Digital Sort-Enabled Counting Allows Absolute Electrical Quantification of Target Nucleic Acid.

Quantitative nucleic acid amplification tests are of great importance for diagnostics, but current approaches require complex and costly optical setups that limit their nonlaboratory applications. Herein we describe the implementation of a microfluidics platform that can perform binary DNA-amplification-activated droplet sorting. The digital sort-enabled counting (DISCO) platform enables label-free absolute quantification of the nucleic acid. This is achieved by provoking a pH change in droplets through a loop-mediated isothermal amplification (LAMP) reaction, followed by using sorting by interfacial tension (SIFT) to direct positive and negative droplets to different outlets. With the use of on-chip electrodes at both outlets, we demonstrate that the digital electrical counting of target DNA and RNA can be realized. DISCO is a promising approach for realizing sensitive nucleic acid quantification in point-of-care settings.

Surface-Area Enhanced Raman Spectroscopy of DNA in Porous Silica: A Quantitative and Reproducible Alternative to Plasmonic-Based SERS.

Despite the success of surface-enhanced Raman spectroscopy (SERS) for detecting DNA immobilized on plasmonic metal surfaces, its quantitative response is limited by the rapid falloff of enhancement with distance from the metal surface and variations in sensitivity that depend on orientation and proximity to plasmonic "hot spots". In this work, we assess an alternative approach for enhancing detection by immobilizing DNA on the interior surfaces of porous silica particles. These substrates provide over a 1000-fold greater surface area for detection compared to a planar support. The porous silica substrate is a purely dielectric material with randomly oriented internal surfaces, where scattering is independent of proximity and orientation of oligonucleotides relative to the silica surface. We characterize the quantitative response of Raman scattering from DNA in porous silica particles with sequences used in previous SERS investigations of DNA for comparison. The results show that Raman scattering of DNA in porous silica is independent of distance of nucleotides from the silica surface, allowing detection of longer DNA strands with constant sensitivity. The surface area enhancement within particles is reproducible (<4% particle-to-particle variation) owing to the uniform internal pore structure and surface chemistry of the silica support. DNA immobilization with a bis-thiosuccinimide linker provides a Raman-active internal standard for quantitative interpretation of Raman scattering results. Despite the high (30 mM) concentrations of immobilized DNA within porous silica particles, they can be used to measure nanomolar binding affinities of target molecules to DNA by equilibrating a very small number of particles with a sufficiently large volume of low-concentration solution of target molecules.

Ion detection in a DNA nanopore FET device.

An ion detection device that combines a DNA-origami nanopore and a field-effect transistor (FET) was designed and modeled to determine sensitivity of the nanodevice to the local cellular environment. Such devices could be integrated into a live cell, creating an abiotic-biotic interface integrated with semiconductor electronics. A continuum model is used to describe the behavior of ions in an electrolyte solution. The drift-diffusion equations are employed to model the ion distribution, taking into account the electric fields and concentration gradients. This was matched to the results from electric double layer theory to verify applicability of the model to a bio-sensing environment. The FET device combined with the nanopore is shown to have high sensitivity to ion concentration and nanopore geometry, with the electrical double layer behavior governing the device characteristics. A logarithmic relationship was found between ion concentration and a single FET current, generating up to 200 nA of current difference with a small applied bias.

Nucleic acid-based electrochemical biosensors.

Colorectal cancer, breast cancer, oxidative DNA damage, and viral infections are all significant and major health threats to human health, presenting substantial challenges in early diagnosis. In this regard, a wide range of nucleic acid-based electrochemical platforms have been widely employed as point-of-care diagnostics in health care and biosensing technologies. This review focuses on biosensor design strategies, underlying principles involved in the development of advanced electrochemical genosensing devices, approaches for immobilizing DNA on electrode surfaces, as well as their utility in early disease diagnosis, with a particular emphasis on cancer, leukaemia, oxidative DNA damage, and viral pathogen detection. Notably, the role of biorecognition elements and nanointerfaces employed in the design and development of advanced electrochemical genosensors for recognizing biomarkers related to colorectal cancer, breast cancer, leukaemia, oxidative DNA damage, and viral pathogens has been extensively reviewed. Finally, challenges associated with the fabrication of nucleic acid-based biosensors to achieve high sensitivity, selectivity, a wide detection range, and a low detection limit have been addressed. We believe that this review will provide valuable information for scientists and bioengineers interested in gaining a deeper understanding of the fabrication and functionality of nucleic acid-based electrochemical biosensors for biomedical diagnostic applications.

