Journal: bioRxiv

Article Title: A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

doi: 10.1101/2025.10.16.682745

Figure Lengend Snippet: Schematic representation of the O PCR based VSR method for cloning and site-directed mutagenesis. ( A ) DNA or cDNA serves as the initial template. ( B ) The desired gene/fragment is amplified using overlapping primers to generate overlapping sequences towards the 5’ and 3’ ends complementary to the plasmid DNA. The strategy enables the cloning of single or multiple gene fragments, including mutated products into plasmid DNA. In the case of multiple gene fragments, the first fragment contains a 5′ overlap with the plasmid, the last fragment contains a 3′ overlap with the plasmid, and the intermediate fragments carry 15 bp overlaps with their neighbouring fragments. ( C ) Overlap PCR reaction, the amplified gene fragments and linearized plasmid are combined with a high-fidelity polymerase, which extends the overlapping ends in the 5′ to 3′ direction, thereby filling the gaps and joining the adjacent fragments to generate a circular plasmid with a single nick at each junction.

Article Snippet: Either 100 ng of PCR product or 200 ng of plasmid DNA template was added to the reaction mix consisting of 0.5 μl of BigDye Terminator 3.1 ready reaction mix (Applied Biosystems, #4336697), 1.75 μl of BigDye Terminator v1.1 & v3.1 5x sequencing buffer, and 1 μl (10 μM) of forward primer or reverse primer, adjusted to 10 μl using nuclease-free water.

Techniques: Cloning, Mutagenesis, Amplification, Plasmid Preparation

Journal: bioRxiv

Article Title: A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

doi: 10.1101/2025.10.16.682745

Figure Lengend Snippet: Ligase-free cloning of diverse genes using the VSR method ( A ) Schematic representation of gene cloning using O PCR , depicting the types of template DNA, plasmid vector, sizes of the gene, and total time employed in the method. Electrophoretic analysis of restriction digestion products loaded on 1% agarose gel ( B ) cloned using purified DNA products as templates ( C ) clones generated using un-purified PCR products as templates; Lane M–1 kb DNA molecular weight ladder. ( D ) Bar graph representing the comparison of cloning efficiency between blunt-end and cohesive-end digested plasmid DNA is plotted. ( E ) Comparison of insert size and cloning efficiency in cohesive-end and blunt-end cloning plotted as a slope graph.

Article Snippet: Either 100 ng of PCR product or 200 ng of plasmid DNA template was added to the reaction mix consisting of 0.5 μl of BigDye Terminator 3.1 ready reaction mix (Applied Biosystems, #4336697), 1.75 μl of BigDye Terminator v1.1 & v3.1 5x sequencing buffer, and 1 μl (10 μM) of forward primer or reverse primer, adjusted to 10 μl using nuclease-free water.

Techniques: Cloning, Plasmid Preparation, Agarose Gel Electrophoresis, Clone Assay, Purification, Generated, Molecular Weight, Comparison

Journal: bioRxiv

Article Title: A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

doi: 10.1101/2025.10.16.682745

Figure Lengend Snippet: Cloning of genes using the VSR method. Agarose gel electrophoresis (1% agarose) of ( A ) PCR amplified product of 13 genes using specific overlapping primer sets. Restriction digestion analysis for the confirmation of the generated clones ( B ) IL2 ( C ) G-chain ( D ) IL4 ( E ) IFN-alpha ( F ) IFN-beta ( G ) FcGRIII; Lane M-1 kb DNA molecular weight marker. SDS-PAGE gel confirming protein expression of cloned genes ( H ) IFN-alpha ( I ) IFN-beta ( J ) IL-2 ( K ) IL4; Lane L-250 kDa protein size marker ( L ) Immunofluorescence staining of cells transiently expressing eGFP, NRP1, SARS-CoV-2 S1, mGluR2 and human FcGRIII using protein specific antibodies; scale bar= 10 um (NRP1), 20 um (mGluR2), 50 um (eGFP, SARS-CoV-2, Human FcGRIII).

Article Snippet: Either 100 ng of PCR product or 200 ng of plasmid DNA template was added to the reaction mix consisting of 0.5 μl of BigDye Terminator 3.1 ready reaction mix (Applied Biosystems, #4336697), 1.75 μl of BigDye Terminator v1.1 & v3.1 5x sequencing buffer, and 1 μl (10 μM) of forward primer or reverse primer, adjusted to 10 μl using nuclease-free water.

