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

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          Abstract

          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.

          Supplementary Information

          The online version contains supplementary material available at 10.1186/s13568-022-01471-1.

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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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            High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences.

            Isolation of unknown DNA sequences flanked by known sequences is an important task in molecular biology research. Thermal asymmetric interlaced PCR (TAIL-PCR) is an effective method for this purpose. However the success rate of the original TAIL-PCR needs to be increased, and it is more desirable to obtain target products with larger sizes. Here we present a substantially improved TAIL-PCR procedure with special primer design and optimized thermal conditions. This high-efficiency TAIL-PCR (hiTAIL-PCR) combines the advantages of the TAIL-cycling and suppression-PCR, thus it can block the amplification of nontarget products and suppress small target ones, but allow efficient amplification of large target sequences. Using this method, we isolated genomic flanking sequences of T-DNA insertions from transgenic rice lines. In our tests, the success rates of the reactions were higher than 90%, and in most cases the obtained major products had sizes of 1-3 kb.
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              An improved PCR method for walking in uncloned genomic DNA.

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                Author and article information

                Contributors
                hxli@ncu.edu.cn
                Journal
                AMB Express
                AMB Express
                AMB Express
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                2191-0855
                12 October 2022
                12 October 2022
                2022
                : 12
                : 131
                Affiliations
                [1 ]GRID grid.260463.5, ISNI 0000 0001 2182 8825, Key Laboratory of Poyang Lake Environment and Resource Utilization of Ministry of Education, School of Environmental and Chemical Engineering, , Nanchang University, ; 330031 Nanchang, PR China
                [2 ]GRID grid.260463.5, ISNI 0000 0001 2182 8825, State Key Laboratory of Food Science and Technology, , Nanchang University, ; 330047 Nanchang, PR China
                [3 ]GRID grid.260463.5, ISNI 0000 0001 2182 8825, Sino-German Joint Research Institute, , Nanchang University, ; 330047 Nanchang, PR China
                [4 ]GRID grid.186587.5, ISNI 0000 0001 0722 3678, Charles W. Davidson College of Engineering, San Jose State University, ; 95192 San Jose, CA USA
                Article
                1471
                10.1186/s13568-022-01471-1
                9556680
                36224448
                618d21fe-32c5-4aec-bd18-6aa751b48b61
                © The Author(s) 2022

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 10 February 2022
                : 22 September 2022
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 32160014
                Award ID: 31570070
                Award Recipient :
                Categories
                Original Article
                Custom metadata
                © The Author(s) 2022

                Biotechnology
                genome walking,differential annealing,intra-strand annealing,racket-like dna,primer root,primer bud

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