Identification of the insertion site of transgenic DNA based on cyclization of the target gene with the flanking sequence and nested inverse PCR

Wang, Jiayu; Bi, Xuetong; Chen, Wei; Zhao, Qinyue; Yang, Jinqi; Tong, Xiangjun; Zhao, Meiping

doi:10.1016/j.talo.2021.100033

Cited by 3 publications

(3 citation statements)

References 33 publications

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“…These researchers obtained the full-length DNA sequence of TaCKX1 by cloning the TaCKX1 fragment from a conserved sequence of wheat cytokinin oxidase/dehydrogenase (CKX). Wang et al (2021) used reverse PCR to amplify and detect the FSTA gene in transgenic zebrafish genomic DNA. This method may be used for gene doping detection in blood samples or to assess the safety of gene therapy and GMOs.…”

Section: Reverse Pcrmentioning

confidence: 99%

Application of transposon insertion site sequencing method in the exploration of gene function in microalgae

Fan

Mao

et al. 2023

Front. Microbiol.

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Microalgae are a large group of organisms that can produce various useful substances through photosynthesis. Microalgae need to be genetically modified at the molecular level to become “Chassis Cells” for food, medicine, energy, and environmental protection and, consequently, obtain benefits from microalgae resources. Insertional mutagenesis of microalgae using transposons is a practical possibility for understanding the function of microalgae genes. Theoretical and technical support is provided in this manuscript for applying transposons to microalgae gene function by summarizing the sequencing method of transposon insertion sites.

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Section: Reverse Pcrmentioning

confidence: 99%

Application of transposon insertion site sequencing method in the exploration of gene function in microalgae

Fan

Mao

et al. 2023

Front. Microbiol.

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“…The genomic DNA library-based walking technique is unpopular owing to the heavy workload and high cost. PCR-based approaches have been favored because placed on both ends of this unknown hybrid (Triglia et al 1988;Wang et al 2021). This cyclized DNA serves as a template for PCR using two sequence-specific primers (SSPs) having opposite orientations.…”

Section: Introductionmentioning

confidence: 99%

“…Inverse PCR requires the endonuclease digestion of genomic DNA and the subsequent self-cyclization of the digested DNA. It thus produces cyclized DNA in which unidentified upstream and downstream regions are situated adjacent to each other, with the known DNA being placed on both ends of this unknown hybrid (Triglia et al 1988 ; Wang et al 2021 ). This cyclized DNA serves as a template for PCR using two sequence-specific primers (SSPs) having opposite orientations.…”

Section: Introductionmentioning

confidence: 99%

DAR-PCR: a new tool for efficient retrieval of unknown flanking genomic DNA

Sun

Jia²,

Wang³

et al. 2022

AMB Expr

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Various PCR-based genome-walking methods have been developed to acquire unknown flanking DNA sequences. However, the specificity and efficacy levels, and the operational processes, of the available methods are unsatisfactory. This work proposes a novel walking approach, termed differential annealing-mediated racket PCR (DAR-PCR). The key to DAR-PCR is the use of primer-mediated intra-strand annealing (ISA). An ISA primer consists of a 5’ root homologous to the known sequence and a heterologous 3’ bud. In the single low-stringency cycle, the ISA primer anneals to a site on an unknown region and extends towards the sequence-specific primer (SSP) 1 site, thereby forming a target single-stranded DNA bound by the SSP1 complement and the ISA primer. In the subsequent more stringent cycles, its complementary strand is accumulated, owing to the differential annealing between the moderate-stringency ISA primer and the high-stringency SSP1. The accumulation of this strand provides an opportunity for ISA mediated by the ISA primer root. A loop-back extension subsequent to ISA occurs, creating a racket-like DNA with the known region positioned at both ends of the unknown sequence. This DNA is exponentially amplified during the secondary PCR driven by an SSP pair inner to SSP1. DAR-PCR was validated as an efficient walking method by determining unknown flanking sequences in Lactobacillus brevis and Oryza sativa.

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Gene doping detection in the era of genomics

Li,

Su,

Shi

et al. 2024

Drug Testing and Analysis

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Recent progress in gene editing has enabled development of gene therapies for many genetic diseases, but also made gene doping an emerging risk in sports and competitions. By delivery of exogenous transgenes into human body, gene doping not only challenges competition fairness but also places health risk on athletes. World Anti‐Doping Agency (WADA) has clearly inhibited the use of gene and cell doping in sports, and many techniques have been developed for gene doping detection. In this review, we will summarize the main tools for gene doping detection at present, highlight the main challenges for current tools, and elaborate future utilizations of high‐throughput sequencing for unbiased, sensitive, economic and large‐scale gene doping detections. Quantitative real‐time PCR assays are the widely used detection methods at present, which are useful for detection of known targets but are vulnerable to codon optimization at exon–exon junction sites of the transgenes. High‐throughput sequencing has become a powerful tool for various applications in life and health research, and the era of genomics has made it possible for sensitive and large‐scale gene doping detections. Non‐biased genomic profiling could efficiently detect new doping targets, and low‐input genomics amplification and long‐read third‐generation sequencing also have application potentials for more efficient and straightforward gene doping detection. By closely monitoring scientific advancements in gene editing and sport genetics, high‐throughput sequencing could play a more and more important role in gene detection and hopefully contribute to doping‐free sports in the future.

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Identification of the insertion site of transgenic DNA based on cyclization of the target gene with the flanking sequence and nested inverse PCR

Cited by 3 publications

References 33 publications

Application of transposon insertion site sequencing method in the exploration of gene function in microalgae

Application of transposon insertion site sequencing method in the exploration of gene function in microalgae

DAR-PCR: a new tool for efficient retrieval of unknown flanking genomic DNA

Gene doping detection in the era of genomics

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