Techniques: Cloning, Agarose Gel Electrophoresis, Amplification, Generated, Clone Assay, Molecular Weight, Marker, SDS Page, Expressing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

doi: 10.1101/2025.10.16.682745

Figure Lengend Snippet: Introduction of a short linker fragment into a gene using VSR method ( A ) Schematic representation of cloning the eGFP11 gene fragment with the addition of multiple cloning sites (MCS), glycine linker (GL), and IgG-Fc tag into the pMCE expression vector. Agarose gel electrophoresis (1% agarose) of PCR-amplified product of ( B ) eGFP11, ( C ) IgG-Fc, ( D ) eGFP11-IgG-Fc, using overlapping primer sets; Lane M–100 bp DNA molecular weight marker ( E ) Restriction digestion analysis of eGFP11-IgG-Fc; Lane M-1 kb DNA molecular weight marker ( F ) Fluorescence imaging of cells transiently expressing eGFP11-IgG-Fc compared to the un-transfected control; scale bar = 50 µm ( G ) Western blot analysis confirming the protein expression of eGFP11-IgG-Fc; Lane L–250 kDa protein size marker. ( H ) Schematic representation of cloning kanamycin resistance gene (kan) by addition of 200 bp homologous arms upstream (left arm, LA) and downstream (right arm, RA) into pMCE expression vector. Agarose gel electrophoresis (1% agarose) of PCR amplified product of ( I ) Kanamycin; Lane M-1 kb DNA molecular weight marker ( J ) Left Arm (LA) and Right Arm (RA); Lane M-100 bp DNA molecular weight marker ( K ) LA-Kan-RA fragment; Lane M-1 kb DNA molecular weight marker ( L ) overlap PCR product of pMCE-LA-Kan-RA; Lane M-1 kb DNA molecular weight marker ( M ) Restriction digestion product of pMCE-LA-Kan-RA positive clone; Lane M-1 kb DNA molecular weight marker.

Article Snippet: Either 100 ng of PCR product or 200 ng of plasmid DNA template was added to the reaction mix consisting of 0.5 μl of BigDye Terminator 3.1 ready reaction mix (Applied Biosystems, #4336697), 1.75 μl of BigDye Terminator v1.1 & v3.1 5x sequencing buffer, and 1 μl (10 μM) of forward primer or reverse primer, adjusted to 10 μl using nuclease-free water.

Techniques: Cloning, Expressing, Plasmid Preparation, Agarose Gel Electrophoresis, Amplification, Molecular Weight, Marker, Fluorescence, Imaging, Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

doi: 10.1101/2025.10.16.682745

Figure Lengend Snippet: Stitching and cloning of more than three fragments of varying sizes in a single tube and generation of minigenome plasmids of VSV. Schematic representation of ( A ) plasmid maps of VSVΔG/GFP and hepatitis delta virus ribozyme (HDVr). ( B ) Cloning of vesicular stomatitis virus (VSV) minigenome plasmids-VSV-Nucleoprotein-Phosphoprotein (VSV-NP). Agarose gel electrophoresis (1% agarose) of PCR amplified product of ( C ) VSV-NP gene; Lane M–1 kb DNA molecular weight marker ( D ) trailer and HDVr; Lane M-100 bp DNA molecular weight marker. ( E ) Restriction digestion mapping of pMCE-VSV-NP; Lane M–1 kb DNA molecular weight marker. ( F ) Schematic representation of cloning the VSV-Large Polymerase protein (VSV-L) into the pMCE expression vector. Agarose gel electrophoresis (1%) of PCR amplified product of ( G ) VSV-L1, VSV-L2, VS-L3; Lane M-1 kb DNA molecular weight marker ( H ) Leader and HDVr gene fragments; M–100 bp DNA molecular weight marker. ( I ) Restriction digestion analysis of pMCE-VSV-L plasmid; Lane M–1 kb DNA molecular weight marker. Distinct lanes cropped from one gel image have been assembled as a composite image and separated by white lines and correspond to the marker run on the same gel.

Article Snippet: Either 100 ng of PCR product or 200 ng of plasmid DNA template was added to the reaction mix consisting of 0.5 μl of BigDye Terminator 3.1 ready reaction mix (Applied Biosystems, #4336697), 1.75 μl of BigDye Terminator v1.1 & v3.1 5x sequencing buffer, and 1 μl (10 μM) of forward primer or reverse primer, adjusted to 10 μl using nuclease-free water.

