ptr uf11  (Addgene inc)

Journal: Human Gene Therapy Methods

Article Title: Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors

doi: 10.1089/hgtb.2019.031

Figure Lengend Snippet: Measured values of qPCR titer depend on the secondary structure of the reference standards. (A) Genome structure of rAAV2RSM. Thin arrows and thick arrows indicate the positions of the amplification target sites on ITR and SV40, respectively. (B) Vector genome titer of rAAV2RSM determined by qPCR showed variation when using reference standards with different secondary structures in pTR-UF11 plasmid DNA. Circular plasmid DNA; linearized plasmid DNA linearized by ScaI digestion; Sma I digested plasmid DNA. A 95% CI of rAAV2RSM determined by the AAVRSM Working Group is 2.7–4.75 × 10 vg/mL. (C) Calibration curves for ITR qPCR ( upper panel ) and SV40 qPCR ( lower panel ) obtained using reference standards with different structures. R 2 of each calibration curve was >0.99. qPCR of RSM was performed after DNase I and proteinase K treatment as described in section. Because SDS in proteinase K buffer inhibits the qPCR and thus the signal could not be detected when the sample was diluted <100-fold, thus only five points were measured in the RSM sample. CI, confidence interval; Ct, threshold cycle; ITR, inverted terminal repeat; qPCR, quantitative PCR; rAAV, recombinant adeno-associated virus; rAAV2RSM, rAAV reference standard material for serotype 2; RSM, reference standard material; SDS, sodium dodecyl sulfate; vg, vector genome.

Article Snippet: Linearized plasmid was obtained by digesting pTR-UF11 with ScaI (within the ampicillin-resistant gene in the plasmid backbone), and plasmid without the ITR structure was prepared by digestion with Sma I (within the ITR region of rAAV) (Takara Bio Inc.).

Techniques: Amplification, Plasmid Preparation, Real-time Polymerase Chain Reaction, Recombinant

Journal: Human Gene Therapy Methods

Article Title: Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors

doi: 10.1089/hgtb.2019.031

Figure Lengend Snippet: The secondary structure of the plasmid DNA tail disturbed the amplification of ITR by qPCR. (A) The illustrations show the left part of the rAAV2RSM positive strand and the different secondary structures of pTR-UF11 plasmid DNAs. Thin arrows and thick arrows indicate the position of ITR and SV40 target sites, respectively. The arrowhead indicates the cutting site of Sma I. The treatment of pTR-UF11 with Sma I disrupted the ITR secondary structure. qPCR amplification was more efficient in the absence of hindrance by the plasmid tail, in Sma I-digested plasmid, and in the rAAV genome compared with linearized plasmid or circular plasmid. This resulted in up shift or down shift of the standard curves and thus overestimation or underestimation of the values measured by qPCR as shown in (B) .

Article Snippet: Linearized plasmid was obtained by digesting pTR-UF11 with ScaI (within the ampicillin-resistant gene in the plasmid backbone), and plasmid without the ITR structure was prepared by digestion with Sma I (within the ITR region of rAAV) (Takara Bio Inc.).

Techniques: Plasmid Preparation, Amplification

Journal: Human Gene Therapy Methods

Article Title: Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors

doi: 10.1089/hgtb.2019.031

Figure Lengend Snippet: 2D ddPCR detection to evaluate the integrity of rAAV2RSM. Dot plot profiles of FAM-labeled SV40 primer and probe (channel 1, amplitude) and HEX-labeled ITR primer and probe (channel 2, amplitude). Droplets emitting 2D signals were separated into four groups as indicated. The number of droplets in each single or double positive group was calculated based on the Poisson distribution. The ratios of each group are indicated as percentages. (A) rAAV2RSM. (C) , Sma I-digested pTR-UF11. (B) The dynamic linear range of each group was confirmed by detecting emission from a series of diluted samples. 2D, two-dimensional; FAM, fluorescein; HEX, hexachloro-6-carboxy-fluorescine.

Article Snippet: Linearized plasmid was obtained by digesting pTR-UF11 with ScaI (within the ampicillin-resistant gene in the plasmid backbone), and plasmid without the ITR structure was prepared by digestion with Sma I (within the ITR region of rAAV) (Takara Bio Inc.).

Techniques: Labeling

Journal: Human Gene Therapy Methods

Article Title: Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors

doi: 10.1089/hgtb.2019.031

Figure Lengend Snippet: Correlation between the integrity of rAAV2RSM evaluated by ddPCR and transfection activity. (A) rAAV2RSM was incubated at 37°C for 0, 1, 3, or 7 days and activity was detected as TU toward Vero cell by FACS analysis after 48 h of infection. The activity of rAAV was demonstrated as the ratio to day 0. (B) Correlation between ddPCR or qPCR titer and TU activity. The vector genome titers of rAAV2RSM were simultaneously detected by qPCR (SV40 titer using ScaI-digested pTR-UF11 as standard) and ddPCR (ITR + /SV40 + group). Values are indicated as mean ± SD (activity n = 3, titer n = 4). (C) The percentage of incomplete vector genome of treated rAAV2RSM. TU, transduction activity.

Article Snippet: Linearized plasmid was obtained by digesting pTR-UF11 with ScaI (within the ampicillin-resistant gene in the plasmid backbone), and plasmid without the ITR structure was prepared by digestion with Sma I (within the ITR region of rAAV) (Takara Bio Inc.).

