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taq dna ligase  (New England Biolabs)


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    New England Biolabs taq dna ligase
    a) Schematic depicting the advantages offered by High Temperature (HighT) ligation in comparison to low temperature ligation in producing exposed overhangs for assembly. b) NUPACK simulated melt curves for all 10 bp duplexes with unpaired intramolecular secondary structures at 50°C. c) Violin plot and nested box and whisker plot depicting the distribution of melting temperatures in the form of T m-n for the simulated data presented in b . The n demarcates the proportion of species bound in the duplex for a 10 nM solution. Summary statistics can be found in Source data . d ) Schematic depicting the qPCR assay used to measure ligation activity of 4 bp and 10 bp overhangs under different ligase and temperature conditions. e) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 4 bp and 10 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no <t>ligase,</t> <t>T4</t> <t>DNA</t> Ligase, or Taq DNA Ligase. f) Bar plot depicting the mean ± SD of relative ligation product for 4 bp and 10 bp overhangs at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. The starting quantity of T4 DNA Ligase at 16°C was defined as 100% and used to normalize all samples.
    Taq Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/taq+dna+ligase/Taq+DNA+Ligase/bio_rxiv__64898__2026__10__09__758000-254-46-49
    Average 97 stars, based on 1408 article reviews
    taq dna ligase - by Bioz Stars, 2026-11
    97/100 stars

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    1) Product Images from "High temperature ligation enables in vitro assembly of eukaryotic genes"

    Article Title: High temperature ligation enables in vitro assembly of eukaryotic genes

    Journal: bioRxiv

    doi: 10.64898/2026.10.09.758000

    a) Schematic depicting the advantages offered by High Temperature (HighT) ligation in comparison to low temperature ligation in producing exposed overhangs for assembly. b) NUPACK simulated melt curves for all 10 bp duplexes with unpaired intramolecular secondary structures at 50°C. c) Violin plot and nested box and whisker plot depicting the distribution of melting temperatures in the form of T m-n for the simulated data presented in b . The n demarcates the proportion of species bound in the duplex for a 10 nM solution. Summary statistics can be found in Source data . d ) Schematic depicting the qPCR assay used to measure ligation activity of 4 bp and 10 bp overhangs under different ligase and temperature conditions. e) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 4 bp and 10 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. f) Bar plot depicting the mean ± SD of relative ligation product for 4 bp and 10 bp overhangs at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. The starting quantity of T4 DNA Ligase at 16°C was defined as 100% and used to normalize all samples.
    Figure Legend Snippet: a) Schematic depicting the advantages offered by High Temperature (HighT) ligation in comparison to low temperature ligation in producing exposed overhangs for assembly. b) NUPACK simulated melt curves for all 10 bp duplexes with unpaired intramolecular secondary structures at 50°C. c) Violin plot and nested box and whisker plot depicting the distribution of melting temperatures in the form of T m-n for the simulated data presented in b . The n demarcates the proportion of species bound in the duplex for a 10 nM solution. Summary statistics can be found in Source data . d ) Schematic depicting the qPCR assay used to measure ligation activity of 4 bp and 10 bp overhangs under different ligase and temperature conditions. e) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 4 bp and 10 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. f) Bar plot depicting the mean ± SD of relative ligation product for 4 bp and 10 bp overhangs at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. The starting quantity of T4 DNA Ligase at 16°C was defined as 100% and used to normalize all samples.

    Techniques Used: Ligation, Comparison, Whisker Assay, Activity Assay, Amplification, Fluorescence, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    a) NUPACK simulated melt curves for DNA with secondary structures. b) NUPACK simulated melting curves for 4 bp and 10 bp overhangs used in the overhang ligation qPCR experiment at discrete temperatures 4°C to 85°C. c) qPCR amplification curves depicting changes in fluorescence over PCR cycles for all individual replicates of the 4 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. d) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 4 bp overhang ligation reactions at conditions 16°C with no ligase, 16°C with T4 DNA Ligase, 50°C with no ligase, and 50°C with Taq DNA Ligase. e) qPCR standard curves for 4 bp overhang ligation reactions, showing Cq as a function of log10 starting quantity across all temperatures and each temperature individually. Dashed lines indicate extrapolation beyond the standard range. f) qPCR amplification curves depicting changes in fluorescence over PCR cycles for all individual replicates of the 10 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. g) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 10 bp overhang ligation reactions at conditions 16°C with no ligase, 16°C with T4 DNA Ligase, 50°C with no ligase, and 50°C with Taq DNA Ligase. h) qPCR standard curves for 10 bp overhang ligation reactions, showing Cq as a function of log10 starting quantity across all temperatures and each temperature individually. Dashed lines indicate extrapolation beyond the standard range.
    Figure Legend Snippet: a) NUPACK simulated melt curves for DNA with secondary structures. b) NUPACK simulated melting curves for 4 bp and 10 bp overhangs used in the overhang ligation qPCR experiment at discrete temperatures 4°C to 85°C. c) qPCR amplification curves depicting changes in fluorescence over PCR cycles for all individual replicates of the 4 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. d) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 4 bp overhang ligation reactions at conditions 16°C with no ligase, 16°C with T4 DNA Ligase, 50°C with no ligase, and 50°C with Taq DNA Ligase. e) qPCR standard curves for 4 bp overhang ligation reactions, showing Cq as a function of log10 starting quantity across all temperatures and each temperature individually. Dashed lines indicate extrapolation beyond the standard range. f) qPCR amplification curves depicting changes in fluorescence over PCR cycles for all individual replicates of the 10 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. g) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 10 bp overhang ligation reactions at conditions 16°C with no ligase, 16°C with T4 DNA Ligase, 50°C with no ligase, and 50°C with Taq DNA Ligase. h) qPCR standard curves for 10 bp overhang ligation reactions, showing Cq as a function of log10 starting quantity across all temperatures and each temperature individually. Dashed lines indicate extrapolation beyond the standard range.