Techniques: Cloning, Plasmid Preparation, Virus, Agarose Gel Electrophoresis, Amplification, Molecular Weight, Marker, Expressing

Journal: bioRxiv

Article Title: A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

doi: 10.1101/2025.10.16.682745

Figure Lengend Snippet: Cloning of large genomic fragments using O PCR ( A ) Schematic representation of cloning a 10.3 kb sized VSVΔG/GFP genome into a pMCE expression vector. Electrophoretic analysis of PCR-amplified products ( B ) VSV fragments–VSV-F5, VSV-F4, VSV-F3, VSV-F2, and VSV-F1 loaded on 1% agarose gel; M–1 kb DNA molecular weight marker; ( C ) hepatitis delta virus ribozyme (HDVr) loaded on 1.5% agarose gel; M–100 bp DNA molecular weight marker (D ) O PCR product mix of VSV fragments (F1-F5), HDVr and pMCE loaded on 1% agarose gel; M–1 kb DNA molecular weight marker ( E,F ) restriction digestion mapping of the plasmid DNA isolated from the positive clone loaded on 1% agarose gel; M–1 kb DNA molecular weight marker. ( G ) Schematic representation of cloning a 32.7 kb sized Ad5 genome by replacing the fiber gene with a kanamycin resistance gene. Electrophoretic analysis of PCR-amplified products ( H ) Adenovirus fragments–AdV-F1, AdV-F2, AdV-F3, AdV-F4, and AdV-F5 ( I ) Kanamycin resistance gene loaded on 1% agarose gel; M–1 kb DNA molecular weight marker ( J ) O PCR product mix of AdV fragments (F1-F5) and kanamycin gene; restriction mapping analysis of plasmid DNA isolated from the positive clone using ( K ) dual cutter and ( L ) unique cutter enzyme; loaded on 0.8% agarose gel; M–1 kb extended DNA molecular weight ladder. Distinct lanes cropped from a single gel image have been assembled as a composite image. In such grouped images, the lanes are separated by ‘white’ dividing lines and correspond to the marker run on the same gel.

Article Snippet: Either 100 ng of PCR product or 200 ng of plasmid DNA template was added to the reaction mix consisting of 0.5 μl of BigDye Terminator 3.1 ready reaction mix (Applied Biosystems, #4336697), 1.75 μl of BigDye Terminator v1.1 & v3.1 5x sequencing buffer, and 1 μl (10 μM) of forward primer or reverse primer, adjusted to 10 μl using nuclease-free water.

Techniques: Cloning, Expressing, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker, Virus, Isolation

Journal: bioRxiv

Article Title: A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

doi: 10.1101/2025.10.16.682745

Figure Lengend Snippet: Incorporation of single and multiple substitution mutations using O PCR ( A ) Schematic representation of introduction of single substitution mutation and multiple substitution mutations at single and multiple loci. ( B ) Tabulation depicting the detailed information of the number of mutations, mutagenesis efficiency, and Sanger trace analysis in comparison with wild-type DNA. Nucleotides marked with red asterisk marks represent mutated nucleotides.

Article Snippet: Either 100 ng of PCR product or 200 ng of plasmid DNA template was added to the reaction mix consisting of 0.5 μl of BigDye Terminator 3.1 ready reaction mix (Applied Biosystems, #4336697), 1.75 μl of BigDye Terminator v1.1 & v3.1 5x sequencing buffer, and 1 μl (10 μM) of forward primer or reverse primer, adjusted to 10 μl using nuclease-free water.

