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      Fusion primer driven racket PCR: A novel tool for genome walking

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          Abstract

          The limitations of the current genome-walking strategies include strong background and cumbersome experimental processes. Herein, we report a genome-walking method, fusion primer-driven racket PCR (FPR-PCR), for the reliable retrieval of unknown flanking DNA sequences. Four sequence-specific primers (SSP1, SSP2, SSP3, and SSP4) were sequentially selected from known DNA (5'→3′) to perform FPR-PCR. SSP3 is the fragment that mediates intra-strand annealing (FISA). The FISA fragment is attached to the 5′ end of SSP1, generating a fusion primer. FPR-PCR comprises two rounds of amplification reactions. The single-fusion primary FPR-PCR begins with the selective synthesis of the target first strand, then allows the primer to partially anneal to some place(s) on the unknown region of this strand, producing the target second strand. Afterward, a new first strand is synthesized using the second strand as the template. The 3′ end of this new first strand undergoes intra-strand annealing to the FISA site, followed by the formation of a racket-like DNA by a loop-back extension. This racket-like DNA is exponentially amplified in the secondary FPR-PCR performed using SSP2 and SSP4. We validated this FPR-PCR method by identifying the unknown flanks of Lactobacillus brevis CD0817 glutamic acid decarboxylase genes and the rice hygromycin gene.

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          Most cited references35

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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 procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences.

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              Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking.

              Isolation of DNA segments adjacent to known sequences is a tedious task in genome-related research. We have developed an efficient PCR strategy that overcomes the shortcomings of existing methods and can be automated. This strategy, thermal asymmetric interlaced (TAIL)-PCR, utilizes nested sequence-specific primers together with a shorter arbitrary degenerate primer so that the relative amplification efficiencies of specific and nonspecific products can be thermally controlled. One low-stringency PCR cycle is carried out to create annealing site(s) adapted for the arbitrary primer within the unknown target sequence bordering the known segment. This sequence is then preferentially and geometrically amplified over nontarget ones by interspersion of high-stringency PCR cycles with reduced-stringency PCR cycles. We have exploited the efficiency of this method to expedite amplification and sequencing of insert end segments from P1 and YAC clones for chromosome walking. In this study we present protocols that are amenable to automation of amplification and sequencing of insert end sequences directly from cells of P1 and YAC clones.
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                Author and article information

                Contributors
                Journal
                Front Genet
                Front Genet
                Front. Genet.
                Frontiers in Genetics
                Frontiers Media S.A.
                1664-8021
                18 October 2022
                2022
                : 13
                : 969840
                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 Key Laboratory of Poyang Lake Environment and Resource Utilization of Ministry of Education , School of Chemistry and Chemical Engineering , Nanchang University , Nanchang, China
                Author notes

                Edited by: Youri I. Pavlov, University of Nebraska Medical Center, United States

                Reviewed by: Birgit Henrich, Heinrich Heine University of Düsseldorf, Germany

                Kazutoshi Okuno, University of Tsukuba, Japan

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

                This article was submitted to Genomic Assay Technology, a section of the journal Frontiers in Genetics

                Article
                969840
                10.3389/fgene.2022.969840
                9623105
                36330444
                50e5e93b-ebf5-4533-9a9d-8b030a01133c
                Copyright © 2022 Pei, Sun, Wang, Pan, Guo 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
                : 16 June 2022
                : 26 September 2022
                : 26 September 2022
                Categories
                Genetics
                Methods

                Genetics
                fusion primer,walking primer,partial annealing,racket-like dna,genome-walking
                Genetics
                fusion primer, walking primer, partial annealing, racket-like dna, genome-walking

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