    Techniques Used: Ligation, Amplification, Fluorescence, Polymerase Chain Reaction

    a) Schematic depicting circular HighT assembly genomically integrated using a circular recombination system. LuxABCDE is divided into 9 fragments, which are HighT ligated to an integration backbone fragment containing two terminators, an attB sequence, and a transcriptionally inactive antibiotic resistance marker +1 ( i ). LuxABCDE is divided into 19 fragments, which are HighT ligated and amplified into an Assembly Amplicon that is subsequently assembled with the same integration backbone ( ii ). The final assembly product of both constructions ( iii ) are electroporated into recipient cells with a plasmid containing the attB integrase, antibiotic resistance marker +2 and a pre-engineered genome containing a promoter, attP site, transcriptionally active GFP, and antibiotic resistance marker +3 ( iv ). Through protein mediated DNA crossover ( v ) the recipient genome gains transcriptionally active antibiotic resistance marker +1, the cargo insert, and losses fluorescence expression in the post-integrated genome ( vi ). b) Phenotypic spot assay where pre-integration attBP circular recipient is compared to post-integration circular attBP cells on various growth media (+1 = kanamycin, +2 = carbenicillin, +3 = chloramphenicol). c) Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 10-piece scheme ( i ) Average accuracy=19.1%, Standard deviation=10.7%, Average yield=1.37 × 10 3 Lux + colonies / µg DNA, Standard deviation= 5.51 × 10 2 Lux + colonies/ µg DNA) and the 19-piece Assembly Amplicon scheme ( ii ) (Average accuracy=30.6%, Standard deviation=2.8%, Average yield=1.43 × 10 4 Lux + colonies/ µg DNA, Standard deviation=1.33× 10 3 Lux + colonies/ µg DNA). d) Schematic depicting LoxP (L1) and Lox2272 (L2) recombination sites onto distal ends of integrated cargo to enable Cre recombinase mediated genome integration. e) Phenotypic spot assay where pre-integration LoxP linear recipient is compared to post-integration linear replaced LoxP cells on various growth media (+1* = chloramphenicol, +2* = tetracycline, +3* = spectinomycin, −1* = sacB sucrose sensitivity). f) Transformation results for LoxP d i 10-piece assemblies (blue) relative to d ii 20-piece Assembly Amplicon (pink). Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 10-piece scheme ( i ) (Average accuracy=58.6%, Standard deviation=5.1%, Average yield=1.22× 10 3 Lux + colonies/ µg DNA, Standard deviation=5.61× 10 2 Lux + colonies/ µg DNA) and the 19-piece Assembly Amplicon scheme ( ii ) (Average accuracy=76.6%, Standard deviation=4.5%, Average yield=3.22× 10 3 Lux + colonies/ µg DNA, Standard deviation=2.68× 10 2 Lux + colonies/ µg DNA).
    Figure Legend Snippet: a) Schematic depicting circular HighT assembly genomically integrated using a circular recombination system. LuxABCDE is divided into 9 fragments, which are HighT ligated to an integration backbone fragment containing two terminators, an attB sequence, and a transcriptionally inactive antibiotic resistance marker +1 ( i ). LuxABCDE is divided into 19 fragments, which are HighT ligated and amplified into an Assembly Amplicon that is subsequently assembled with the same integration backbone ( ii ). The final assembly product of both constructions ( iii ) are electroporated into recipient cells with a plasmid containing the attB integrase, antibiotic resistance marker +2 and a pre-engineered genome containing a promoter, attP site, transcriptionally active GFP, and antibiotic resistance marker +3 ( iv ). Through protein mediated DNA crossover ( v ) the recipient genome gains transcriptionally active antibiotic resistance marker +1, the cargo insert, and losses fluorescence expression in the post-integrated genome ( vi ). b) Phenotypic spot assay where pre-integration attBP circular recipient is compared to post-integration circular attBP cells on various growth media (+1 = kanamycin, +2 = carbenicillin, +3 = chloramphenicol). c) Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 10-piece scheme ( i ) Average accuracy=19.1%, Standard deviation=10.7%, Average yield=1.37 × 10 3 Lux + colonies / µg DNA, Standard deviation= 5.51 × 10 2 Lux + colonies/ µg DNA) and the 19-piece Assembly Amplicon scheme ( ii ) (Average accuracy=30.6%, Standard deviation=2.8%, Average yield=1.43 × 10 4 Lux + colonies/ µg DNA, Standard deviation=1.33× 10 3 Lux + colonies/ µg DNA). d) Schematic depicting LoxP (L1) and Lox2272 (L2) recombination sites onto distal ends of integrated cargo to enable Cre recombinase mediated genome integration. e) Phenotypic spot assay where pre-integration LoxP linear recipient is compared to post-integration linear replaced LoxP cells on various growth media (+1* = chloramphenicol, +2* = tetracycline, +3* = spectinomycin, −1* = sacB sucrose sensitivity). f) Transformation results for LoxP d i 10-piece assemblies (blue) relative to d ii 20-piece Assembly Amplicon (pink). Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 10-piece scheme ( i ) (Average accuracy=58.6%, Standard deviation=5.1%, Average yield=1.22× 10 3 Lux + colonies/ µg DNA, Standard deviation=5.61× 10 2 Lux + colonies/ µg DNA) and the 19-piece Assembly Amplicon scheme ( ii ) (Average accuracy=76.6%, Standard deviation=4.5%, Average yield=3.22× 10 3 Lux + colonies/ µg DNA, Standard deviation=2.68× 10 2 Lux + colonies/ µg DNA).

    Techniques Used: Sequencing, Marker, Amplification, Plasmid Preparation, Fluorescence, Expressing, Spot Test, Transformation Assay, Standard Deviation, Sensitivity

    a) Assembly Amplicon is HighT assembled to two oriT containing conjugation backbones. This assembly is transformed into high efficiency commercial electrocompetent cells. A conjugation plasmid is then added to make this cell a Donor. Conjugation between the two oriT sites on the donor plasmid is performed and delivered into the same Recipient as in . After integrating the conjugated DNA, the Chimeric result has a unique phenotype that enables positive and negative selection to kill off both parents, Donor and Recipient. b) Phenotypic results of 5 µL of OD 600 = 0.2 cells from Donor conjugator (D), Recipient integrator (R), and Chimeric result (C) spotted on selective agar. Selection markers and concentrations: +1 kanamycin Resistance (100 µg/mL), −1 streptinomycin Resistance (300 µg/mL), +2 Hygromycin Resistance (200 µg/mL), −2 Cl-Phe Sensitivity (5 mM p-chloro-DL-phenylalanine), +3 Apramycin Resistance (200 µg/mL), −3 Sucrose Sensitivity (7.5% w/v), +4 Chloramphenicol Resistance (20 µg/mL), and +5 Spectinomycin Resistance (60 µg/mL). GFP was taken for cells grown on LB only plates.
    Figure Legend Snippet: a) Assembly Amplicon is HighT assembled to two oriT containing conjugation backbones. This assembly is transformed into high efficiency commercial electrocompetent cells. A conjugation plasmid is then added to make this cell a Donor. Conjugation between the two oriT sites on the donor plasmid is performed and delivered into the same Recipient as in . After integrating the conjugated DNA, the Chimeric result has a unique phenotype that enables positive and negative selection to kill off both parents, Donor and Recipient. b) Phenotypic results of 5 µL of OD 600 = 0.2 cells from Donor conjugator (D), Recipient integrator (R), and Chimeric result (C) spotted on selective agar. Selection markers and concentrations: +1 kanamycin Resistance (100 µg/mL), −1 streptinomycin Resistance (300 µg/mL), +2 Hygromycin Resistance (200 µg/mL), −2 Cl-Phe Sensitivity (5 mM p-chloro-DL-phenylalanine), +3 Apramycin Resistance (200 µg/mL), −3 Sucrose Sensitivity (7.5% w/v), +4 Chloramphenicol Resistance (20 µg/mL), and +5 Spectinomycin Resistance (60 µg/mL). GFP was taken for cells grown on LB only plates.

