Quantification of DNA in a forensic sample is of major importance for proper DNA amplification and STR profiling. Several methods have been developed to quantify DNA, from basic UV spectrometry, through gel-based techniques, to dye staining, blotting techniques, and, very recently, DNA amplification methods (polymerase chain reaction, PCR). Early techniques simply measured total DNA, but newer techniques can specifically measure human DNA while excluding non-human DNA (foodstuff, animal, or bacterial contamination). These newer assays can be faster and less expensive than traditional methods, making them ideal for the busy forensic laboratory. This paper reviews classic and newer quantification techniques and presents methods recently developed by the authors on the basis of PCR of Alu sequences.
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http://dx.doi.org/10.1007/s00216-003-1924-z | DOI Listing |
Anal Chem
January 2025
School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China.
Accurate identification and quantification of 5-hydroxymethylcytosine (5hmC) can help elucidate its function in gene expression and disease pathogenesis. Current 5hmC analysis methods still present challenges, especially for clinical applications, such as having a risk of false-positive results and a lack of sufficient sensitivity. Herein, a 5hmC quantification method for fragment-specific DNA sequences with extreme specificity, high sensitivity, and clinical applicability was established using a quantitative real-time PCR (qPCR)-based workflow through the combination of enzymatic digestion and biological deamination strategy (EDD-5hmC assay).
View Article and Find Full Text PDFJ Vis Exp
December 2024
Institute for Biological and Medical Engineering, Pontificia Universidad Católica de Chile;
Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy.
View Article and Find Full Text PDFAnal Chem
January 2025
Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Aptamer Engineering Center of Hunan Province, Hunan University, Changsha, Hunan 410082, China.
DNA reaction equilibrium-based calculations have great potential in thermodynamic characterization, but their widespread applications are hindered by significant measurement deviation of equilibrium concentration. Here, we report the advantages of metastable DNA hybridization in reducing quantification deviation of equilibrium concentration and propose a universal and standardized strategy for measuring aptamer binding energy, termed metastable DNA reference calorimetry (MDRC). We built different MDRC-based algorithms tailored to different aptamer binding models, enabling the calculation of thermodynamic parameters for aptamers with one or more binding sites.
View Article and Find Full Text PDFAnal Chim Acta
February 2025
Institute of Microfluidic Chip Development in Biomedical Engineering, College of Information Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China. Electronic address:
Background: Digital recombinase polymerase amplification (dRPA) is an effective tool for the absolute quantification of nucleic acids and the detection of rare mutations. Due to the high viscosity or other physical properties of the reagent, this can compromise the accuracy and reproducibility of detection results, which limits the broader adoption and practical application of this technology. In this study, we developed an asymmetric contact angle digital isothermal detection (ACA-DID) chip and optimized the ACA-DID chip structure to achieve rapid digital recombinase polymerase amplification.
View Article and Find Full Text PDFFoods
December 2024
Grain Research Laboratory, Canadian Grain Commission, Winnipeg, MB R3P1N1, Canada.
The number of genetically modified (GMO) events for canola, corn, and soybean is steadily increasing. Some countries, including those in the EU, have regulatory requirements for the approval and use of plant ingredients containing GMOs. Multiplex digital PCR (dPCR) has been used for the simultaneous detection and quantification of various GMO events.
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