Primer group, kit and sequencing library for detecting large fragment deletion of rare beta thalassemia gene with unclear fracture site

Abstract

The invention relates to the field of gene detection, in particular to a primer group, a kit and a sequencing library for detecting large fragment deletion of a rare beta thalassemia gene with an unclear fracture site. A pair of complementary primers which are far away from each other are designed in upstream and downstream regions of a beta-globin gene cluster, a pair of normal internal control complementary primers which are moderate in distance is designed in an HBB gene region, long-fragment PCR amplification is performed, and a third-generation sequencing library is constructed and sequenced; the accurate and rapid detection of the large-fragment deletion variation of the rare beta thalassemia gene with the unclear fracture site is realized. Besides, according to the method, DNA of different sequences of 5-50 nt can be added to the 5'end of the designed primer, namely a DNA barcode (Barcode) for distinguishing different samples and achieving simultaneous detection of multiple samples, and the method is easy and convenient to operate, reliable in result and high in repeatability and has very high clinical applicability and generalizability.

Description

technical field [0001] The present application relates to the field of gene detection, in particular to a primer set, a kit and a sequencing library for detecting the deletion of a large fragment of a rare β-thalassemia gene with an unknown breakpoint. Background technique [0002] Thalassemia (Thalassemia) is a hereditary hemolytic disease caused by globin synthesis disorder caused by globin gene defect. It is divided into two types: alpha-thalassemia and beta-thalassemia. Thalassemia is the most widespread and most common single-gene genetic disease in the world. There are at least 345 million people in the world who carry thalassemia gene variants, and the thalassemia gene variant carrying rate in different countries and regions is about 1-5%, especially in Southeast Asia, southern China, the Mediterranean region, India, the Middle East, and North Africa. When parents are carriers of the same type of alpha or beta thalassemia gene variant, their offspring have a 25% chan...

AI Technical Summary

Problems solved by technology

[0004] For the rare variants of thalassemia genes, Sanger sequencing combined with multiple ligation probe amplification (MLPA) and other technologies can be used to detect further, but the variants covered by these technologies are still very limited, and the throughput is low, the cost is high, and the operation is cumbersome

For the complex structural variation of the thalassemia gene cluster, such as large fragment deletions with known breakpoint ranges, it can be detected by cross-breakpoint PCR (Gap-PCR) combined with multiple ligation probe amplification (MLPA) technology, but it is still unable to accurately detect The specific location of the break site, and it is impossible to detect the complex structural variation of the thalassemia gene cluster whose range of the break site is unknown

[0005] The application of next-generation sequencing (next-generation sequencing, NGS) for thalassemia gene detection can significantly improve the detection timeliness, detection accuracy and detection rate of gene variation, but NGS sequencing reads are short and can only Detect point mutations, small fragment insertions / deletions, and copy number variations, but still cannot detect complex structural variations of thalassemia gene clusters such as large fragment heterozygous deletions

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