    Techniques Used: Amplification, Conjugation Assay, Transformation Assay, Plasmid Preparation, Selection, Sensitivity

    a) Schematic depicting isolation of isoform splicing variants from eukaryotic systems from error prone cDNA or amplified from low error gDNA and assembled with HighT. b) Coverage plot of cDNA MAPT amplicon reads aligned to MAPT genomic chromosomal location. c) PacBio sequencing to compare isoform variation in amplicons of MAPT generated from human heart cDNA or gDNA HighT Assembly Amplicons. Target isoform denoted in red, all isoforms or sequencing artifacts with >1% representation are given unique identification numbers. d) Comparison of isoform variation in amplicons of VEGFA generated from human heart cDNA compared to two different HighT assemblies, one gDNA HighT Assembly Amplicons and the second entirely from oligo based UPDOG & HighT Assembly Amplicon. e) Comparison of isoform variation in amplicons of BRCA1 generated from human heart cDNA or a hybrid gDNA amplicon and oligo based HighT assembly. f) DNA agarose gel depicting 50 ng of either cDNA amplicon or HighT Assembly Amplicons from panels c-e for MAPT, VEGFA, and BRCA1.
    Figure Legend Snippet: a) Schematic depicting isolation of isoform splicing variants from eukaryotic systems from error prone cDNA or amplified from low error gDNA and assembled with HighT. b) Coverage plot of cDNA MAPT amplicon reads aligned to MAPT genomic chromosomal location. c) PacBio sequencing to compare isoform variation in amplicons of MAPT generated from human heart cDNA or gDNA HighT Assembly Amplicons. Target isoform denoted in red, all isoforms or sequencing artifacts with >1% representation are given unique identification numbers. d) Comparison of isoform variation in amplicons of VEGFA generated from human heart cDNA compared to two different HighT assemblies, one gDNA HighT Assembly Amplicons and the second entirely from oligo based UPDOG & HighT Assembly Amplicon. e) Comparison of isoform variation in amplicons of BRCA1 generated from human heart cDNA or a hybrid gDNA amplicon and oligo based HighT assembly. f) DNA agarose gel depicting 50 ng of either cDNA amplicon or HighT Assembly Amplicons from panels c-e for MAPT, VEGFA, and BRCA1.

    Techniques Used: Isolation, Amplification, PacBio Sequencing, Generated, Sequencing, Comparison, Agarose Gel Electrophoresis, Reverse Transcription, Polymerase Chain Reaction

    a) Schematic depicting the de novo assembly pipeline integrating Sidewinder with HighT 2WJ assembly for in vitro hierarchical assembly of large constructs. b) DNA agarose gel depicting 50 ng of the ten 1 kb Sidewinder assemblies post PCR amplification with dU incorporation. c) DNA agarose gel depicting 50 ng of the 10 kb HighT assembly post PCR amplification with in vitro hierarchical assembly (left) and a comparative PCR from the in vivo assembled confirmed SNP free template (right). d) Fragment level PacBio sequencing analysis each of the primary assembly step using Sidewinder. Reads are analyzed considering the connections between Sidewinder fragments composing the each of the 1 kb sub-assemblies with correct products (green), partially aligned products (subset of fragments in the correct order) (light green), and PCR or sequencing artifacts or incorrect assemblies (grey). e) Junction level PacBio sequencing analysis from unfiltered data counting all observed Sidewinder junctions for each of the ten 1 kb amplicons depicting total junctions, correctly ligated junctions, and incorrectly ligated junctions with the global count (left) and the count by sequence (right). f) Nucleotide level PacBio sequencing analysis as a violin plot and nested box and whisker plot depicting the distribution of per-base accuracies for each position for each of reads containing the full 10 kb assembly (left) and all reads aligned to each of the ten 1 kb segments. Summary statistics available in Source Data . g) Fragment level PacBio sequencing analysis the secondary assembly step using HighT. Reads are analyzed considering the connections between the ten HighT fragments with correct products (green), partially aligned products (subset of fragments in the correct order) (light green), and PCR or sequencing artifacts or incorrect assemblies (grey). h) Coverage plots of the 10 kb amplicons for the in vitro hierarchical assembly sequenced with PacBio (left) and the in vivo assembled confirmed SNP free template.
    Figure Legend Snippet: a) Schematic depicting the de novo assembly pipeline integrating Sidewinder with HighT 2WJ assembly for in vitro hierarchical assembly of large constructs. b) DNA agarose gel depicting 50 ng of the ten 1 kb Sidewinder assemblies post PCR amplification with dU incorporation. c) DNA agarose gel depicting 50 ng of the 10 kb HighT assembly post PCR amplification with in vitro hierarchical assembly (left) and a comparative PCR from the in vivo assembled confirmed SNP free template (right). d) Fragment level PacBio sequencing analysis each of the primary assembly step using Sidewinder. Reads are analyzed considering the connections between Sidewinder fragments composing the each of the 1 kb sub-assemblies with correct products (green), partially aligned products (subset of fragments in the correct order) (light green), and PCR or sequencing artifacts or incorrect assemblies (grey). e) Junction level PacBio sequencing analysis from unfiltered data counting all observed Sidewinder junctions for each of the ten 1 kb amplicons depicting total junctions, correctly ligated junctions, and incorrectly ligated junctions with the global count (left) and the count by sequence (right). f) Nucleotide level PacBio sequencing analysis as a violin plot and nested box and whisker plot depicting the distribution of per-base accuracies for each position for each of reads containing the full 10 kb assembly (left) and all reads aligned to each of the ten 1 kb segments. Summary statistics available in Source Data . g) Fragment level PacBio sequencing analysis the secondary assembly step using HighT. Reads are analyzed considering the connections between the ten HighT fragments with correct products (green), partially aligned products (subset of fragments in the correct order) (light green), and PCR or sequencing artifacts or incorrect assemblies (grey). h) Coverage plots of the 10 kb amplicons for the in vitro hierarchical assembly sequenced with PacBio (left) and the in vivo assembled confirmed SNP free template.