Techniques: Mutagenesis, Comparison

Journal: bioRxiv

Article Title: A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

doi: 10.1101/2025.10.16.682745

Figure Lengend Snippet: Introduction of multiple substitution mutations in the hDPP4 and hACE2 genes. ( A ) Schematic representation of introducing H750E substitution mutation in the human DPP4 receptor (mutant-1). Agarose gel electrophoresis of PCR amplified products of ( B ) hDPP4 F-1; Lane M–1 kb DNA molecular weight marker ( C ) hDPP4 F-2; Lane M–100 bp DNA molecular weight marker. ( D ) Sanger trace analysis of pMCE-hDPP4-Mutant-1 with reference to hDPP4 WT. ( E ) Schematic representation of introducing F713A, W734A, and Y735A mutations in the human DPP4 receptor (mutant-2). Agarose gel electrophoresis of PCR amplified products of ( F ) hDPP4 F-1; Lane M–1 kb DNA molecular weight marker ( G ) hDPP4 F-2, F-3; Lane M–100 bp DNA molecular weight marker. ( H ) Sanger trace analysis of pMCE-hDPP4-Mutant-2 with reference to hDPP4 WT. ( I ) Schematic representation of introducing Q139A, R652A, Q653A, S709A, R710A, and D713A mutations in the human ACE-2 receptor. Agarose gel electrophoresis of PCR amplified products of ( J ) hDPP4 F-1 ( K ) hDPP4 F-2, F-3; Lane M–1 kb DNA molecular weight marker. ( L ) Sanger trace analysis of pMCE-hACE-2-Mutant with reference to hACE-2 WT. Distinct lanes cropped from one gel image have been assembled as a composite image and separated by white lines and correspond to the marker run on the same gel.

Article Snippet: Either 100 ng of PCR product or 200 ng of plasmid DNA template was added to the reaction mix consisting of 0.5 μl of BigDye Terminator 3.1 ready reaction mix (Applied Biosystems, #4336697), 1.75 μl of BigDye Terminator v1.1 & v3.1 5x sequencing buffer, and 1 μl (10 μM) of forward primer or reverse primer, adjusted to 10 μl using nuclease-free water.

Techniques: Mutagenesis, Agarose Gel Electrophoresis, Amplification, Molecular Weight, Marker

Journal: bioRxiv

Article Title: A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

doi: 10.1101/2025.10.16.682745

Figure Lengend Snippet: Incorporation of insertion and deletion mutations using O PCR . ( A ) Schematic representation of introduction of short insertion, short and large deletion mutations. ( B ) Tabulation depicting the detailed information of number of mutations, mutagenesis efficiency and Sanger trace analysis in comparison with the wild type DNA. Nucleotides highlighted in red colour are mutated. ( C ) Correlation of mutagenesis efficiency with the number of mutations incorporated using O PCR. Each coloured circle and the square in the graph represent the number of mutations incorporated corresponding to the mutagenesis efficiency.

Article Snippet: Either 100 ng of PCR product or 200 ng of plasmid DNA template was added to the reaction mix consisting of 0.5 μl of BigDye Terminator 3.1 ready reaction mix (Applied Biosystems, #4336697), 1.75 μl of BigDye Terminator v1.1 & v3.1 5x sequencing buffer, and 1 μl (10 μM) of forward primer or reverse primer, adjusted to 10 μl using nuclease-free water.

Techniques: Mutagenesis, Comparison

Journal: bioRxiv

Article Title: A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

doi: 10.1101/2025.10.16.682745

Figure Lengend Snippet: Synthesis of a short gene fragment using O PCR. ( A ) Schematic representation of the synthesis of SARS-CoV-2 RBD using oligo-assembly. Agarose gel electrophoresis of ( B ) PCR product of oligonucleotides assembled of sizes ranging from 100 bp to 700 bp; Lane M–100 bp DNA molecular weight marker ( C ) Assembly of oligonucleotides encoding the RBD gene of SARS-CoV-2-Omicron variant; Lane M–100 bp DNA molecular weight marker ( D ) O PCR product of assembled RBD cloned into the pMCE vector; Lane M–1 kb DNA molecular weight marker ( E ) Sanger sequencing result of the assembled RBD gene. Blue and orange arrows indicate forward and reverse primers, respectively. Distinct lanes cropped from one gel image have been assembled as a composite image and separated by white lines and correspond to the marker run on the same gel.

Article Snippet: Either 100 ng of PCR product or 200 ng of plasmid DNA template was added to the reaction mix consisting of 0.5 μl of BigDye Terminator 3.1 ready reaction mix (Applied Biosystems, #4336697), 1.75 μl of BigDye Terminator v1.1 & v3.1 5x sequencing buffer, and 1 μl (10 μM) of forward primer or reverse primer, adjusted to 10 μl using nuclease-free water.

Techniques: Agarose Gel Electrophoresis, Molecular Weight, Marker, Variant Assay, Clone Assay, Plasmid Preparation, Sequencing