Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability

Gill, Peter; Bleka, Øyvind; Fonneløp, Ane Elida

doi:10.1016/j.fsigen.2022.102777

Cited by 9 publications

(13 citation statements)

References 20 publications

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“…However, the current quantification methods are stated to be too inaccurate to give the “true” amount of DNA, which complicates the assessment of homogeneity. As Gill et al [45] recently described, a quantification method could overestimate the amount of amplifiable DNA (especially for degraded samples). In their research, they outlined that a posterior quantification (using RFUs of amplified products) seems to provide a better estimate, which, however, remains an estimate.…”

Section: Discussionmentioning

confidence: 99%

Collecting touch DNA from glass surfaces using different sampling solutions and volumes: Immediate and storage effects on genetic STR analysis

Schulte,

Rittiner,

Seiberle

et al. 2023

Journal of Forensic Sciences

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Touch DNA has become increasingly important evidence in todays' forensic casework. However, due to its invisible nature and typically minute amounts of DNA, the collection of biological material from touched objects remains a particular challenge that underscores the importance of the best collection methods for maximum recovery efficiency. So far, swabs moistened with water are often utilized in forensic crime scene investigations for touch DNA sampling, even though an aqueous solution provokes osmosis, endangering the cell's integrity. The aim of the research presented here was to systematically determine whether DNA recovery from touched glass items can be significantly increased by varying swabbing solutions and volumes compared with water‐moistened swabs and dry swabbing. A second objective was to investigate the possible effects of storage of swab solutions prior to genetic analysis on DNA yield and profile quality when stored for 3 and 12 months, as is often the case with crime scene samples. Overall, the results indicate that adapting volumes of the sampling solutions had no significant effect on DNA yield, while the detergent‐based solutions performed better than water and dry removal, with the SDS reagent yielding statistically significant results. Further, stored samples showed an increase in degradation indices for all solutions tested, but no deterioration in DNA content and profile quality, allowing for unrestricted processing of touch DNA samples stored for at least 12 months. One further finding was a strong intraindividual change in DNA amounts observed over the 23 deposition days which may be related to the donor's menstrual cycle.

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Section: Discussionmentioning

confidence: 99%

Collecting touch DNA from glass surfaces using different sampling solutions and volumes: Immediate and storage effects on genetic STR analysis

Schulte,

Rittiner,

Seiberle

et al. 2023

Journal of Forensic Sciences

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“…Linear regression is used to estimate the constant value (number of molecules present at C t ) by averaging the results for the three species. Then K≃ 7.6 : 10 12 (equation 8) is inferred from observed relative fluorescence unit (RFU) values, assuming within-replicate proportionality between RFU and DNA copy number (Gill et al, 2022), although RFU values are not standardized and depend on many experimental factors (Svec et al, 2015). Then, the efficiencies Λ s were estimated for each replicate from this constant value of (equation 9).…”

Section: Measure Of the Amplification Efficienciesmentioning

confidence: 99%

“…For this study, however, only the relative values of the efficiencies are important. A commonly used formula (equation 9, Gill, Bleka, & Fonneløp, 2022) can be derived from the exponential model to estimate amplification efficiencies from a series of qPCRs performed on successive dilutions. However, a major limitation of this formula that has been identified here is that the estimation of the slope is very sensitive to small variations in C t , resulting in a large variance of the estimator of the efficiency Λ.…”

Section: Measure Of the Amplification Efficienciesmentioning

confidence: 99%

“…The estimate of K is given by equation 10. This equation is established from observed relative fluorescence unit (RFU) values, assuming within-replicate proportionality between RFU and DNA copy number (Gill et al, 2022), although RFU values are not standardized, and fluorescence saturates at the end of amplification and depends on many experimental factors (Svec et al, 2015). This approach only provides an estimate: K = 7.6 × 10 12 here (see Discussion).…”

Section: Measure Of the Amplification Efficienciesmentioning

confidence: 99%

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Towards quantitative DNA Metabarcoding: A method to overcome PCR amplification bias

Moinard,

Piau,

Laporte

et al. 2023

Preprint

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Metabarcoding analyses have recently undergone significant development due to the power of this technique in biodiversity monitoring. However, it is still difficult to draw accurate quantitative conclusions about the ecosystems studied, mainly because of biases inherent in the environmental DNA or introduced during the experimental process. These biases alter the relationship between the amount of DNA observed and the biomass or number of individuals of the species detected. Two of the biases inherent in metabarcoding have been measured: the ratio between total DNA and target DNA concentrations, and the PCR amplification bias. A method for their correction is proposed. All experimental tests were performed on mock alpine plant communities using the marker Sper01, which is expected to have low amplification bias due to its highly conserved priming sites. Our approach combines standard quantitative PCR techniques (qPCR and digital droplet PCR) with a realistic stochastic model of PCR dynamics that accounts for PCR saturation. The model was used to estimate PCR efficiencies for each species and to infer the true species proportions of the mock communities from the read relative frequencies. The corrections are easy to implement and can be applied to previously generated DNA metabarcoding data. This work demonstrates the relative importance of the two biases considered and is an open door to quantitative metabarcoding data, although many other biases remain to be considered.

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“…In contrast, qPCR permits the rapid and accurate measurement of the DNA concentrations. Consequently, optimization and validation performed to ascertain the specificity and sensitivity of an assay are rarely reported in the scientific literature . However, the extent to which qPCR can efficiently detect DNA damage not usually considered to be polymerase-blocking, such as 8-hydroxydeoxyguanosine (8-OHdG), has not been rigorously investigated .…”

Section: Introductionmentioning

confidence: 99%

Simple and Practical DNA Quantification Method for DNA-Encoded Library Synthesis

Ma,

Sun,

Xue

et al. 2023

ACS Omega

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Over the past three decades, DNA-encoded library (DEL) technologies have become one of the most relevant strategies for hit-finding. Recent advances in synthetic methodologies for DNA-encoded libraries rendered the increased chemical space available, but it is unknown how every variety of chemistry affects DNA’s integrity. Available assays to quantify DNA damage are restricted to electrophoresis, ligation efficiency, and mostly qPCR quantification and sequencing, which may contain predisposition and inconsistency. We developed an external standard method through LC-MS analysis to accurately quantify DNA damage throughout the chemical transformations. An assessment was conducted on on-DNA chemical reactions that are frequently employed in DEL synthesis, and these results were compared to traditional qPCR measurements. Our study provides a simple, practicable, and accurate measurement for DNA degradation during DEL synthesis. Our finding reveals substantial disagreement among the usual DNA-damaging assessment methods, which have been largely neglected so far.

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Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability

Cited by 9 publications

References 20 publications

Collecting touch DNA from glass surfaces using different sampling solutions and volumes: Immediate and storage effects on genetic STR analysis

Collecting touch DNA from glass surfaces using different sampling solutions and volumes: Immediate and storage effects on genetic STR analysis

Towards quantitative DNA Metabarcoding: A method to overcome PCR amplification bias

Simple and Practical DNA Quantification Method for DNA-Encoded Library Synthesis

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