    Techniques Used: In Vitro, Construct, Agarose Gel Electrophoresis, Amplification, In Vivo, PacBio Sequencing, Analysis, Sequencing, Counting, Whisker Assay

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    other:

    Article Title: A non-canonical redox family protein regulates bacterial interaction machineries.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER BD Bacto Agar BD Cat#214010 Luria Broth HiMedia Laboratories, LLC Cat#M575 Tartoff - Hobbs Broth (Terrific Broth) HiMedia Laboratories, LLC Cat#M1250 Critical commercial assays Crystallization Screens (Crystal Screen) Hampton Research Cat#HR2-110 Crystallization Screens (Crystal Screen 2) Hampton Research Cat#HR2-112 Crystallization Screens (Index) Hampton Research Cat#HR2-144 Crystallization Screens (PEG/Ion Screen) Hampton Research Cat#HR2-126 Crystallization Screens (PEG/Ion 2 Screen) Hampton Research Cat#HR2-098 Q5 High-Fidelity DNA Polymerase New England Biolabs Cat#M0491S T5 Exonuclease New England Biolabs Cat#M0663S Taq DNA Ligase New England Biolabs Cat#M0208S Isothermal Assembly Master Mix Gibson et al.58 N/A DNase I recombinant, RNase-free Roche Diagnostics Cat#04716728001 One-Step TB Green PrimeScript RT-PCR Kit II Takara Cat#RR086A Ni-NTA Agarose Qiagen Cat#30230 QIAprep Spin Miniprep Kit Qiagen Cat#27106 MinElute PCR Purification Kit Qiagen Cat#28004 QIAquick Gel Extraction Kit Qiagen Cat#28706 Nuclei Lysis Solution Promega Cat#A7941 Protein Precipitation Solution Promega Cat#A795A TB Green Premix Ex Taq II (Tli RNase H Plus) Takara Cat#RR820L Deposited data Mass spectrometry (YmdB immunepulldown) PXD069961 PRIDE X-ray structures (reduced state) 9X3C, pdb_00009x3c RCSB PDB X-ray structures (oxidized state) 9WVG, pdb_00009wvg RCSB PDB Raw X-ray diffraction images (reduced form) https://doi.org/10.5281/zenodo.17372066 Zenodo Raw X-ray diffraction images (oxidized form) https://doi.org/10.5281/zenodo.17201831 Zenodo Phylogenetic relationships dataset and script (FASTA) https://doi.org/10.5281/zenodo.21918644 Zenodo Oligonucleotides Strain construction primers This study Table S4 Plasmid construction primers This study Table S4 Assay-specific primers This study Table S5 Recombinant DNA pDR111 (amyE::Phyper-spank-spec) Britton et al. 59 Sigal Ben-Yehuda’s Laboratory stock pHB201 (cat, erm) Bron et al.60 Sigal Ben-Yehuda’s Laboratory stock pMD20-YdzFBa (Bacillus anthracis YdzF TA vector) This study Synthesized by Barcode Biosciences pMD20-YdzFCb (Clostridium botulinum YdzF TA vector) This study Synthesized by Barcode Biosciences pRB30 (amyE::Phyper-spankydzFBacillus-subtilis-spec) This study N/A (Continued on next page) 18 Cell Reports 45, 118071, October 27, 2026

    Plasmid Preparation:

    Article Title: Salmonella effector SteE reprograms the macrophage regulatory network to drive specific hyperactivation of STAT3 target genes.
    Article Snippet: .. STAT1 and STAT3 gRNAs (Table S15) were designed using CHOPCHOP67 and inserted in px330-GFP plasmids using BbsI (NEB) digestion followed by ligation with Taq DNA ligase (NEB) according to the manufacturer’s instructions. iBMDMs were nucleofected with 2.5 ng of STAT1 or STAT3 gRNA-containing px330-GFP plasmid using Amaxa Mouse Macrophage Nucleofection Kit (Lonza) following manufacturer’s instructions. ..



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    Image Search Results


    a) Schematic depicting the advantages offered by High Temperature (HighT) ligation in comparison to low temperature ligation in producing exposed overhangs for assembly. b) NUPACK simulated melt curves for all 10 bp duplexes with unpaired intramolecular secondary structures at 50°C. c) Violin plot and nested box and whisker plot depicting the distribution of melting temperatures in the form of T m-n for the simulated data presented in b . The n demarcates the proportion of species bound in the duplex for a 10 nM solution. Summary statistics can be found in Source data . d ) Schematic depicting the qPCR assay used to measure ligation activity of 4 bp and 10 bp overhangs under different ligase and temperature conditions. e) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 4 bp and 10 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. f) Bar plot depicting the mean ± SD of relative ligation product for 4 bp and 10 bp overhangs at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. The starting quantity of T4 DNA Ligase at 16°C was defined as 100% and used to normalize all samples.

    Journal: bioRxiv

    Article Title: High temperature ligation enables in vitro assembly of eukaryotic genes

    doi: 10.64898/2026.10.09.758000

    Figure Lengend Snippet: a) Schematic depicting the advantages offered by High Temperature (HighT) ligation in comparison to low temperature ligation in producing exposed overhangs for assembly. b) NUPACK simulated melt curves for all 10 bp duplexes with unpaired intramolecular secondary structures at 50°C. c) Violin plot and nested box and whisker plot depicting the distribution of melting temperatures in the form of T m-n for the simulated data presented in b . The n demarcates the proportion of species bound in the duplex for a 10 nM solution. Summary statistics can be found in Source data . d ) Schematic depicting the qPCR assay used to measure ligation activity of 4 bp and 10 bp overhangs under different ligase and temperature conditions. e) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 4 bp and 10 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. f) Bar plot depicting the mean ± SD of relative ligation product for 4 bp and 10 bp overhangs at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. The starting quantity of T4 DNA Ligase at 16°C was defined as 100% and used to normalize all samples.

    Article Snippet: Ligation mixtures were prepared with 1 μL ligase per 100μL reaction with 0.1 pmol (for 4 base experiment) or 0.4 pmol (for 10 base experiment) ssDNA oligo with either 1X T4 DNA ligase buffer with T4 ligase (New England Biolabs), 1X Hifi Taq ligase buffer with Taq DNA Ligase (New England Biolabs), or only 1X Hifi Taq ligase buffer (control).