Techniques: Transfection, Activity Assay, Incubation, Infection, Plasmid Preparation, Transduction

Journal: Human Gene Therapy Methods

Article Title: Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors

doi: 10.1089/hgtb.2019.031

Figure Lengend Snippet: Measured values of qPCR titer depend on the secondary structure of the reference standards. (A) Genome structure of rAAV2RSM. Thin arrows and thick arrows indicate the positions of the amplification target sites on ITR and SV40, respectively. (B) Vector genome titer of rAAV2RSM determined by qPCR showed variation when using reference standards with different secondary structures in pTR-UF11 plasmid DNA. Circular plasmid DNA; linearized plasmid DNA linearized by ScaI digestion; Sma I digested plasmid DNA. A 95% CI of rAAV2RSM determined by the AAVRSM Working Group is 2.7–4.75 × 10 vg/mL. (C) Calibration curves for ITR qPCR ( upper panel ) and SV40 qPCR ( lower panel ) obtained using reference standards with different structures. R 2 of each calibration curve was >0.99. qPCR of RSM was performed after DNase I and proteinase K treatment as described in section. Because SDS in proteinase K buffer inhibits the qPCR and thus the signal could not be detected when the sample was diluted <100-fold, thus only five points were measured in the RSM sample. CI, confidence interval; Ct, threshold cycle; ITR, inverted terminal repeat; qPCR, quantitative PCR; rAAV, recombinant adeno-associated virus; rAAV2RSM, rAAV reference standard material for serotype 2; RSM, reference standard material; SDS, sodium dodecyl sulfate; vg, vector genome.

Article Snippet: rAAV2RSM, rAAV8RSM, and vector plasmid pTR-UF11, each containing AAV2 ITR, GFP, and the SV40 polyA sequence (SV40), were purchased from ATCC.

Techniques: Amplification, Plasmid Preparation, Real-time Polymerase Chain Reaction, Recombinant

Journal: Human Gene Therapy Methods

Article Title: Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors

doi: 10.1089/hgtb.2019.031

Figure Lengend Snippet: The secondary structure of the plasmid DNA tail disturbed the amplification of ITR by qPCR. (A) The illustrations show the left part of the rAAV2RSM positive strand and the different secondary structures of pTR-UF11 plasmid DNAs. Thin arrows and thick arrows indicate the position of ITR and SV40 target sites, respectively. The arrowhead indicates the cutting site of Sma I. The treatment of pTR-UF11 with Sma I disrupted the ITR secondary structure. qPCR amplification was more efficient in the absence of hindrance by the plasmid tail, in Sma I-digested plasmid, and in the rAAV genome compared with linearized plasmid or circular plasmid. This resulted in up shift or down shift of the standard curves and thus overestimation or underestimation of the values measured by qPCR as shown in (B) .

Article Snippet: rAAV2RSM, rAAV8RSM, and vector plasmid pTR-UF11, each containing AAV2 ITR, GFP, and the SV40 polyA sequence (SV40), were purchased from ATCC.

Techniques: Plasmid Preparation, Amplification

Journal: Human Gene Therapy Methods

Article Title: Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors

doi: 10.1089/hgtb.2019.031

Figure Lengend Snippet: 2D ddPCR detection to evaluate the integrity of rAAV2RSM. Dot plot profiles of FAM-labeled SV40 primer and probe (channel 1, amplitude) and HEX-labeled ITR primer and probe (channel 2, amplitude). Droplets emitting 2D signals were separated into four groups as indicated. The number of droplets in each single or double positive group was calculated based on the Poisson distribution. The ratios of each group are indicated as percentages. (A) rAAV2RSM. (C) , Sma I-digested pTR-UF11. (B) The dynamic linear range of each group was confirmed by detecting emission from a series of diluted samples. 2D, two-dimensional; FAM, fluorescein; HEX, hexachloro-6-carboxy-fluorescine.

Article Snippet: rAAV2RSM, rAAV8RSM, and vector plasmid pTR-UF11, each containing AAV2 ITR, GFP, and the SV40 polyA sequence (SV40), were purchased from ATCC.

Techniques: Labeling

Journal: Human Gene Therapy Methods

Article Title: Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors

doi: 10.1089/hgtb.2019.031

Figure Lengend Snippet: Correlation between the integrity of rAAV2RSM evaluated by ddPCR and transfection activity. (A) rAAV2RSM was incubated at 37°C for 0, 1, 3, or 7 days and activity was detected as TU toward Vero cell by FACS analysis after 48 h of infection. The activity of rAAV was demonstrated as the ratio to day 0. (B) Correlation between ddPCR or qPCR titer and TU activity. The vector genome titers of rAAV2RSM were simultaneously detected by qPCR (SV40 titer using ScaI-digested pTR-UF11 as standard) and ddPCR (ITR + /SV40 + group). Values are indicated as mean ± SD (activity n = 3, titer n = 4). (C) The percentage of incomplete vector genome of treated rAAV2RSM. TU, transduction activity.

Article Snippet: rAAV2RSM, rAAV8RSM, and vector plasmid pTR-UF11, each containing AAV2 ITR, GFP, and the SV40 polyA sequence (SV40), were purchased from ATCC.

Techniques: Transfection, Activity Assay, Incubation, Infection, Plasmid Preparation, Transduction