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Genetic reference material for gene detection of alpha-thalassemia and preparation method thereof

A thalassemia and reference material technology, applied in the field of genetic reference material for alpha thalassemia gene detection and its preparation, can solve the problems of difficult quantitative determination, precious sample quantity, and high requirements for preservation conditions, and achieves easy calculation of genomic DNA copy number, The DNA extraction method is simple and easy, and the gene operation is simple and fast.

Pending Publication Date: 2020-11-06
重庆市人口和计划生育科学技术研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Analyzing the advantages and disadvantages of existing genetic reference standards, we found that patient samples are closest to the test sample form and have a definite genetic background, but due to the precious number of samples, inconvenient acquisition, and poor uniformity, it is impossible to prepare a large number of standards; cell samples It is also relatively close to the form of clinical samples, and the pretreatment and extraction methods are similar, but the establishment of immortalized cell lines requires high storage conditions, and the construction and screening process of genetically modified cell lines is complicated, and it is necessary to distinguish between homozygous and heterozygous mutation types; the construction process of plasmid & PCR products is simple Fast, not limited by rare mutation samples, but the high copy number concentration of the sample is easy to cause diffusion and contamination between different genotypes, resulting in false positives in experimental results; similarly, synthetic DNA samples are easy to obtain and are not restricted by rare mutation samples limit
However, it is quite different from clinical samples, and it is difficult to determine and quantify

Method used

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  • Genetic reference material for gene detection of alpha-thalassemia and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Selection and design of guide RNA target sites

[0037] 1) Selection and design of guide RNA target sites

[0038] Three gRNAs targeting URA3 were designed on the http: / / crispr.dbcls.jp / website, and the guide RNA target sites are as follows:

[0039] URA-g1: cattacgaatgcacacggtgTGG (351-373bp in SEQ ID NO: 1, the uppercase base is PAM, and the lowercase base is protospacers);

[0040] URA-g2:gttagcagaattgtcatgcaAGG (447-469bp in SEQ ID NO:1, the uppercase base is PAM, and the lowercase base is protospacers);

[0041] URA-g3: gaagagatgaaggttacgatTGG (569-591bp in SEQ ID NO: 1, the uppercase base is PAM, and the lowercase base is protospacers);

[0042] Design two reverse complementary primers according to the guide RNA target site, and add protective bases at both ends of the primers. The primer sequences are as follows (the protective bases are all three bases at the 5' end):

[0043] URA-g1F: +5'ATCcattacgaatgcacacggtg3'

[0044] URA-g1R: -5'aacCACCGTGT...

Embodiment 2

[0056] Example 2: Construction of Saccharomyces cerevisiae homologous recombination template

[0057] Design the homology arm based on the Cas9 system to target the cleavage site, and connect the upstream homology arm and the downstream homology arm into the multiple cloning site region of the pBluescript vector through the molecular cloning method of restriction enzyme digestion or the recombination of the homology arm , Restriction sites are reserved between homologous sequences for ligation into knock-in sequences. The successful construction of the recombination template obtains the double-stranded homologous recombination template containing the upstream and downstream homology arms and the target sequence by restriction enzyme digestion or PCR amplification.

[0058] Design the homology arm based on the Cas9 system targeting the cleavage site (guide RNA target site), the upstream homology arm 134bp (214-347bp in SEQ ID NO: 1), the downstream homology arm 141bp (in SEQ ID...

Embodiment 3

[0069] Embodiment 3: the acquisition of genetic reference material

[0070] The gRNA1-p425-Sap-TEF1p-Cas9-CYC1t-2xSap plasmid targeting URA3 and the double-stranded homologous recombination template containing upstream and downstream homology arms and genetic reference material genes obtained in Example 2 were transformed into yeast using the lithium acetate method . After culturing, PCR and first-generation sequencing were performed on the single clone to identify the recombination of the single clone.

[0071] Lithium acetate (LiAC) conversion system:

[0072] 1) Take 1ml of YPD liquid medium cultured overnight, discard the supernatant at 12000rpm for 30sec;

[0073] 2) Resuspend and wash the cells with 1ml of pure water, discard the supernatant at 12000rpm for 30sec;

[0074] 3) Resuspend and wash the cells with 1ml of 1×TE / LiAC, 12000rpm for 30sec, and remove the supernatant completely;

[0075] 4) Add 600ng of gRNA1-p425-Sap-TEF1p-Cas9-CYC1t-2xSap plasmid, 4μl of Carr...

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Abstract

The invention relates to the technical field of biology, and particularly discloses a genetic reference material for gene detection of alpha-thalassemia and a preparation method thereof. The genetic reference material of the invention is to insert yeast cells of a genetic reference material gene required for gene detection of the alpha-thalassemia into a URA3 gene. The invention selects the yeastcells as the background and carriers of genetic mutation, utilizes the advantages that the yeast cells are convenient to storage and culture, simple in genetic operation, high in homologous recombination efficiency, convenient to count and the genome DNA copy number is convenient to calculate and the like, and combines a CRISPR / Cas9 technology to target the URA3 gene to prepare the genetic reference material for gene detection of the alpha-thalassemia, and consistency with human cell sample treatment operations can be achieved through protoplast formation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a genetic reference material for α-thalassemia gene detection and a preparation method thereof. Background technique [0002] Thalassemia is the most common and most harmful genetic disease in southern my country. There is no effective treatment for this disease. Therefore, the development of genetic diagnosis technology for clinical and prevention has become the first choice and has important clinical significance. Genetic testing has gradually become the gold standard for diagnosing thalassemia, and genetic reference materials are essential for genetic testing, which can provide negative and positive controls for genetic testing experiments to ensure the accuracy and reliability of experimental results. Thalassemia genetic reference materials can be used as positive and negative quality control products for thalassemia gene detection, as well as daily quality control and performanc...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6806C12N1/19C12N15/81C12N15/90C12R1/865
CPCC12Q1/6883C12Q1/6806C07K14/805C12N15/81C12N15/905C12Q2600/156C12Q2600/166
Inventor 朱一剑卢大儒陈红岩何鑫
Owner 重庆市人口和计划生育科学技术研究院
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