    Techniques: Ligation, Comparison, Whisker Assay, Activity Assay, Amplification, Fluorescence, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    a) NUPACK simulated melt curves for DNA with secondary structures. b) NUPACK simulated melting curves for 4 bp and 10 bp overhangs used in the overhang ligation qPCR experiment at discrete temperatures 4°C to 85°C. c) qPCR amplification curves depicting changes in fluorescence over PCR cycles for all individual replicates of the 4 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. d) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 4 bp overhang ligation reactions at conditions 16°C with no ligase, 16°C with T4 DNA Ligase, 50°C with no ligase, and 50°C with Taq DNA Ligase. e) qPCR standard curves for 4 bp overhang ligation reactions, showing Cq as a function of log10 starting quantity across all temperatures and each temperature individually. Dashed lines indicate extrapolation beyond the standard range. f) qPCR amplification curves depicting changes in fluorescence over PCR cycles for all individual replicates of the 10 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. g) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 10 bp overhang ligation reactions at conditions 16°C with no ligase, 16°C with T4 DNA Ligase, 50°C with no ligase, and 50°C with Taq DNA Ligase. h) qPCR standard curves for 10 bp overhang ligation reactions, showing Cq as a function of log10 starting quantity across all temperatures and each temperature individually. Dashed lines indicate extrapolation beyond the standard range.

    Journal: bioRxiv

    Article Title: High temperature ligation enables in vitro assembly of eukaryotic genes

    doi: 10.64898/2026.10.09.758000

    Figure Lengend Snippet: a) NUPACK simulated melt curves for DNA with secondary structures. b) NUPACK simulated melting curves for 4 bp and 10 bp overhangs used in the overhang ligation qPCR experiment at discrete temperatures 4°C to 85°C. c) qPCR amplification curves depicting changes in fluorescence over PCR cycles for all individual replicates of the 4 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. d) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 4 bp overhang ligation reactions at conditions 16°C with no ligase, 16°C with T4 DNA Ligase, 50°C with no ligase, and 50°C with Taq DNA Ligase. e) qPCR standard curves for 4 bp overhang ligation reactions, showing Cq as a function of log10 starting quantity across all temperatures and each temperature individually. Dashed lines indicate extrapolation beyond the standard range. f) qPCR amplification curves depicting changes in fluorescence over PCR cycles for all individual replicates of the 10 bp overhang ligation reactions at 16°C, 22°C, 30°C, and 50°C with no ligase, T4 DNA Ligase, or Taq DNA Ligase. g) qPCR amplification curves depicting mean fluorescence ± SD over PCR cycles for the 10 bp overhang ligation reactions at conditions 16°C with no ligase, 16°C with T4 DNA Ligase, 50°C with no ligase, and 50°C with Taq DNA Ligase. h) qPCR standard curves for 10 bp overhang ligation reactions, showing Cq as a function of log10 starting quantity across all temperatures and each temperature individually. Dashed lines indicate extrapolation beyond the standard range.

    Article Snippet: Ligation mixtures were prepared with 1 μL ligase per 100μL reaction with 0.1 pmol (for 4 base experiment) or 0.4 pmol (for 10 base experiment) ssDNA oligo with either 1X T4 DNA ligase buffer with T4 ligase (New England Biolabs), 1X Hifi Taq ligase buffer with Taq DNA Ligase (New England Biolabs), or only 1X Hifi Taq ligase buffer (control).

    Techniques: Ligation, Amplification, Fluorescence, Polymerase Chain Reaction

    a) Schematic depicting circular HighT assembly genomically integrated using a circular recombination system. LuxABCDE is divided into 9 fragments, which are HighT ligated to an integration backbone fragment containing two terminators, an attB sequence, and a transcriptionally inactive antibiotic resistance marker +1 ( i ). LuxABCDE is divided into 19 fragments, which are HighT ligated and amplified into an Assembly Amplicon that is subsequently assembled with the same integration backbone ( ii ). The final assembly product of both constructions ( iii ) are electroporated into recipient cells with a plasmid containing the attB integrase, antibiotic resistance marker +2 and a pre-engineered genome containing a promoter, attP site, transcriptionally active GFP, and antibiotic resistance marker +3 ( iv ). Through protein mediated DNA crossover ( v ) the recipient genome gains transcriptionally active antibiotic resistance marker +1, the cargo insert, and losses fluorescence expression in the post-integrated genome ( vi ). b) Phenotypic spot assay where pre-integration attBP circular recipient is compared to post-integration circular attBP cells on various growth media (+1 = kanamycin, +2 = carbenicillin, +3 = chloramphenicol). c) Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 10-piece scheme ( i ) Average accuracy=19.1%, Standard deviation=10.7%, Average yield=1.37 × 10 3 Lux + colonies / µg DNA, Standard deviation= 5.51 × 10 2 Lux + colonies/ µg DNA) and the 19-piece Assembly Amplicon scheme ( ii ) (Average accuracy=30.6%, Standard deviation=2.8%, Average yield=1.43 × 10 4 Lux + colonies/ µg DNA, Standard deviation=1.33× 10 3 Lux + colonies/ µg DNA). d) Schematic depicting LoxP (L1) and Lox2272 (L2) recombination sites onto distal ends of integrated cargo to enable Cre recombinase mediated genome integration. e) Phenotypic spot assay where pre-integration LoxP linear recipient is compared to post-integration linear replaced LoxP cells on various growth media (+1* = chloramphenicol, +2* = tetracycline, +3* = spectinomycin, −1* = sacB sucrose sensitivity). f) Transformation results for LoxP d i 10-piece assemblies (blue) relative to d ii 20-piece Assembly Amplicon (pink). Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 10-piece scheme ( i ) (Average accuracy=58.6%, Standard deviation=5.1%, Average yield=1.22× 10 3 Lux + colonies/ µg DNA, Standard deviation=5.61× 10 2 Lux + colonies/ µg DNA) and the 19-piece Assembly Amplicon scheme ( ii ) (Average accuracy=76.6%, Standard deviation=4.5%, Average yield=3.22× 10 3 Lux + colonies/ µg DNA, Standard deviation=2.68× 10 2 Lux + colonies/ µg DNA).

    Journal: bioRxiv

    Article Title: High temperature ligation enables in vitro assembly of eukaryotic genes

