N7-Ended Walker PCR: An Efficient Genome-Walking Tool.

As a tool for acquiring uncharacterized genomic DNA adjacent to characterized DNA, genome-walking is integral to bioscience-related research works. Herein, a new genome-walking tool known as N7-ended walker PCR (polymerase chain reaction) is presented. The key aspect for this method is the use of a degenerate walker primer in secondary/tertiary PCR. The 7 nt 5' tail of this primer completely degenerates to N7 relative to its corresponding primary walker primer. The degeneracy reduces the efficiency of annealing this primer to its predecessor site. Clearly, primary nontarget DNA defined by the primary walker primer prefers to form a hairpin structure via the inverted ends rather than hybridizing with the degenerate primer. As a result, N7-ended walker PCR achieves genome-walking by selectively boosting the DNA of interest. The feasibility of the N7-ended walker PCR method was proven by acquiring uncharacterized DNAs flanking several characterized DNAs.

Rice transcription factor OsWRKY37 positively regulates flowering time and grain fertility under copper deficiency.

Efficient uptake, translocation, and distribution of Cu to rice (Oryza sativa) spikelets is crucial for flowering and yield production. However, the regulatory factors involved in this process remain unidentified. In this study, we isolated a WRKY transcription factor gene induced by Cu deficiency, OsWRKY37, and characterized its regulatory role in Cu uptake and transport in rice. OsWRKY37 was highly expressed in rice roots, nodes, leaf vascular bundles, and anthers. Overexpression of OsWRKY37 promoted the uptake and root-to-shoot translocation of Cu in rice under -Cu condition but not under +Cu condition. While mutation of OsWRKY37 significantly decreased Cu concentrations in the stamen, the root-to-shoot translocation and distribution ratio in brown rice affected pollen development, delayed flowering time, decreased fertility, and reduced grain yield under -Cu condition. yeast one-hybrid, transient co-expression and EMSAs, together with in situ RT-PCR and RT-qPCR analysis, showed that OsWRKY37 could directly bind to the upstream promoter region of OsCOPT6 (copper transporter) and OsYSL16 (yellow stripe-like protein) and positively activate their expression levels. Analyses of oscopt6 mutants further validated its important role in Cu uptake in rice. Our study demonstrated that OsWRKY37 acts as a positive regulator involved in the uptake, root-to-shoot translocation, and distribution of Cu through activating the expression of OsCOPT6 and OsYSL16, which is important for pollen development, flowering, fertility, and grain yield in rice under Cu deficient conditions. Our results provide a genetic strategy for improving rice yield under Cu deficient condition.

Single primer site-specific nested PCR for accurate and rapid genome-walking.

Genome-walking is a molecular tool used to unveil uncharacterized DNA regions flanking a known DNA, which has been widely used in bioscience and related areas. This study developed a reliable and efficient PCR-based genome-walking approach, named as single primer site-specific nested PCR (SPN-PCR). A SPN-PCR set sequentially consists of three single-primer nested PCR amplifications. The primary relaxed thermal cycle promotes outmost nested site-specific primer (NSSP) to partially combine with numerous places on DNA template, synthesizing many single-stranded DNAs (ssDNA). Among them, the target ssDNA is exponentially amplified in the subsequent stringent cycles, as its 3' part possesses the outmost NSSP complement; but a non-target ssDNA cannot be amplified, because it does not possess such a complement. Stringent secondary and tertiary PCRs also exclusively enrich this target DNA. Finally, the target DNA product becomes predominant. The feasibility of SPN-PCR was validated by genome-walking several selected genes from two divergent species.

Primer extension refractory PCR: an efficient and reliable genome walking method.

Genome walking, a molecular technique for obtaining unknown flanking genomic sequences from a known genomic sequence, has been broadly applied to determine transgenic sites, mine new genetic resources, and fill in chromosomal gaps. This technique has advanced genomics, genetics, and related disciplines. Here, an efficient and reliable genome walking technique, called primer extension refractory PCR (PER-PCR), is presented. PER-PCR uses a set of primary, secondary, and tertiary walking primers. The middle 15 nt of the primary walking primer overlaps with the 3' parts of the secondary and tertiary primers. The 5' parts of the three primers are heterologous to each other. The short overlap allows the walking primer to anneal to its predecessor only in a relaxed-stringency PCR cycle, resulting in a series of single-stranded DNAs; however, the heterologous 5' part prevents the creation of a perfect binding site for the walking primer. In the next stringent cycle, the target single strand can be extended into a double-stranded DNA molecule by the sequence-specific primer and thus can be exponentially amplified by the remaining stringent cycles. The nontarget single strand fails to be enriched due to the lack of a perfect binding site for any primer. PER-PCR was validated by extension into unknown flanking regions of the hyg gene in rice and the gadR gene in Levilactobacillus brevis CD0817. In summary, in this study, a new practical PER-PCR method was constructed as a potential alternative to existing genome walking methods.

