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Kit and method for detecting copy number of human 5S ribosome DNA

A detection method, ribosome technology, applied in the field of molecular biology and medical detection, to achieve the effect of small system error, simple operation and high accuracy

Pending Publication Date: 2022-04-12
中国疾病预防控制中心职业卫生与中毒控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few high-throughput detection methods for determining the copy number of 5S ribosomal DNA

Method used

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  • Kit and method for detecting copy number of human 5S ribosome DNA
  • Kit and method for detecting copy number of human 5S ribosome DNA
  • Kit and method for detecting copy number of human 5S ribosome DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A kit for detecting the copy number of human 5S ribosomal DNA

[0035] There are 8 products in this kit, including:

[0036] 1. One standard product

[0037] The DNA concentration was 90 ng / μL, and the samples came from the mixed whole blood DNA samples of 3000 people (aged 20-60 years old).

[0038] 2. 4 primers

[0039] Include the upstream and downstream primers of 5S ribosomal gene and single-copy gene (albumin) respectively (add corresponding volume of ddH according to the tube body prompts) 2 After O the concentration was 100 μM). Specific primer sequences are as follows

[0040]

[0041] 3. One SYBR qPCR mix contains reaction buffer, GreenⅠ, dNTPs, Mg 2+ 2× master mix of rTaq DNA polymerase and anti-DNA polymerase antibody.

[0042] 4. ROX reference dye 1 piece

[0043] 5. ddH 2 O 1 stick.

Embodiment 2

[0045] A method for detecting the copy number of human 5S ribosomal DNA for non-diagnostic purposes, using the kit described in Example 1, comprising the following steps:

[0046] 1. Extract the genomic DNA to be tested, and prepare the sample to be tested with a concentration of 5 ng / μL;

[0047]2. Take the standard in the kit, prepare a standard series gradient according to the concentration of the DNA sample to be tested, so that the sample DNA concentration is included in the standard series concentration (for example, prepare 20, 10, 5, 2.5, 1.25, 0.625ng / ul composition standard series of gradients).

[0048] 3. The genomic DNA to be tested and the standard product are detected by real-time fluorescence quantitative PCR (RT-PCR) method, and the albumin (ALB) gene is used as an internal reference gene, and the primers in the kit are used; the method of adding samples is specifically Add the sample / standard substance to be tested and each reagent in the kit to the same rea...

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Abstract

The invention provides a human 5S ribosome DNA copy number detection method which comprises the following steps: extracting genome DNA of a sample to be detected, respectively detecting the genome DNA of the sample to be detected and a reference standard by using a fluorescent quantitative PCR (Polymerase Chain Reaction) method, and collecting a ribosome gene fluorescence signal (R) and a single copy gene fluorescence signal (S) through PCR reaction, calculating the relative quantity of the copy number of the human ribosome DNA of the sample to be detected based on the standard curve and the R / S ratio obtained by detecting the genome DNA of the sample to be detected; in the PCR reaction, an albumin gene is taken as a reference gene, a proper primer is used, and every 10 mu L of reaction system is controlled to contain 0.5 mu L of a 10 mu M upstream primer, 0.5 mu L of a 10 mu M downstream primer and 1 mu L of a to-be-detected sample DNA or a reference standard DNA. The invention also provides a kit for detecting the copy number of the human 5S ribosome DNA. The detection method and the kit are high in detection accuracy, simple and convenient to operate, low in sample demand and suitable for detection of crowds with large sample sizes.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and medical detection, in particular to a 5S ribosomal DNA copy number detection kit and a detection method for detecting human 5S ribosomal DNA copy number using the kit. Background technique [0002] Ribosomal DNA (rDNA) is the most abundant and important housekeeping gene in cells, and its main function is to encode ribosomal RNA to complete subsequent ribosome assembly and protein processing. Due to its repetitive nature, ribosomal DNA plays an important role in maintaining genome stability and is one of the most vulnerable regions in the eukaryotic genome, affecting cellular functions such as aging. More and more studies have shown that the abnormality of ribosome biosynthesis, including the increase in the number of ribosomes, is closely related to the occurrence and development of tumors. The 5S rDNA sequence in the human body is distributed on chromosome 1. At present, there a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/11
Inventor 段化伟王艳华王婷张丽雅
Owner 中国疾病预防控制中心职业卫生与中毒控制所
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