    doi: 10.64898/2026.10.09.758000

    Figure Lengend Snippet: a) Schematic depicting circular HighT assembly genomically integrated using a circular recombination system. LuxABCDE is divided into 9 fragments, which are HighT ligated to an integration backbone fragment containing two terminators, an attB sequence, and a transcriptionally inactive antibiotic resistance marker +1 ( i ). LuxABCDE is divided into 19 fragments, which are HighT ligated and amplified into an Assembly Amplicon that is subsequently assembled with the same integration backbone ( ii ). The final assembly product of both constructions ( iii ) are electroporated into recipient cells with a plasmid containing the attB integrase, antibiotic resistance marker +2 and a pre-engineered genome containing a promoter, attP site, transcriptionally active GFP, and antibiotic resistance marker +3 ( iv ). Through protein mediated DNA crossover ( v ) the recipient genome gains transcriptionally active antibiotic resistance marker +1, the cargo insert, and losses fluorescence expression in the post-integrated genome ( vi ). b) Phenotypic spot assay where pre-integration attBP circular recipient is compared to post-integration circular attBP cells on various growth media (+1 = kanamycin, +2 = carbenicillin, +3 = chloramphenicol). c) Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 10-piece scheme ( i ) Average accuracy=19.1%, Standard deviation=10.7%, Average yield=1.37 × 10 3 Lux + colonies / µg DNA, Standard deviation= 5.51 × 10 2 Lux + colonies/ µg DNA) and the 19-piece Assembly Amplicon scheme ( ii ) (Average accuracy=30.6%, Standard deviation=2.8%, Average yield=1.43 × 10 4 Lux + colonies/ µg DNA, Standard deviation=1.33× 10 3 Lux + colonies/ µg DNA). d) Schematic depicting LoxP (L1) and Lox2272 (L2) recombination sites onto distal ends of integrated cargo to enable Cre recombinase mediated genome integration. e) Phenotypic spot assay where pre-integration LoxP linear recipient is compared to post-integration linear replaced LoxP cells on various growth media (+1* = chloramphenicol, +2* = tetracycline, +3* = spectinomycin, −1* = sacB sucrose sensitivity). f) Transformation results for LoxP d i 10-piece assemblies (blue) relative to d ii 20-piece Assembly Amplicon (pink). Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 10-piece scheme ( i ) (Average accuracy=58.6%, Standard deviation=5.1%, Average yield=1.22× 10 3 Lux + colonies/ µg DNA, Standard deviation=5.61× 10 2 Lux + colonies/ µg DNA) and the 19-piece Assembly Amplicon scheme ( ii ) (Average accuracy=76.6%, Standard deviation=4.5%, Average yield=3.22× 10 3 Lux + colonies/ µg DNA, Standard deviation=2.68× 10 2 Lux + colonies/ µg DNA).

    Article Snippet: Ligation mixtures were prepared with 1 μL ligase per 100μL reaction with 0.1 pmol (for 4 base experiment) or 0.4 pmol (for 10 base experiment) ssDNA oligo with either 1X T4 DNA ligase buffer with T4 ligase (New England Biolabs), 1X Hifi Taq ligase buffer with Taq DNA Ligase (New England Biolabs), or only 1X Hifi Taq ligase buffer (control).

    Techniques: Sequencing, Marker, Amplification, Plasmid Preparation, Fluorescence, Expressing, Spot Test, Transformation Assay, Standard Deviation, Sensitivity

    a) Assembly Amplicon is HighT assembled to two oriT containing conjugation backbones. This assembly is transformed into high efficiency commercial electrocompetent cells. A conjugation plasmid is then added to make this cell a Donor. Conjugation between the two oriT sites on the donor plasmid is performed and delivered into the same Recipient as in . After integrating the conjugated DNA, the Chimeric result has a unique phenotype that enables positive and negative selection to kill off both parents, Donor and Recipient. b) Phenotypic results of 5 µL of OD 600 = 0.2 cells from Donor conjugator (D), Recipient integrator (R), and Chimeric result (C) spotted on selective agar. Selection markers and concentrations: +1 kanamycin Resistance (100 µg/mL), −1 streptinomycin Resistance (300 µg/mL), +2 Hygromycin Resistance (200 µg/mL), −2 Cl-Phe Sensitivity (5 mM p-chloro-DL-phenylalanine), +3 Apramycin Resistance (200 µg/mL), −3 Sucrose Sensitivity (7.5% w/v), +4 Chloramphenicol Resistance (20 µg/mL), and +5 Spectinomycin Resistance (60 µg/mL). GFP was taken for cells grown on LB only plates.

    Journal: bioRxiv

    Article Title: High temperature ligation enables in vitro assembly of eukaryotic genes

    doi: 10.64898/2026.10.09.758000

    Figure Lengend Snippet: a) Assembly Amplicon is HighT assembled to two oriT containing conjugation backbones. This assembly is transformed into high efficiency commercial electrocompetent cells. A conjugation plasmid is then added to make this cell a Donor. Conjugation between the two oriT sites on the donor plasmid is performed and delivered into the same Recipient as in . After integrating the conjugated DNA, the Chimeric result has a unique phenotype that enables positive and negative selection to kill off both parents, Donor and Recipient. b) Phenotypic results of 5 µL of OD 600 = 0.2 cells from Donor conjugator (D), Recipient integrator (R), and Chimeric result (C) spotted on selective agar. Selection markers and concentrations: +1 kanamycin Resistance (100 µg/mL), −1 streptinomycin Resistance (300 µg/mL), +2 Hygromycin Resistance (200 µg/mL), −2 Cl-Phe Sensitivity (5 mM p-chloro-DL-phenylalanine), +3 Apramycin Resistance (200 µg/mL), −3 Sucrose Sensitivity (7.5% w/v), +4 Chloramphenicol Resistance (20 µg/mL), and +5 Spectinomycin Resistance (60 µg/mL). GFP was taken for cells grown on LB only plates.

    Article Snippet: Ligation mixtures were prepared with 1 μL ligase per 100μL reaction with 0.1 pmol (for 4 base experiment) or 0.4 pmol (for 10 base experiment) ssDNA oligo with either 1X T4 DNA ligase buffer with T4 ligase (New England Biolabs), 1X Hifi Taq ligase buffer with Taq DNA Ligase (New England Biolabs), or only 1X Hifi Taq ligase buffer (control).

    Techniques: Amplification, Conjugation Assay, Transformation Assay, Plasmid Preparation, Selection, Sensitivity

    a) Schematic depicting isolation of isoform splicing variants from eukaryotic systems from error prone cDNA or amplified from low error gDNA and assembled with HighT. b) Coverage plot of cDNA MAPT amplicon reads aligned to MAPT genomic chromosomal location. c) PacBio sequencing to compare isoform variation in amplicons of MAPT generated from human heart cDNA or gDNA HighT Assembly Amplicons. Target isoform denoted in red, all isoforms or sequencing artifacts with >1% representation are given unique identification numbers. d) Comparison of isoform variation in amplicons of VEGFA generated from human heart cDNA compared to two different HighT assemblies, one gDNA HighT Assembly Amplicons and the second entirely from oligo based UPDOG & HighT Assembly Amplicon. e) Comparison of isoform variation in amplicons of BRCA1 generated from human heart cDNA or a hybrid gDNA amplicon and oligo based HighT assembly. f) DNA agarose gel depicting 50 ng of either cDNA amplicon or HighT Assembly Amplicons from panels c-e for MAPT, VEGFA, and BRCA1.