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

Here, we describe a protocol based on semi-site-specific primer PCR (3SP-PCR) to access unknown flanking DNA sequences. We specify the guidelines for designing primers for 3SP-PCR. We also describe experimental procedures for the 3SP-PCR, along with PCR product purification and subsequent sequencing and analysis. For complete details on the use and execution of this protocol, please refer to Wei et al.1.

Robust binding between secondary amines and Au electrodes.

While primary amines are one of the most widely used linker groups for forming single-molecule junctions, it remains elusive whether and how the substitution of one hydrogen in a primary amine with a methyl group (secondary amine) can alter its functional properties as a linker group. Here we show that a robust binding between a secondary amine and an Au electrode is absent with the use of a non-coated Au tip and is achieved when in contact with a wax-coated Au tip, which we propose is catalyzed by the more frequent formation of Au adatoms in measurements with a wax-coated tip.

Fork PCR: a universal and efficient genome-walking tool.

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.

Small molecule linoleic acid inhibiting whey syneresis via interact with milk proteins in the fermentation of set yogurt fortified with c9,t11-conjugated linoleic acid.

The study aimed to investigate the impact of fermentation conditions on c9,t11-conjugated linoleic acid (CLA) synthesis by Lactobacillus casei, as well as its effects on whey syneresis, water holding capacity (WHC), and texture characteristics of set yogurt. The amount of whey syneresis decreased about 30% with the adding of 0.1% linoleic acid (LA). The interaction between LA and casein (CS), ß-lactoglobulin (ß-Lg) and bovine serum albumin (BSA) was observed by UV-Vis absorption spectroscopy, 3D fluorescence spectroscopy and CD spectroscopy. It found that LA changed the microenvironment and polarity around amino acids, as well as the conformation of the three milk proteins. Scanning electron microscope (SEM) analysis revealed that the addition of LA resulted in a more uniform and compact microstructure of the set yogurt. It indicates that LA can promote the crosslink of milk proteins, which may be the reason for the reduction of whey syneresis in set yogurt.

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

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.

Extraction, structural characterization, and antioxidant activity of polysaccharides derived from Arctium lappa L.

Introduction: Arctium lappa L. root has high nutritional and medicinal values and has been identified as a healthy food raw material by the Ministry of Health of the People's Republic of China. Methods: In the present study, an aqueous two-phase system (ATPS) of polyethylene glycol (PEG)-(NH4)2SO4 was used to extract Arctium lappa L. polysaccharides (ALPs) from the Arctium lappa L. roots, the optimal extraction conditions of crude ALPs were optimized by using the single-factor experiment and response surface methodology. The structure and composition of ALPs were determined by fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and high-performance liquid chromatography (HPLC). At the same time, the antioxidant activity of ALPs was investigated by in vitro antioxidant experiment. Results: The optimized extraction parameters for extraction ALPs were as follows: the PEG relative molecular weight of 6,000, a quality fraction of PEG 25%, a quality fraction of (NH4)2SO4 18%, and an extraction temperature of 80°C. Under these conditions, the extraction rate of ALPs could reach 28.83%. FTIR, SEM and HPLC results showed that ALPs were typical acidic heteropolysaccharides and had uneven particle size distribution, an irregular shape, and a rough surface. The ALPs were chiefly composed of glucose, rhamnose, arabinose, and galactose with a molar ratio of 70.19:10.95:11.16:6.90. In addition, the ALPs had intense antioxidant activity in vitro with IC50 values in the ·OH radical (1.732 mg/ml), DPPH radical (0.29 mg/ml), and superoxide anion (0.15 mg/ml) scavenging abilities. Discussion: The results showed that ATPS was an efficient method to extract polysaccharides and could be used for the extraction of other polysaccharides. These results indicated that ALPs had great prospects as a functional food and could be exploited in multiple fields.

Sensitivity Intensified Ninhydrin-Based Chromogenic System by Ethanol-Ethyl Acetate: Application to Relative Quantitation of GABA.

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.

Contributions of Support Point Number to Mirror Assembly Thermal Sensitivity Control.

Due to the extreme environmental temperature variations, solutions that enable ultra-low thermal sensitivity in a mirror assembly are crucial for high-performance aerial optical imaging sensors (AOIS). Strategies such as the elimination of the coefficient of thermal expansion (CTE) mismatch and the employment of a flexure connection at the interface cannot be simply duplicated for the application involved, demanding specific design constraints. The contributions of support point number to the surface thermal sensitivity reduction and support stiffness improvement have been studied. A synthetic six-point support system that integrates equally spaced multiple ultra-low radial stiffness mirror flexure units and assembly external interface flexure units has been demonstrated on a 260 mm apertured annular mirror that involves significant CTE mismatch and demanding support stiffness constraint. The surface deformation RMS, due to the 35 °C temperature variation, is 16.7 nm.

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

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.

Bridging PCR: An Efficient and Reliable Scheme Implemented for Genome-Walking.

