Protocol to access unknown flanking DNA sequences using Wristwatch-PCR for genome-walking

Wang, Lingqin; Jia, Mengya; Li, Zhaoqin; Liu, Xiaohua; Sun, Tianyi; Pei, Jinfeng; Wei, Cheng; Lin, Zhiyu; Li, Haixing

doi:10.1016/j.xpro.2022.102037

Cited by 6 publications

(6 citation statements)

References 18 publications

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“…It is expected that at least one fork PCR set can generate a positive walking outcome if parallel ones are performed. Based on the walking rationale, the success rate of fork PCR is anticipated to be equal to that of wristwatch PCR ( Wang et al, 2022 , 2023 ).…”

Section: Discussionmentioning

confidence: 99%

“…The aforementioned specificity guarantee design for fork PCR rationalizes the unnecessity of tertiary amplification. This feature facilitates fork PCR rapidly acquiring walking data ( Wang et al, 2022 , 2023 ). In this regard, fork PCR method surpasses most the existing methods that have to proceed three rounds of amplifications.…”

Section: Discussionmentioning

confidence: 99%

“…The latter type is completely free of genomic template pretreatment preceding PCR. As a result, random priming-based PCR has occupied a major position in genome-walking ( Chang et al, 2018 ; Wang et al, 2023 ).…”

Section: Introductionmentioning

confidence: 99%

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Fork PCR: a universal and efficient genome-walking tool

Pan,

Guo,

Pan

et al. 2023

Front. Microbiol.

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The reported genome-walking methods still suffer from some deficiencies, such as cumbersome experimental steps, short target amplicon, or deep background. Here, a simple and practical fork PCR was proposed for genome-walking. The fork PCR employs a fork primer set of three random oligomers to implement walking task. In primary fork PCR, the low-stringency amplification cycle mediates the random binding of primary fork primer to some places on genome, producing a batch of single-stranded DNAs. In the subsequent high-stringency amplification, the target single-strand is processed into double-strand by the site-specific primer, but a non-target single-stranded DNA cannot be processed by any primer. As a result, only the target DNA can be exponentially amplified in the remaining high-stringency cycles. Secondary/tertiary nested fork PCR(s) further magnifies the amplification difference between the both DNAs by selectively enriching target DNA. The applicability of fork PCR was validated by walking several gene loci. The fork PCR could be a perspective substitution for the existing genome-walking schemes.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Fork PCR: a universal and efficient genome-walking tool

Pan,

Guo,

Pan

et al. 2023

Front. Microbiol.

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“… 2 , 3 , 4 , 5 , 6 , 7 , 8 Recently, we have devised Wristwatch PCR for genome-walking. 9 The Wristwatch PCR involves a set of three random walking primers that form a wristwatch-like structure with each other. Although this wristwatch-like structure helps selectively enrich target molecules, it may reduce the personalized random annealing of walking primers.…”

Section: Before You Beginmentioning

confidence: 99%

Protocol to retrieve unknown flanking DNA sequences using semi-site-specific PCR-based genome walking

Chen,

Wei,

Lin

et al. 2024

STAR Protocols

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“…Many target strains have been isolated using these methods [ 15 , 16 , 17 , 18 ]. However, DNA-sequence-based methods are attractive methods in this field [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ].…”

Section: Introductionmentioning

confidence: 99%

Sodium-Ion-Free Fermentative Production of GABA with Levilactobacillus brevis CD0817

Pei

Wei

et al. 2023

Metabolites

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Gamma-aminobutyric acid (GABA) has positive effects on many physiological processes. Lactic acid bacterial production of GABA is a future trend. This study aimed to produce a sodium-ion-free GABA fermentation process for Levilactobacillus brevis CD0817. In this fermentation, both the seed and fermentation media used L-glutamic acid instead of monosodium L-glutamate as the substrate. We optimized the key factors influencing GABA formation, adopting Erlenmeyer flask fermentation. The optimized values of the key factors of glucose, yeast extract, Tween 80, manganese ion, and fermentation temperature were 10 g/L, 35 g/L, 1.5 g/L, 0.2 mM, and 30 °C, respectively. Based on the optimized data, a sodium-ion-free GABA fermentation process was developed using a 10-L fermenter. During the fermentation, L-glutamic acid powder was continuously dissolved to supply substrate and to provide the acidic environment essential for GABA synthesis. The current bioprocess accumulated GABA at up to 331 ± 8.3 g/L after 48 h. The productivity of GABA was 6.9 g/L/h and the molar conversion rate of the substrate was 98.1%. These findings demonstrate that the proposed method is promising in the fermentative preparation of GABA by lactic acid bacteria.

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Protocol to access unknown flanking DNA sequences using Wristwatch-PCR for genome-walking

Cited by 6 publications

References 18 publications

Fork PCR: a universal and efficient genome-walking tool

Fork PCR: a universal and efficient genome-walking tool

Protocol to retrieve unknown flanking DNA sequences using semi-site-specific PCR-based genome walking

Sodium-Ion-Free Fermentative Production of GABA with Levilactobacillus brevis CD0817

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