Enhancement of PCRs by Partial Restriction Digestion of Genomic Templates

Vázquez, Raúl; Steinberg, Mark L.

doi:10.2144/99261st02

Cited by 5 publications

(4 citation statements)

References 5 publications

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“…Prior to actual analysis, we first evaluated template amplification in digital panels by comparing results with undigested genomic DNA and genomic DNA digested with either Taq I or Msp I restriction endonuclease. Enhancement of amplification efficiency has been observed previously following fragmentation of target genomic DNA with restriction enzymes . The results confirm that digestion of lambda genomic DNA with either Taq I or Msp I improves efficiency of target amplification and reduces the proportion of positive partitions with a Ct >30 compared to undigested DNA.…”

Section: Resultssupporting

confidence: 85%

Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units

et al. 2010

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Accurate estimation of total DNA concentration (mass concentration, e.g., ng/muL) that is traceable to the International System of Units (SI) is a crucial starting point for improving reproducible measurements in many applications involving nucleic acid testing and requires a DNA reference material which has been certified for its total DNA concentration. In this study, the concentrations of six different lambda DNA preparations were determined using different measurement platforms: UV Absorbance at 260 nm (A(260)) with and without prior sodium hydroxide (NaOH) treatment of the DNA, PicoGreen assay, and digital polymerase chain reaction (dPCR). DNA concentration estimates by A(260) with and without prior NaOH treatment were significantly different for five of the six samples tested. There were no significant differences in concentration estimates based on A(260) with prior NaOH treatment, PicoGreen analysis, and dPCR for two of the three samples tested using dPCR. Since the measurand in dPCR is amount (copy number) concentration (copies/muL), the results suggest that accurate estimation of DNA mass concentration based on copy number concentration is achievable provided the DNA is fully characterized and in the double-stranded form or amplification is designed to be initiated from only one of the two complementary strands.

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Section: Resultssupporting

confidence: 85%

Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units

et al. 2010

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“…An investigation of the impact of circular versus linear DNA template was also performed using microfluidic dPCR (Figure ). It has previously been demonstrated that the type of template can affect quantification values when using qPCR. ,− DNA fragmentation or plasmid linearization can increase amplification efficiency and minimize bias in estimation of the true copy number. ,− In the experiment described here, directly comparable aliquots of ADH template were evaluated in both circular and linearized forms. The same eluate was either digested ( n = 3 reactions) or undigested ( n = 3 negative control digest reactions) prior to dilution to ∼57 copies/panel, based on Nanodrop UV spectrophotometry estimations.…”

Section: Resultsmentioning

confidence: 99%

Evaluation of Digital PCR for Absolute DNA Quantification

Sanders¹,

Huggett²,

Bushell³

et al. 2011

Anal. Chem.

294

237

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The emerging technique of microfluidic digital PCR (dPCR) offers a unique approach to real-time quantitative PCR for measuring nucleic acids that may be particularly suited for low-level detection. In this study, we evaluated the quantitative capabilities of dPCR when measuring small amounts (<200 copies) of DNA and investigated parameters influencing technical performance. We used various DNA templates, matrixes, and assays to evaluate the precision, sensitivity and reproducibility of dPCR, and demonstrate that this technique can be highly reproducible when performed at different times and when different primer sets are targeting the same molecule. dPCR exhibited good analytical sensitivity and was reproducible outside the range recommended by the instrument manufacturer; detecting 16 estimated targets with high precision. The inclusion of carrier had no effect on this estimated quantity, but did improve measurement precision. We report disagreement when using dPCR to measure different template types and when comparing the estimated quantities by dPCR and UV spectrophotometry. Finally, we also demonstrate that preamplification can impose a significant measurement bias. These findings provide an independent assessment of low copy molecular measurement using dPCR and underline important factors for consideration in dPCR experimental design.

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“…Dilutions into a ddPCR reaction that are less than 20-fold would require a genome concentration in the lysed AAV sample that is even lower than 80 fM to get 2,500 copies/μL. Note that the pAV-CMV-GFP data uses HindIII, which has a single cleavage site in pAV-CMV-GFP that is located outside of all the milepost assays, instead of a no enzyme condition, to convert the plasmid to a linear conformation and minimize any potential quantification bias resulting from amplicon access in supercoiled plasmids [ 9 , 25 , 26 , 54 , 55 ].…”

Section: Discussionmentioning

confidence: 99%

Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

Prantner

Maar

2023

PLoS ONE

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Precise, reproducible characterization of AAV is critical for comparing preclinical results between laboratories and determining a safe and effective clinical dose for gene therapy applications. In this study, we systematically evaluated numerous parameters to produce a simple and robust ddPCR protocol for AAV characterization. The protocol uses a low ionic strength buffer containing Pluronic-F68 and polyadenylic acid to dilute the AAV into the ddPCR concentration range and a 10-minute thermal capsid lysis prior to assembling ddPCR reactions containing MspI. A critical finding is that the buffer composition affected the ITR concentration of AAV but not the ITR concentration of a double stranded plasmid, which has implications when using a theoretical, stoichiometric conversion factor to obtain the titer based on the ITR concentration. Using this protocol, a more comprehensive analysis of an AAV vector formulation was demonstrated with multiple ddPCR assays distributed throughout the AAV vector genome. These assays amplify the ITR, regulatory elements, and eGFP transgene to provide a more confident estimate of the vector genome concentration and a high-resolution characterization of the vector genome identity. Additionally, we compared two methods of genome integrity analysis for three control sample types at eight different concentrations for each sample. The genome integrity was independent of sample concentration and the expected values were obtained when integrity was determined based on the excess number of positive droplets relative to the number of double positive droplets expected by chance co-encapsulation of two DNA targets. The genome integrity was highly variable and produced unexpected values when the double positive droplet percentage was used to calculate the genome integrity. A protocol using a one-minute thermal capsid lysis prior to assembling ddPCR reactions lacking a restriction enzyme used the non-ITR assays in a duplex ddPCR milepost experiment to determine the genome integrity using linkage analysis.

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Enhancement of PCRs by Partial Restriction Digestion of Genomic Templates

Cited by 5 publications

References 5 publications

Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units

Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units

Evaluation of Digital PCR for Absolute DNA Quantification

Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

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