Search

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.

Summary: Here we describe a protocol for wristwatch PCR, an approach based on wristwatch-like structure formed between walking primers to obtain unknown flanks. We specify the criteria for designing wristwatch primers and gene-specific primers. We detail how to set wristwatch primer permutations to obtain personalized walking outcomes and improve walking efficiency. We describe experimental procedures for isolating a DNA of interest using three rounds of nested wristwatch PCR as well as the subsequent steps for DNA purification, cloning, and sequencing.For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.

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.

Gamma-aminobutyric acid (GABA) is a functional metabolite in various organisms. Herein, a sensitivity intensified ninhydrin-based chromogenic system (SINICS), achieved by ethanol and ethyl acetate, is described for the reliable relative quantitation of GABA. A 2.9 mL SINICS kit comprises 1% ninhydrin, 40% ethanol, 25% ethyl acetate, and 35 μL 0.2 M sodium acetate buffer (pH 5.0). In practice, following the addition of a 0.1 mL sample to the kit, the chromogenic reaction is completed by heating at 70 °C for 30 min. The kit increased the color development sensitivity of L-glutamic acid and GABA, with the detection limits being reduced from 20 mM and 200 mM to 5 mM and 20 mM, respectively. The chromophore was stable for at least 2 h at room temperature, which was sufficient for a routine colorimetric analysis. The absorbance at 570 nm with the deduction of background directly represents the content of amino acid. For a proof-of-concept, the SINICS was adopted to optimize the GABA fermentation process of Levilactobacillus brevis CD0817. The results demonstrated that SINICS is an attractive alternative to the available ninhydrin-based colorimetric methods.

There is a recent trend of using lactic acid bacteria for the production of gamma-aminobutyric acid (GABA). This study described a method that combines fermentation and self-buffered whole-cell catalysis for the efficient production of GABA using Levilactobacillus brevis CD0817. Upon the completion of GABA fermentation, cells were recovered to conduct whole-cell catalysis by which the substrate L-glutamic acid was catalytically decarboxylated to GABA. L-glutamic acid itself maintained the acidity essential for decarboxylation. To maximize the whole-cell catalysis ability, the effects of the cell culture method, catalysis temperature, catalysis time, cell concentration, and L-glutamic acid dosage were investigated. The results illustrate that the cells that were cultivated for 16 h in a fermentation medium supplemented with 20.0 g/L of glucose were the most suitable for the whole-cell catalytic production of GABA. At 16 h, the fermentative GABA content reached 204.2 g/L. Under optimized whole-cell catalytic conditions (temperature 45.0 °C, time 12.0 h, wet cells 25.0 g/L, and L-glutamic acid 120.0 g/L), 85.1 g/L of GABA was obtained, with 3.7 ± 0.9 g/L of substrate residue. GABA was recovered from the system by sequentially performing rotary vacuum evaporation, precipitation with ethanol, filtration with filter paper, and drying. The purity of the GABA product reached 97.1%, with a recovery rate of 87.0%. These data suggest that the proposed method has potential applications in the production of GABA.

Gamma-aminobutyric acid (GABA) plays a role in several physiological functions. GABA production by lactic acid bacteria has attracted considerable interest; however, there is need to improve production. This study aimed to develop a pH auto-sustain (PAS)-based GABA fermentation process for Lactobacillus brevis CD0817, with L-glutamic acid (solubility ~6.0 g/L and isoelectric point 3.22) as the substrate. Firstly, we determined the optimum levels of vital factors affecting GABA synthesis using Erlenmeyer flask experiments. The results showed that optimal levels of sugar, yeast extract, Tween-80, manganese ion, and temperature were 5.0 g/L, 35.0 g/L, 1.0 g/L, 16.0 mg/L, and 30.0 °C, respectively. The added L-glutamic acid (650 g per liter of medium) mostly existed in the form of solid powder was slowly released to supply the substrate and acidity essential for GABA production with the progress of fermentation. Based on the optimizations, the PAS-based GABA fermentation was performed using a 10 L fermenter. The PAS-based strategy promoted GABA synthesis by the strain of up to 321.9 ± 6.7 g/L after 48 h, with a productivity of 6.71 g/L/h and a substrate molar conversion rate of 99.6%. The findings suggest that the PAS-based fermentation is a promising method for GABA production by lactic acid bacteria.