The efficacy of the available genome-walking methods is restricted by low specificity, high background, or composite operations. We herein conceived bridging PCR, an efficient genome-walking approach. Three primers with random sequences, inner walker primer (IWP), bridging primer (BP), and outer walker primer (OWP), are involved in bridging PCR. The BP is fabricated by splicing OWP to the 5'-end of IWP's 5'-part. A bridging PCR set is constituted by three rounds of amplification reactions, sequentially performed by IWP, BP plus OWP, and OWP, respectively pairing with three nested sequence-specific primers (SSP). A non-target product arising from IWP alone undergoes end-lengthening mediated by BP. This modified non-target product is a preferentially formed hairpin between the lengthened ends, instead of binding with shorter OWP. Meanwhile, a non-target product, triggered by SSP alone or SSP plus IWP, is removed by nested SSP. As a result, only the target DNA is accumulated. The efficacy of bridging PCR was validated by walking the gadA/R genes of Levilactobacillus brevis CD0817 and the hyg gene of rice.

Semi-Site-Specific Primer PCR: A Simple but Reliable Genome-Walking Tool.

Genome-walking has been frequently applied to molecular biology and related areas. Herein, a simple but reliable genome-walking technique, termed semi-site-specific primer PCR (3SP-PCR), is presented. The key to 3SP-PCR is the use of a semi-site-specific primer in secondary PCR that partially overlaps its corresponding primary site-specific primer. A 3SP-PCR set comprises two rounds of nested amplification reactions. In each round of reaction, any primer is allowed to partially anneal to the DNA template once only in the single relaxed-stringency cycle, creating a pool of single-stranded DNAs. The target single-stranded DNA can be converted into a double-stranded molecule directed by the site-specific primer, and thus can be exponentially amplified by the subsequent high-stringency cycles. The non-target one cannot be converted into a double-strand due to the lack of a perfect binding site to any primer, and thus fails to be amplified. We validated the 3SP-PCR method by using it to probe the unknown DNA regions of rice hygromycin genes and Levilactobacillus brevis CD0817 glutamic acid decarboxylase genes.

Multiple biological activities and biosynthesis mechanisms of specific conjugated linoleic acid isomers and analytical methods for prospective application.

Conjugated linoleic acid (CLA) is a collective term for the octadecadienoic acid isomers containing conjugated double bonds. This article reviewed CLA isomers from biological activities, biosynthesis mechanisms and analytical methods. The biological activities of CLA isomers in anti-obesity, cardiovascular protection, diabetes management and anti-cancer in vitro and in vivo were mainly reviewed. More attention has been paid to the production of the specific CLA isomer due to its biological activity. The biosynthesis methods of CLA isomers, such as dietary modification in ruminants and fermentation by microorganisms & enzymes, were systematically introduced. A rapid, accurate and economic analysis method will promote the research in both biological activities and biosynthesis mechanisms of CLA isomers. The merits of UV spectrometry, GC, HPLC, MS and CE used in the analysis of CLA isomers were also compared in detail. This paper aims to put into perspective the current status and future trends on CLA isomers.

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

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.

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

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.

Quantitative determination of environmental factors governing the snow melting: a geodetector case study in the central Tienshan Mountains.

Because of the distinctive vertical climate and topography gradients in the alpine region, the snow cover of the Tienshan Mountains possesses complex spatiotemporal heterogeneity, particularly during the melting process. Quantifying the environmental factors is therefore crucial to understanding the melting process and for predicting and managing snowmelt runoff. Herein, the snow cover area, grain size, and contamination extent were determined to characterize the detailed melting status based on surface reflectance data of MOD09A1 in the central Tienshan Mountains from 2013 to 2017. The environmental factors collected include relief (elevation, slope, and aspect); meteorology (surface air temperature, land surface temperature, solar radiation, and wind speed); and land surface vegetation. Analysis of the geodetector results indicated the following. (1) Patterns of changes in the overall dominant environmental variables were consistent for the pre-, mid-, and post-melting periods defined according to the decline of snow cover area over five years. (2) The overall major environmental factors were wind speed and radiation (pre-period), land surface temperature and elevation (mid-period), and elevation and land surface types (post-period), respectively. (3) Regional distinctions were detected of the dominant environmental factors. In the pre-melting period, the effects of solar radiation and wind speed were noticeable in the north and south regions, respectively. The effects of elevation, land surface temperature, and land cover types became more prominent in all regions during the mid- and post-melting periods. (4) Interaction between the major environmental factors was significantly enhanced on both the overall and regional scales, thus affecting the snow-melting process. Finally, the energy distribution mismatch resulted in the snowmelt. Multiple environmental factors substantially affect heat redistribution at different spatiotemporal scales, resulting in the snowmelt as a complex manifestation of the factors and their interactions. The findings highlight regional differences in various environmental factors of the melting process and offer a theoretical foundation for the melting process at various scales over multiple years.

Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy.

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.