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      Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy

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          Abstract

          Genome walking is a method used to retrieve unknown flanking DNA. Here, we reported wristwatch (WW) PCR, an efficient genome walking technique mediated by WW primers (WWPs). WWPs feature 5′- and 3′-overlap and a heterologous interval. Therefore, a wristwatch-like structure can be formed between WWPs under relatively low temperatures. Each WW-PCR set is composed of three nested (primary, secondary, and tertiary) PCRs individually performed by three WWPs. The WWP is arbitrarily annealed somewhere on the genome in the one low-stringency cycle of the primary PCR, or directionally to the previous WWP site in one reduced-stringency cycle of the secondary/tertiary PCR, producing a pool of single-stranded DNAs (ssDNAs). A target ssDNA incorporates a gene-specific primer (GSP) complementary at the 3′-end and the WWP at the 5′-end and thus can be exponentially amplified in the next high-stringency cycles. Nevertheless, a non-target ssDNA cannot be amplified as it lacks a perfect binding site for any primers. The practicability of the WW-PCR was validated by successfully accessing unknown regions flanking Lactobacillus brevis CD0817 glutamate decarboxylase gene and the hygromycin gene of rice. The WW-PCR is an attractive alternative to the existing genome walking techniques.

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          Most cited references48

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          Haplotype-resolved diverse human genomes and integrated analysis of structural variation

          Long-read and strand-specific sequencing technologies together facilitate the de novo assembly of high-quality haplotype-resolved human genomes without parent–child trio data. We present 64 assembled haplotypes from 32 diverse human genomes. These highly contiguous haplotype assemblies (average contig N50: 26 Mbp) integrate all forms of genetic variation even across complex loci. We identify 107,590 structural variants (SVs), of which 68% are not discovered by short-read sequencing, and 278 SV hotspots (spanning megabases of gene-rich sequence). We characterize 130 of the most active mobile element source elements and find that 63% of all SVs arise by homology-mediated mechanisms. This resource enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1,526 expression quantitative trait loci as well as SV candidates for adaptive selection within the human population.
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            Genetic applications of an inverse polymerase chain reaction.

            A method is presented for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. The method uses the polymerase chain reaction (PCR), but it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle. This procedure of inverse PCR (IPCR) has many applications in molecular genetics, for example, the amplification and identification of sequences flanking transposable elements. In this paper we show the feasibility of IPCR by amplifying the sequences that flank an IS1 element in the genome of a natural isolate of Escherichia coli.
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              A high-throughput Arabidopsis reverse genetics system.

              A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.
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                Author and article information

                Contributors
                Journal
                Front Bioeng Biotechnol
                Front Bioeng Biotechnol
                Front. Bioeng. Biotechnol.
                Frontiers in Bioengineering and Biotechnology
                Frontiers Media S.A.
                2296-4185
                12 April 2022
                2022
                : 10
                : 792848
                Affiliations
                [1] 1 State Key Laboratory of Food Science and Technology , Nanchang University , Nanchang, China
                [2] 2 Sino-German Joint Research Institute , Nanchang University , Nanchang, China
                [3] 3 Charles W. Davidson College of Engineering , San Jose State University , San Jose, CA, United States
                [4] 4 Key Laboratory of Poyang Lake Environment and Resource Utilization , Ministry of Education , School of Environmental and Chemical Engineering , Nanchang University , Nanchang, China
                Author notes

                Edited by: Zhi-Qiang Liu, Zhejiang University of Technology, China

                Reviewed by: Nagarjun Vijay, Indian Institute of Science Education and Research, India

                Vivek Sharma, Chandigarh University, India

                *Correspondence: Haixing Li, hxli@ 123456ncu.edu.cn

                This article was submitted to Industrial Biotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology

                Article
                792848
                10.3389/fbioe.2022.792848
                9039356
                35497369
                e7cf7502-87d6-431b-a29f-b9d9e336515f
                Copyright © 2022 Wang, Jia, Li, Liu, Sun, Pei, Wei, Lin and Li.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 11 October 2021
                : 08 March 2022
                Funding
                Funded by: National Natural Science Foundation of China , doi 10.13039/501100001809;
                Award ID: 31570070
                Funded by: Jiangxi Provincial Department of Science and Technology , doi 10.13039/501100010857;
                Categories
                Bioengineering and Biotechnology
                Methods

                wristwatch primer,partially annealing,wristwatch-like dna,wristwatch pcr,genome walking

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