- •qPCR tends to underestimate the quantity of degraded DNA.
- •The average RFU value can be converted into DNA quantity.
- •The method can be standardised across different laboratories.
- •Applications include activity level and direct PCR.
- •A software is provided to facilitate calculations.
2. Use of qPCR to measure DNA quantity
3. An outline of factors that affect the outcome
- 1.The sampling method: The swabbing or tape technique used to remove cells/ free DNA from the evidence.
- 2.The extraction method: Technique/ reagents used (solid, magnetic beads etc.)
- 3.The elution volume () : The volume of the eluant in l that contains the total DNA from the swabs.
- 4.Quantification: Concentration () of DNA in the eluant, using short () and long () fragments respectively, measured in ng/l. Variable measures the total amount of DNA in ng that is recovered from the eluant (), hence
- 5.Degradation factor (): Measured as the proportion: and since DNA is subject to varying degrees of degradation, that increasingly affects the high molecular weight loci (see [], Section 4.5.1).
- 6.The volume of the eluant in l that is taken for PCR () : Laboratories will seek to optimise the amount of DNA that is subject to PCR. A typical target amount may be a total of 1ng. For high template recovery, it is necessary to dilute the sample to obtain the optimum amount, whereas for low template, it may not be possible to achieve the target amount, hence the profile will be lower quality and/or partial.
- 7.The pre-PCR set-up will consist of:(a) Volume : The reaction mix of primers for a given multiplex, Taq polymerase and buffer.(b) Volume : Taken from the eluant and added to volume of buffer or water () so that a constant volume is always achieved irrespective of the sample analysed. Hence the total PCR volume is and is also a constant volume across all experiments.
- 8.PCR amplification: The number of cycles; the multiplex used; volumes of reaction buffer; the PCR machine, all affect the outcome.
- 9.Post-PCR analysis: The analytical platform; manufacturer and model; the injection parameters used; analytical threshold.
4. Relationship between DNA concentration and
4.2 Calibration protocol
where is the sum of the peak heights per locus , with loci in the multiplex. The formula can further be expanded to include replicates:
where is the replicate index and is the total number of replicates and replicates are based upon the same multiplex.
- Hill C.R.
- Duewer D.L.
- Kline M.C.
- Sprecher C.J.
- McLaren R.S.
- Rabbach D.R.
- Krenke B.E.
- Ensenberger M.G.
- Fulmer P.M.
- Storts D.R.
- et al.
where is independent identically distributed as normal with expectation zero and constant variance.
where intercept and regression coefficient, which must be estimated. Since the control samples comprise pristine DNA (Fig. 1), they provide regressions with very high R-squared values . The coefficient is very close to one in both regressions (and standard errors are low). Consequently, by fixing , the prediction equation can be simplified to ; alternatively, to predict the amount of amplifiable DNA for a given , this is simply:
All that is needed to carry out the conversion to DNA quantity is a multiplex specific intercept regression coefficient () (Table 1).
To conclude, provided pristine samples are analysed with minimal degradation, the amplicon provides an excellent method to estimate the actual amount of amplifiable DNA represented by the multiplex in question and this underpins the basis of the calibration.
4.2.1 Calibration of the regression slope coefficient and intercept
|Coefficients||Standard error||95% CI||R-squared|
|Fusion 6C||5.21 (5.2)||0.08||0.98|
|Vaginal mucosa||5.08 (5.25)||0.21||0.57|
|Skin cells||4.70 (4.84)||0.20||0.49|
4.3 Comparison of casework samples
5. Calculation of the dilution factor
5.1 Calculation of the dilution factor when low template DNA is recovered
5.2 Calculation of the dilution factor when high template DNA is recovered
6. Normalisation of data to compare results generated from different protocols
where , and are the Fusion 6C observation and pre-determined coefficients; , and are the ESX17 observation and pre-determined coefficients. To normalise ESX17 to the Fusion 6C expectation, for any given value of :
By rearrangement, is normalised:
or without logs:
i.e., substitutes in Eq. (14).
Because is used as the standard volume, everything is normalised against this (Fig. 2, Fig. 4, Fig. 6 all show adjusted values).