    Journal: bioRxiv

    Article Title: High temperature ligation enables in vitro assembly of eukaryotic genes

    doi: 10.64898/2026.10.09.758000

    Figure Lengend Snippet: a) Schematic depicting isolation of isoform splicing variants from eukaryotic systems from error prone cDNA or amplified from low error gDNA and assembled with HighT. b) Coverage plot of cDNA MAPT amplicon reads aligned to MAPT genomic chromosomal location. c) PacBio sequencing to compare isoform variation in amplicons of MAPT generated from human heart cDNA or gDNA HighT Assembly Amplicons. Target isoform denoted in red, all isoforms or sequencing artifacts with >1% representation are given unique identification numbers. d) Comparison of isoform variation in amplicons of VEGFA generated from human heart cDNA compared to two different HighT assemblies, one gDNA HighT Assembly Amplicons and the second entirely from oligo based UPDOG & HighT Assembly Amplicon. e) Comparison of isoform variation in amplicons of BRCA1 generated from human heart cDNA or a hybrid gDNA amplicon and oligo based HighT assembly. f) DNA agarose gel depicting 50 ng of either cDNA amplicon or HighT Assembly Amplicons from panels c-e for MAPT, VEGFA, and BRCA1.

    Article Snippet: Ligation mixtures were prepared with 1 μL ligase per 100μL reaction with 0.1 pmol (for 4 base experiment) or 0.4 pmol (for 10 base experiment) ssDNA oligo with either 1X T4 DNA ligase buffer with T4 ligase (New England Biolabs), 1X Hifi Taq ligase buffer with Taq DNA Ligase (New England Biolabs), or only 1X Hifi Taq ligase buffer (control).

    Techniques: Isolation, Amplification, PacBio Sequencing, Generated, Sequencing, Comparison, Agarose Gel Electrophoresis, Reverse Transcription, Polymerase Chain Reaction

    a) Schematic depicting the de novo assembly pipeline integrating Sidewinder with HighT 2WJ assembly for in vitro hierarchical assembly of large constructs. b) DNA agarose gel depicting 50 ng of the ten 1 kb Sidewinder assemblies post PCR amplification with dU incorporation. c) DNA agarose gel depicting 50 ng of the 10 kb HighT assembly post PCR amplification with in vitro hierarchical assembly (left) and a comparative PCR from the in vivo assembled confirmed SNP free template (right). d) Fragment level PacBio sequencing analysis each of the primary assembly step using Sidewinder. Reads are analyzed considering the connections between Sidewinder fragments composing the each of the 1 kb sub-assemblies with correct products (green), partially aligned products (subset of fragments in the correct order) (light green), and PCR or sequencing artifacts or incorrect assemblies (grey). e) Junction level PacBio sequencing analysis from unfiltered data counting all observed Sidewinder junctions for each of the ten 1 kb amplicons depicting total junctions, correctly ligated junctions, and incorrectly ligated junctions with the global count (left) and the count by sequence (right). f) Nucleotide level PacBio sequencing analysis as a violin plot and nested box and whisker plot depicting the distribution of per-base accuracies for each position for each of reads containing the full 10 kb assembly (left) and all reads aligned to each of the ten 1 kb segments. Summary statistics available in Source Data . g) Fragment level PacBio sequencing analysis the secondary assembly step using HighT. Reads are analyzed considering the connections between the ten HighT fragments with correct products (green), partially aligned products (subset of fragments in the correct order) (light green), and PCR or sequencing artifacts or incorrect assemblies (grey). h) Coverage plots of the 10 kb amplicons for the in vitro hierarchical assembly sequenced with PacBio (left) and the in vivo assembled confirmed SNP free template.

    Journal: bioRxiv

    Article Title: High temperature ligation enables in vitro assembly of eukaryotic genes

    doi: 10.64898/2026.10.09.758000

    Figure Lengend Snippet: a) Schematic depicting the de novo assembly pipeline integrating Sidewinder with HighT 2WJ assembly for in vitro hierarchical assembly of large constructs. b) DNA agarose gel depicting 50 ng of the ten 1 kb Sidewinder assemblies post PCR amplification with dU incorporation. c) DNA agarose gel depicting 50 ng of the 10 kb HighT assembly post PCR amplification with in vitro hierarchical assembly (left) and a comparative PCR from the in vivo assembled confirmed SNP free template (right). d) Fragment level PacBio sequencing analysis each of the primary assembly step using Sidewinder. Reads are analyzed considering the connections between Sidewinder fragments composing the each of the 1 kb sub-assemblies with correct products (green), partially aligned products (subset of fragments in the correct order) (light green), and PCR or sequencing artifacts or incorrect assemblies (grey). e) Junction level PacBio sequencing analysis from unfiltered data counting all observed Sidewinder junctions for each of the ten 1 kb amplicons depicting total junctions, correctly ligated junctions, and incorrectly ligated junctions with the global count (left) and the count by sequence (right). f) Nucleotide level PacBio sequencing analysis as a violin plot and nested box and whisker plot depicting the distribution of per-base accuracies for each position for each of reads containing the full 10 kb assembly (left) and all reads aligned to each of the ten 1 kb segments. Summary statistics available in Source Data . g) Fragment level PacBio sequencing analysis the secondary assembly step using HighT. Reads are analyzed considering the connections between the ten HighT fragments with correct products (green), partially aligned products (subset of fragments in the correct order) (light green), and PCR or sequencing artifacts or incorrect assemblies (grey). h) Coverage plots of the 10 kb amplicons for the in vitro hierarchical assembly sequenced with PacBio (left) and the in vivo assembled confirmed SNP free template.

    Article Snippet: Ligation mixtures were prepared with 1 μL ligase per 100μL reaction with 0.1 pmol (for 4 base experiment) or 0.4 pmol (for 10 base experiment) ssDNA oligo with either 1X T4 DNA ligase buffer with T4 ligase (New England Biolabs), 1X Hifi Taq ligase buffer with Taq DNA Ligase (New England Biolabs), or only 1X Hifi Taq ligase buffer (control).

