Anemia screening kit for CRISPR and CAS9 targeted capture of long fragment DNA and method thereof

Abstract

The embodiment of the invention relates to the technical field of biological detection, in particular to an anemia screening kit for capturing long-fragment DNA in a targeted mode through CRISPR and CAS9. The anemia screening kit comprises sgRNA used for capturing thalassemia related genes in a targeted mode, a Y-type connector used for cyclization amplification and a primer used for cyclization amplification. The target capture range of the sgRNA is as follows: chr11: 5166361 to 5296578 (hg38), and chr16: 48994 to 210817 (hg38), and the target capture range of the sgRNA is as follows: chr11: 5166361 to 5296578 (hg38). According to the anemia screening kit for target capture of long fragment DNA by CRISPR and CAS9 and the method thereof, a large fragment region of thalassemia variation is captured in a targeted manner through CRISPR and CAS9 systems, five types of thalassemia mutation regions including alpha, beta, delta, delta beta and epsilon gamma delta beta are covered, and a three-generation or four-generation long-reading sequencing technology is utilized to identify the thalassemia mutation region of the thalassemia mutation region of the thalassemia mutation region of the thalassemia mutation region of the thalassemia mutation region of the thalassemia mutation region. The kit can effectively detect pathogenic mutation, complex structure variation and large fragment deletion which may cause thalassemia in people, and non-deletion and deletion mutation covering beta globin genes and related regulatory genes including HS-40 and beta LCR genes.

Description

technical field [0001] The present application relates to the technical field of biological detection, and more particularly, to an anemia screening kit for CRISPR and CAS9 targeted capture of long DNA fragments and a method thereof. Background technique [0002] Thalassemias are a group of autosomal recessive blood disorders. There are four kinds of peptide chains that make up globin, namely α, β, γ, and δ chains, which are encoded by their corresponding genes. The deletion or point mutation of these genes can cause the synthesis obstacles of various peptide chains, resulting in the components of hemoglobin. Change. According to the different types of globin peptide chain synthesis disorders, thalassemias are generally divided into α thalassemia, β thalassemia, δ thalassemia, δβ thalassemia and εγδβ thalassemia. Among them, beta and alpha thalassemias are more common. The human α-globin gene cluster is located on chromosome 16 and contains 7 loci: 5'-zeta-pseudozeta-mu-p...

AI Technical Summary

Problems solved by technology

The Sanger sequencing method is cumbersome to operate, which greatly limits its application in clinical diagnosis. The detection throughput of sequencing is also limited, and manual analysis is required for the detection results, which is time-consuming and labor-intensive.

RFLP is to carry out enzyme digestion reaction by identifying specific sequence sites. The method is simple and low-cost, but its limitation is that it can only detect limited mutations that can produce enzyme cutting sites. lead to false positive or false negative results

The result of reverse dot hybridization technology is interpreted by naked eyes, and the error rate is high, often resulting in repeated testing of a sample

ARMS-PCR needs to design corresponding primers for each mutation, and the amplification conditions of each pair of primers need to be optimized. If multiple mutations need to be detected at the same time, the operation is cumbersome, and false positive or false negative results may also occur

At present, common products based on multiplex GapPCR can only achieve 3-4 common deletion-type thalassemia mutations. For rare mutations, including some rare structural variation mutations, the coverage is limited

Next-generation sequencing technology usually requires targeted capture sequencing or multiplex PCR enrichment, complex primer design and complicated procedures, high requirements for operators, and high cost

Next-generation sequencing has a short read length and is often used to detect small fragment insertion mutations, small fragment deletion mutations, and point mutations. There is still a possibility of missing detection of complex structural variations of large fragments

In other technologies, the combined probe-anchored polymerization sequencing method used in the α and β thalassemia gene detection kits has limited detection of large fragment structural variations. Anemia Screening Kit and Method for Capturing Long Fragment DNA to Solve the Problems Raised Above

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