centrifuge at low speed for 10 s.

[0074] (4) Put the PCR tube into the PCR instrument, set the program: 94°C for 5 minutes, and immediately transfer to ice for cooling for 5 minutes after the end.

[0075] (5) Using the 96-well plate matched with the ABI Genetic Analyzer, transfer the solution described in (4) into the wells of the 96-well plate one by one.

[0076] (6) Cover with the matching plug of ABI Genetic Ana...

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Abstract

The invention provides a primer group and a kit containing the same. The primer group according to the embodiment of the invention is a primer group which is designed aiming to 6 STS sites of an Y chromosome. The primer group can be applied to single multiplex polymerase chain reaction (PCR), i.e., the groups of primers is used in one PCR to perform amplification, and multiple PCR products. Compared with conventional PCR, the operation is more simple and convenient, the time consumption of an experiment is also shortened, and amplification of the 6 STS sites in one multiplex polymerase chain reaction is successfully realized. The kit according to the embodiment of the invention is used for detecting Y chromosome microdeletions and has the advantages of higher flexibility, more simplenenssand convenience in operation, more easiness in result interpretion and lower requirements for specimens.

Description

technical field [0001] The invention relates to the field of biological detection, in particular, the invention relates to a primer set and a kit. Background technique [0002] There is a gene related to spermatogenesis on the q11.23 region of the long arm of the Y chromosome, called azoospermia factor (AZF). AZF is divided into three regions: AZFa, AZFb, and AZFc. Microdeletions in any one region can lead to spermatogenesis disorders, oligoasthenospermia or azoospermia. Current studies have shown that there are a large number of palindromic structures and repetitive sequences in the AZF region, making this region very prone to structural abnormalities, such as deletions, inversions, and sequence rearrangements. Causes mutations in related genes in the region, leading to male infertility. Due to the differences in genes in the regions, the differences in male infertility phenotypes caused by deletions in different regions are determined. Studies have shown that the deleti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2537/143
Inventor 李崎马燕琳陈宏健周繇
Owner HAINAN MEDICAL COLLEGE
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