    Techniques: In Vitro, Construct, Agarose Gel Electrophoresis, Amplification, In Vivo, PacBio Sequencing, Analysis, Sequencing, Counting, Whisker Assay

    Optimization of real-time 5hmC-RCA assay. The empty grid corresponds to reactions containing the 5hmC template at a concentration of 50 copies per reaction, whereas grids marked with left-diagonal and right-diagonal lines represent reactions with the 5mC and unmodified C targets, respectively. (A) . Effect of HiFi Taq DNA ligase-mediated ligation cycles from 1 to 5 cycles, with the 5hmC/5mC/C target concentration at 2000 copies/reaction. All data are presented as the mean of three biological/technical replicates, and error bars denote the corresponding standard deviation. (B) . Effect of ligation mediator concentrations from 0.1 to 0.6 μM with the 5hmC and 5mC target concentration at 500 copies/reaction. Data represents the mean of three independent replicates, with error bars indicating the standard deviation. (C) . Effect of HiFi Taq DNA ligase concentration on reaction performance. 1: 2 nM (black squares); 2: 0.5 nM (red disks); 3: 0.1 nM (blue triangles); 4: 0.05 nM (green inverted triangles); (D) Effect of different incubation temperature of HiFi Taq DNA ligase-mediated ligation. 1: 54°C; 2: 57°C; 3: 60°C; 4: 63°C. (E) Real-time fluorescence curves produced by different concentrations of 5hmC target. 1: 1000 copies/reaction (black squares); 2: 250 copies/reaction (red circles); 3: 50 copies/reaction (green triangles); 4: 10 copies/reaction (blue inverted triangles); 5: ddH 2 O as a negative control (NC) (light blue lozenges). (F) Electropherograms of sensitivity of 5hmC-RCA [inset graph in (E) ]. L: 100 bp ladder; 1: 1,000 copies/reaction; 2: 250 copies/reaction; 3: 50 copies/reaction; 4: 10 copies/reaction; 5: NC. (G) Target specificity test for 5hmC-RCA assay with the target concentration at 5000 copies/reaction. 1. 5hmC (red line); 2. 5mC (green line); 3. C (blue line); 4: NC (yellow line). (H) Detection of 5hmC target at varying ratios (50%, 12.5%, 2.5%, 0.5%) against a background of unmodified C target, with a total target concentration fixed at 2,000 copies per reaction [inset graph in (G) ]. Data were the average of three replicates and error bars represent the standard deviation. The data fitted with linear regression (R 2 = 0.99). Differences were considered statistically significant if p < 0.05. *** p < 0.001, ** p < 0.01, and * p < 0.05.

    Journal: Frontiers in Immunology

    Article Title: Selective rolling circle amplification detection of site-specific DNA 5-hydroxymethylcytosine

    doi: 10.3389/fimmu.2026.1960016

    Figure Lengend Snippet: Optimization of real-time 5hmC-RCA assay. The empty grid corresponds to reactions containing the 5hmC template at a concentration of 50 copies per reaction, whereas grids marked with left-diagonal and right-diagonal lines represent reactions with the 5mC and unmodified C targets, respectively. (A) . Effect of HiFi Taq DNA ligase-mediated ligation cycles from 1 to 5 cycles, with the 5hmC/5mC/C target concentration at 2000 copies/reaction. All data are presented as the mean of three biological/technical replicates, and error bars denote the corresponding standard deviation. (B) . Effect of ligation mediator concentrations from 0.1 to 0.6 μM with the 5hmC and 5mC target concentration at 500 copies/reaction. Data represents the mean of three independent replicates, with error bars indicating the standard deviation. (C) . Effect of HiFi Taq DNA ligase concentration on reaction performance. 1: 2 nM (black squares); 2: 0.5 nM (red disks); 3: 0.1 nM (blue triangles); 4: 0.05 nM (green inverted triangles); (D) Effect of different incubation temperature of HiFi Taq DNA ligase-mediated ligation. 1: 54°C; 2: 57°C; 3: 60°C; 4: 63°C. (E) Real-time fluorescence curves produced by different concentrations of 5hmC target. 1: 1000 copies/reaction (black squares); 2: 250 copies/reaction (red circles); 3: 50 copies/reaction (green triangles); 4: 10 copies/reaction (blue inverted triangles); 5: ddH 2 O as a negative control (NC) (light blue lozenges). (F) Electropherograms of sensitivity of 5hmC-RCA [inset graph in (E) ]. L: 100 bp ladder; 1: 1,000 copies/reaction; 2: 250 copies/reaction; 3: 50 copies/reaction; 4: 10 copies/reaction; 5: NC. (G) Target specificity test for 5hmC-RCA assay with the target concentration at 5000 copies/reaction. 1. 5hmC (red line); 2. 5mC (green line); 3. C (blue line); 4: NC (yellow line). (H) Detection of 5hmC target at varying ratios (50%, 12.5%, 2.5%, 0.5%) against a background of unmodified C target, with a total target concentration fixed at 2,000 copies per reaction [inset graph in (G) ]. Data were the average of three replicates and error bars represent the standard deviation. The data fitted with linear regression (R 2 = 0.99). Differences were considered statistically significant if p < 0.05. *** p < 0.001, ** p < 0.01, and * p < 0.05.

    Article Snippet: HiFi Taq DNA Ligase and Bst DNA Polymerase (Large Fragment) were purchased from New England Biolabs Ltd. All oligonucleotide sequences were synthesized by Sangon (Shanghai, China), with details listed in .

    Techniques: Concentration Assay, Ligation, Standard Deviation, Incubation, Fluorescence, Produced, Negative Control, Sensitivity, IF-P

    (A) The mechanism of 5hmC-RCA LFD detection. After HiFi Taq DNA Ligase-mediated ligation, a biotin labeled primer hybridized to the circular ssDNA leading to the generation of RCA product with tandem repeat sequences. Then a 3’end FITC labeled probe binds to the RCA product and finally detected by LFD. (B) The structure of lateral flow dipstick. (C, D) The sensitivity of 5hmC-RCA LFD results using the 5hmC target. 1: 1000 copies/reaction; 2: 250 copies/reaction; 3: 50 copies/reaction; 4: 10 copies/reaction; 5: ddH 2 O as a negative control (NC). Representative photos (C) and the bar graphs for the intensity of test lines (D) for the 5hmC-RCA-LFD assay at variable concentrations.

    Journal: Frontiers in Immunology

    Article Title: Selective rolling circle amplification detection of site-specific DNA 5-hydroxymethylcytosine

    doi: 10.3389/fimmu.2026.1960016

    Figure Lengend Snippet: (A) The mechanism of 5hmC-RCA LFD detection. After HiFi Taq DNA Ligase-mediated ligation, a biotin labeled primer hybridized to the circular ssDNA leading to the generation of RCA product with tandem repeat sequences. Then a 3’end FITC labeled probe binds to the RCA product and finally detected by LFD. (B) The structure of lateral flow dipstick. (C, D) The sensitivity of 5hmC-RCA LFD results using the 5hmC target. 1: 1000 copies/reaction; 2: 250 copies/reaction; 3: 50 copies/reaction; 4: 10 copies/reaction; 5: ddH 2 O as a negative control (NC). Representative photos (C) and the bar graphs for the intensity of test lines (D) for the 5hmC-RCA-LFD assay at variable concentrations.

    Article Snippet: HiFi Taq DNA Ligase and Bst DNA Polymerase (Large Fragment) were purchased from New England Biolabs Ltd. All oligonucleotide sequences were synthesized by Sangon (Shanghai, China), with details listed in .

    Techniques: Ligation, Labeling, Sensitivity, Negative Control