Evaluation of DNA yield from various tissue and sampling sources for use in single nucleotide polymorphism panels.

Genetics studies are used by wildlife managers and researchers to gain inference into a population of a species of interest. To gain these insights, microsatellites have been the primary method; however, there currently is a shift from microsatellites to single nucleotide polymorphisms (SNPs). With the different DNA requirements between microsatellites and SNPs, an investigation into which samples can provide adequate DNA yield is warranted. Using samples that were collected from previous genetic projects from regions in the USA from 2014 to 2021, we investigated the DNA yield of eight sample categories to gain insights into which provided adequate DNA to be used in ddRADseq or already developed high- or medium-density SNP panels. We found seven sample categories that met the DNA requirements for use in all three panels, and one sample category that did not meet any of the three panels requirements; however, DNA integrity was highly variable and not all sample categories that met panel DNA requirements could be considered high quality DNA. Additionally, we used linear random-effects models to determine which covariates would have the greatest influence on DNA yield. We determined that all covariates (tissue type, storage method, preservative, DNA quality, time until DNA extraction and time after DNA extraction) could influence DNA yield.

Liquid nitrogen improves the decellularization effectiveness of whole-ovary.

BACKGROUND: Ovarian tissue cryopreservation for fertility preservation carries a risk of malignant cell re-seeding. Artificial ovary is a promising method to solve such a problem. However, ovary decellularization protocols are limited. Hence, further studies are necessary to get better ovarian decellularization techniques for the construction of artificial ovary scaffolds. OBJECTIVE: To establish an innovative decellularization technique for whole porcine ovaries by integrating liquid nitrogen with chemical agents to reduce the contact time between the scaffolds and chemical reagents. MATERIALS AND METHODS: Porcine ovaries were randomly assigned to three groups: novel decellularized group, conventional decellularized group and fresh group. The ovaries in the novel decellularized group underwent three cycles of freezing by liquid nitrogen and thawing at temperatures around 37 degree C before decellularization. The efficiency of the decellularization procedure was assessed through histological staining and DNA content analysis. The maintenance of ovarian decellularized extracellular matrix(ODECM) constituents was determined by analyzing the content of matrix proteins. Additionally, we evaluated the biocompatibility of the decellularized extracellular matrix(dECM) by observing the growth of granulosa cells on the ODECM scaffold in vitro. RESULTS: Hematoxylin and eosin staining, DAPI staining and DNA quantification techniques collectively confirm the success of the novel decellularization methods in removing cellular and nuclear components from ovarian tissue. Moreover, quantitative assessments of ODECM contents revealed that the novel decellularization technique preserved more collagen and glycosaminoglycan compared to the conventional decellularized group (P<0.05). Additionally, the novel decellularized scaffold exhibited a significantly higher number of granulosa cells than the conventional scaffold during in vitro co-culture (P<0.05). CONCLUSION: The novel decellularized method demonstrated high efficacy in eliminating DNA and cellular structures while effectively preserving the extracellular matrix. As a result, the novel decellularized method holds significant promise as a viable technique for ovarian decellularization in forthcoming studies. Doi.org/10.54680/fr24310110212.

DNA Quantity and Quality Comparisons between Cryopreserved and FFPE Tumors from Matched Pan-Cancer Samples.

Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing in clinical cancer care comes almost universally from formalin-fixed, paraffin-embedded tissue (FFPE). As newer technologies emerge for DNA characterization that requires higher molecular weight DNA, it was necessary to compare the quality of DNA in terms of DNA length between FFPE and cryopreserved samples. We hypothesized that cryopreserved samples would yield higher quantity and superior quality DNA compared to FFPE samples. We analyzed DNA metrics by performing a head-to-head comparison between FFPE and cryopreserved samples from 38 human tumors representing various cancer types. DNA quantity and purity were measured by UV spectrophotometry, and DNA from cryopreserved tissue demonstrated a 4.2-fold increase in DNA yield per mg of tissue (p-value < 0.001). DNA quality was measured on a fragment microelectrophoresis analyzer, and again, DNA from cryopreserved tissue demonstrated a 223% increase in the DNA quality number and a 9-fold increase in DNA fragments > 40,000 bp (p-value < 0.0001). DNA from the cryopreserved tissues was superior to the DNA from FFPE samples in terms of DNA yield and quality.