6.1 Using total quantity
6.2 Extraction efficiency
7. Limitations of qPCR quantification
8. General applicability where prior quantification is precluded
9. A summary of recommendations
- 1.Any given system can be calibrated with a dilution series of approximately six control samples that must be undegraded and of known quantity. They must be carefully selected to be within the log-linear range of fluorescence response as illustrated by Fig. 1. A successful calibration will give regression slope (, allowance for error). The intercept () coefficient is a measurement of the sensitivity of the method.
- 3.The ng/l value is apportioned between contributors by multiplying by the mixture proportion () and the dilution factor (). is calculated using probabilistic genotyping software
- 4.The total quantity () in ng of DNA recovered is calculated with Eq. (17)
- 5.Worked examples are shown in the Supplementary Material S1
10. Software to calculate average RFU
Declaration of Competing Interest
Appendix A. Supplementary data
- MMC S1
Some examples showing how to calculate the dilution factor and DNA quantity.
- Estimating the probability of allelic drop-out of STR alleles in forensic genetics.Forensic Sci. Int. Genet. 2009; 3: 222-226
- Evaluating the weight of evidence by using quantitative short tandem repeat data in DNA mixtures.J. R. Stat. Soc. Ser. C. Appl. Stat. 2010; 59: 855-874
- RFU derived LRs for activity level assignments using Bayesian networks.Forensic Sci. Int. Genet. 2022; 56102608
- Non-self DNA on the neck: a 24 hours time-course study.Forensic Sci. Int. Genet. 2022; 102661
- Who packed the drugs? Application of Bayesian networks to address questions of DNA transfer, persistence, and recovery from plastic bags and tape.Genes. 2021; 13: 18
- EuroForMix: An open source software based on a continuous model to evaluate STR DNA profiles from a mixture of contributors with artefacts.Forensic Sci. Int. Genet. 2016; 21: 35-44
- Evaluation of forensic genetics findings given activity level propositions: A review.Forensic Sci. Int. Genet. 2018; 36: 34-49
- Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant® system.Forensic Sci. Int. Genet. 2016; 23: 166-177
- Developmental validation of a real-time PCR assay for the simultaneous quantification of total human and male DNA.Forensic Sci. Int. Genet. 2008; 3: 14-21
- Direct visualization of the highly polymorphic RNU2 locus in proximity to the BRCA1 gene.PLoS One. 2013; 8e76054
- Direct qPCR quantification using the Quantifiler® Trio DNA quantification kit.Forensic Sci. Int. Genet. 2014; 13: 10-19
- Forensic Practitioner’s Guide to the Interpretation of Complex DNA Profiles.Academic Press, 2020
- RNA/DNA co-analysis from human skin and contact traces–results of a sixth collaborative EDNAP exercise.Forensic Sci. Int. Genet. 2015; 16: 139-147
- Developmental validation of the PowerPlex® Fusion 6C system.Forensic Sci. Int. Genet. 2016; 21: 134-144
- Concordance and population studies along with stutter and peak height ratio analysis for the PowerPlex® ESX 17 and ESI 17 systems.Forensic Sci. Int. Genet. 2011; 5: 269-275
- Quantitating fluorescence intensity from fluorophores: practical use of MESF values.J. Res. Natl. Inst. Stand. Technol. 2002; 107: 339
- Transfer, persistence and recovery of DNA and mRNA vaginal mucosa markers after intimate and social contact with Bayesian network analysis for activity level reporting.Forensic Sci. Int. Genet. 2022; 60: 102750
- Evaluating the performance of five up-to-date DNA/RNA co-extraction methods for forensic application.Forensic Sci. Int. 2021; 328110996
- Helping to distinguish primary from secondary transfer events for trace DNA.Forensic Sci. Int. Genet. 2017; 28: 155-177
- Degradation of forensic DNA profiles.Aust. J. Forensic Sci. 2013; 45: 445-449
- The quality assurance standards for forensic dna testing laboratories, effective july 1, 2020.2022 (https://www.swgdam.org/_files/ugd/4344b0_d73afdd0007c4ed6a0e7e2ffbd6c4eb8.pdf. (Accessed 23 May 2022))
- Recent trends and developments in forensic DNA extraction.Wiley Interdiscip. Rev. Forensic Sci. 2021; 3e1395
User LicenseCreative Commons Attribution (CC BY 4.0) |
- Read, print & download
- Redistribute or republish the final article
- Text & data mine
- Translate the article
- Reuse portions or extracts from the article in other works
- Sell or re-use for commercial purposes
Elsevier's open access license policy