Journal: The Journal of Biological Chemistry

Article Title: Simple in vitro single-stranded linear and circular DNA preparation, functional selection, and validation using phosphor-derived modifications

doi: 10.1016/j.jbc.2025.110874

Figure Lengend Snippet: Preparation of ssPCRP. A , visualization via a 2% agarose gel. Lane M: markers. Lanes 1 and 14: single-stranded amplicon with a final length of 100 bp (80 μM). Lanes 2, 5, 8, and 11: PCR products. Lanes 3, 6, 9, and 12: ssPCRP preparation with lambda exonuclease enzyme from PCR products. Lanes 4, 7, 10, and 13: ssPCRP treatment using exonuclease I enzyme. All PCRs were performed using phosphorylated reverse primers (1 μM). Lanes 2, 3, and 4; 5, 6, and 7; 8, 9, and 10; and 11, 12, and 13 have been done using phosphorothioated, phosphorothioated–phosphorylated, normal, and phosphorylated forward primers, respectively, as indicated in the figure, in a final concentration of 1 μM. B , ssPCRP analysis using 6% denaturing PAGE. Lanes 1 and 2: 32 P-labeled 100-base oligonucleotide (0.1 pmol), intact or treated with lambda exonuclease, respectively. Lanes 3 and 6: PCR with phosphorylated reverse primers (1 μM). Lanes 4 and 7: ssPCRP preparation by lambda exonuclease. Lanes 5 and 8: ssPCRP incubation with exonuclease I enzyme. Lanes 3, 4, and 5; and lanes 6, 7, and 8: PCRs were done by employing normal- 32 P-labeled and phosphorothioated- 32 P-labeled forward primers, respectively, in a final amount of 4 pmol. ssPCRP, single-stranded PCR product.

Article Snippet: For ssPCRP preparation, each 10 μl of double-stranded PCR products were treated with 5 units of lambda exonuclease enzyme (New England Biolabs [NEB]) at 37 °C for 45 min, which is followed by 10 min at 75 °C for enzyme inactivation.

Techniques: Agarose Gel Electrophoresis, Amplification, Concentration Assay, Labeling, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Simple in vitro single-stranded linear and circular DNA preparation, functional selection, and validation using phosphor-derived modifications

doi: 10.1016/j.jbc.2025.110874

Figure Lengend Snippet: PCR product circularization strategy. A , schematic representation of the template used with specific sequences as PCR primer–binding sites ( red line ) at the extremities and random sequences ( dark blue line ) in the middle . B , PCR using reverse primer ( orange line ) with 5′ phosphorylation modification ( green dot ) and forward primer ( light blue line ) with 5′ phosphorylation modification ( green dot ) and five phosphorothioate bonds ( gray line ) at the far 5′ end of the primer. The “A” illustrates the adenine added by the Taq DNA polymerase at the 3′ end of its product. C , exonuclease reaction by utilizing the lambda exonuclease enzyme ( orange semicircle ) for degradation of the nonphosphorothioated reverse strand. D , single-stranded PCR product (ssPCRP) circularization via complementary strand ( black line ). E , head-to-tail sealing of ssPCRP through ligation reaction using a ligase enzyme ( yellow ellipse ). F , finally, nonligated ssPCRP and the complementary strand are degraded by the action of exonuclease I enzyme ( red semicircle ).

Article Snippet: For ssPCRP preparation, each 10 μl of double-stranded PCR products were treated with 5 units of lambda exonuclease enzyme (New England Biolabs [NEB]) at 37 °C for 45 min, which is followed by 10 min at 75 °C for enzyme inactivation.

Techniques: Binding Assay, Phospho-proteomics, Modification, Ligation