Specific Small-Molecule Detection Using Designed Nucleic Acid Nanostructure Carriers and Nanopores.

In the realm of nanopore sensor technology, an enduring challenge lies in achieving the discerning detection of small biomolecules with a sufficiently high signal-to-noise ratio. This study introduces a method for reliably quantifying the concentration of target small molecules, utilizing tetrahedral DNA nanostructures as surrogates for the captured molecules through a magnetic-bead-based competition substitution mechanism. Magnetic Fe3O4-DNA tetrahedron nanoparticles (MNPs) are incorporated into a nanopore electrochemical system for small-molecule sensing. In the presence of the target, the DNA tetrahedron, featuring an aptamer tail acting as a molecular carrier, detaches from the MNPs due to aptamer deformation. Following removal of the MNPs, the DNA tetrahedron bound to the target traversed the nanopore by applying a positive potential. This approach exhibits various advantages, including heightened sensitivity, selectivity, an improved signal-to-noise ratio (SNR), and robust anti-interference capabilities. Our findings demonstrate that this innovative methodology has the potential to significantly enhance the sensing of various small-molecule targets by nanopores, thereby advancing the sensitivity and dynamic range. This progress holds promise for the development of precise clinical diagnostic tools.

DNA flow cytometry for detection of genomic instability as a cancer precursor in the gastrointestinal tract.

One of the earliest applications of flow cytometry was the measurement of DNA content in cells. This method is based on the ability to stain DNA in a stoichiometric manner (i.e., the amount of stain is directly proportional to the amount of DNA within the cell). For more than 40years, a number of studies have consistently demonstrated the utility of DNA flow cytometry as a potential diagnostic and/or prognostic tool in patients with most epithelial tumors, including pre-invasive lesions (such as dysplasia) in the gastrointestinal tract. However, its availability as a clinical test has been limited to few medical centers due to the requirement for fresh tissue in earlier studies and perceived technical demands. However, more recent studies have successfully utilized formalin-fixed paraffin-embedded (FFPE) tissue to generate high-quality DNA content histograms, demonstrating the feasibility of this methodology. This review summarizes step-by-step methods on how to perform DNA flow cytometry using FFPE tissue and analyze DNA content histograms based on the published consensus guidelines in order to assist in the diagnosis and/or risk stratification of many different epithelial tumors, with particular emphasis on dysplasia associated with Barrett's esophagus and inflammatory bowel disease.

Developing centrifugal force real-time digital PCR for detecting extremely low DNA concentration.

Digital PCR (dPCR) is a technique for absolute quantification of nucleic acid molecules. To develop a dPCR technique that enables more accurate nucleic acid detection and quantification, we established a novel dPCR apparatus known as centrifugal force real-time dPCR (crdPCR). This system is efficient than other systems with only 2.14% liquid loss by dispensing samples using centrifugal force. Moreover, we applied a technique for analyzing the real-time graph of the each micro-wells and distinguishing true/false positives using artificial intelligence to mitigate the rain, a persistent issue with dPCR. The limits of detection and quantification were 1.38 and 4.19 copies/µL, respectively, showing a two-fold higher sensitivity than that of other comparable devices. With the integration of this new technology, crdPCR will significantly contribute to research on next-generation PCR targeting absolute micro-analysis.

Implementing L-DNA analogs as mirrors of PCR reactant hybridization state: theoretical and practical guidelines for PCR cycle control.

