Method for establishing sequencing library of PKHD1 gene mutation and kit and use thereof

Abstract

The invention provides a method for establishing a sequencing library of PKHD1 gene mutation and a kit and use thereof. The method for establishing the sequencing library of PKHD1 gene mutation comprises the following steps of: performing amplification in the first round; digesting primers; performing amplification in the second round; purifying and recovering all DNA strips; performing sequencing; and performing analysis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and a kit for constructing a sequencing library of PKHD1 gene mutation through high-throughput sequencing technology and its application. Background technique [0002] Polycystic kidney disease (PKD) is a disease in which multiple cysts develop in the kidney and lead to damage to the kidney's structure and function, and the cysts involve other organs. According to the different ways of inheritance, PKD is divided into autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). Cysts in patients with PKD often show continuous formation and enlargement. [0003] ARPKD is a recessive genetic disorder affecting 1 in 20,000 live births. ARPKD mainly has serious effects on fetuses, newborns and children. ARPKD can cause severe polycystic kidney phenotypes in fetuses, such as biliary defects, oligohydramnios, etc. About half o...

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Problems solved by technology

[0008] Other mutation screening methods: denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism analysis (SSCP), and high-resolution melting curve (HRM) can also be applied to the detection of PKHD1 gene, but the disadvantages of these methods are Mutation sites cannot be directly identified, only mutations can be screened

[0009] Although traditional generation sequencing (Sanger sequencing) is still the gold standard for diagnosing human genetic disease variation, due to the limitations of this technology itself, its defects such as low sequencing efficiency, high cost, and low throughput make this technology incapable of handling huge genes. And large sample size sequencing requirements, if it is necessary to sequence all exons in the entire genome to discover new variant genes or loci, this method is even more powerless

In addition, sanger sequencing requires very complicated preliminary preparations. The entire experimental process includes multiple steps such as amplification, purification, sequencing PCR, and repurification, which is not only time-consuming and labor-intensive, but also expensive. possible

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