A high-throughput, versatile typing technique for multiple types of molecular markers

Abstract

The invention discloses a high-throughput typing technique universal to various molecular markers and belongs to the field of molecular biology. By the use of hybrid template production of 5' terminal biotin labeling, a target area having single-nucleotide polymorphism, simple sequence repeat, insertion deletion, copy number variation and the like and containing various molecular markers and target areas such as exon and species specific single-copy areas can be subjected to capturing and gene typing; meanwhile, allele quantitative typing and precise quantitative detecting are both considered; site selection flexibility of the solution hybridization is combined with the advantages of the high-throughput sequencing technique, such as high throughput and low cost. Compared with the use of the existing molecular marker typing technique, the use of the high-throughput typing technique solves the problems such that the existing fixed-point molecular marker typing technique is available for only typing single molecular markers and unavailable for typing various markers at the same time or precisely and quantitatively detecting alleles, and production of random markers or unlabeled hybrid templates is limited.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a high-throughput, multi-molecular marker general typing technique: HD-Marker (High-throughput Diverse Marker genotyping). Background technique [0002] Molecular markers are polymorphic markers of deoxyribonucleotide (DNA) molecules caused by base insertion, deletion, recombination, mutation, etc. Polymorphism, SNP), simple sequence repeats (Simple Sequence Repeats, SSR), insertion and deletion (Insertion and deletion, Indel), copy number variation (Copy number variation, CNV), species-specific single-copy regions, etc. These molecular markers have excellent characteristics such as large number, comprehensive genome coverage, high polymorphism, and rich genetic information content. field is widely used. The use of typing technology to perform allelic typing, copy number variation, and quantitative detection of species abundance in experimental samples of candidate ...

AI Technical Summary

Problems solved by technology

Most of the current technologies use the random biotin hybridization template labeling method. Biotin has a large steric hindrance. For longer target fragments, it is difficult to achieve simultaneous hybridization of upstream and downstream probes, so that it is impossible to achieve longer target fragments. capture

In order to avoid this problem, some technologies directly use unlabeled DNA as a hybridization template, but the new problem is that the recovery efficiency in the enrichment of the target fragment is not high, and the follow-up needs to increase the polymerase chain reaction (Polymerase Chain Reaction, PCR ) The number of amplification cycles to get enough library samples affects the uniformity of capture sites

Therefore, most of the existing technologies fix the length range of the capture region, and can only type a single type of molecular markers, such as single nucleotide polymorphism sites or microsatellite sites, and cannot realize the detection of multiple molecular markers by one technology. Universal typing, which makes researchers have to try multiple typing systems when trying to analyze multiple types of molecular markers in the same experimental sample, which increases experimental costs and time, and seriously affects typing efficiency and research progress

More importantly, due to the influence of hybridization efficiency and capture uniformity, the existing candidate site capture typing technology can only perform qualitative detection of allele types of polymorphic sites, and cannot achieve such as copy number variation. Quantitative analysis of copy number and specific single-copy regions of different species in samples limits the application of candidate site capture technology in copy number variation detection, species identification and metagenomic species abundance analysis

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