In previous reports, we described a PCR cycle control approach in which the hybridization state of optically labeled L-DNA enantiomers of the D-DNA primers and targets determined when the thermal cycle was switched from cooling to heating and heating to cooling. A consequence of this approach is that it also "adapts" the cycling conditions to compensate for factors that affect the hybridization kinetics of primers and targets. It assumes, however, that the hybridization state of the labeled L-DNA analogs accurately reflects the hybridization state of the D-DNA primers and targets. In this report, the Van't Hoff equation is applied to determine the L-DNA concentration and ratio of L-DNA strands required by this assumption. Simultaneous fluorescence and temperature measurements were taken during L-DNA controlled cycling, and the optical and thermal switch points compared as a function of both total L-DNA concentration and ratio of strands. Based on the Van't Hoff relationship and these experimental results, L-DNA best mirrors the hybridization of PCR primers and targets when total L-DNA concentration is set equal to the initial concentration of the D-DNA primer of interest. In terms of strand ratios, L-DNA hybridization behavior most closely matches the behavior of their D-DNA counterparts throughout the reaction when one of the L-DNA strands is far in excess of the other. The L-DNA control algorithm was then applied to the practical case of the SARS-CoV-2 N2 reaction, which has been shown to fail or have a delayed Cq when PCR was performed without nucleic acid extraction. PCR Cq values for simulated "unextracted" PCR samples in a nasopharyngeal background and in an NaCl concentration similar to that of viral transport media were determined using either the L-DNA control algorithm (N = 6) or preset cycling conditions (N = 3) and compared to water background controls run in parallel. For preset cycling conditions, the presence of nasopharyngeal background or a high salt background concentration significantly increased Cq, but the L-DNA control algorithm had no significant delay. This suggests that a carefully designed L-DNA-based control algorithm "adapts" the cycling conditions to compensate for hybridization errors of the PCR D-DNA reactants that produce false negatives.

Novel, fast, and reliable electrochemical dsDNA biosensor based on O-terminated pristine nanocrystalline boron-doped diamond electrode for DNA interaction studies.

We present a novel application of a nanocrystalline boron-doped diamond electrode (B-NCDE) for the construction of an electrochemical DNA biosensor based on double-stranded DNA (dsDNA) for various bioanalytical applications. Surface characterization of the transducer surface (prior and after the fabrication of negatively charged O-terminated surface - O-B-NCDE) was performed by scanning electron microscopy (SEM), Raman spectroscopy, and linear sweep voltammetry (LSV) that was further used for the voltammetric determination, scan rate dependence investigation, and repeatability examination of dsDNA electrochemical oxidation at the O-B-NCDE. The fabrication of a dsDNA/O-B-NCDE biosensor via electrostatic adsorption of dsDNA involved a thorough optimization process of deposition potential (Edep), deposition time (tdep), and optimal saturation concentration (cg(satur)) with optimal values of 0.3 V, 3 min, and 10 mg/mL. The bioanalytical applicability of the fabricated dsDNA/O-B-NCDE biosensor was verified by examining the nature of the interaction between dsDNA and five selected DNA intercalators - namely thioridazine hydrochloride (TR), trimipramine maleate (TRIM), levomepromazine maleate (LEV), imipramine hydrochloride (IMI), and prochlorperazine maleate (PER) - where intercalation was proven for all of the five tested compounds. Moreover, the proposed novel bioanalytical test offers the possibility to selectively distinguish between the phenothiazine representatives (TR, LEV, and PER) and representatives of tricyclic antidepressants group (TRIM and IMI).

A Measure of the DNA Barcode Gap for Applied and Basic Research.

DNA barcoding has largely established itself as a mainstay for rapid molecular taxonomic identification in both academic and applied research. The use of DNA barcoding as a molecular identification method depends on a "DNA barcode gap"-the separation between the maximum within-species difference and the minimum between-species difference. Previous work indicates the presence of a gap hinges on sampling effort for focal taxa and their close relatives. Furthermore, both theory and empirical work indicate a gap may not occur for related pairs of biological species. Here, we present a novel evaluation approach in the form of an easily calculated set of nonparametric metrics to quantify the extent of proportional overlap in inter- and intraspecific distributions of pairwise differences among target species and their conspecifics. The metrics are based on a simple count of the number of overlapping records for a species falling within the bounds of maximum intraspecific distance and minimum interspecific distance. Our approach takes advantage of the asymmetric directionality inherent in pairwise genetic distance distributions, which has not been previously done in the DNA barcoding literature. We apply the metrics to the predatory diving beetle genus Agabus as a case study because this group poses significant identification challenges due to its morphological uniformity despite both relative sampling ease and well-established taxonomy. Results herein show that target species and their nearest neighbor species were found to be tightly clustered and therefore difficult to distinguish. Such findings demonstrate that DNA barcoding can fail to fully resolve species in certain cases. Moving forward, we suggest the implementation of the proposed metrics be integrated into a common framework to be reported in any study that uses DNA barcoding for identification. In so doing, the importance of the DNA barcode gap and its components for the success of DNA-based identification using DNA barcodes can be better appreciated.

DNA Barcoding for an Undergraduate Class.

DNA technique is a topic mandatorily covered in a biology and biochemistry undergraduate curriculum. Inquiry-based pedagogy is proven to be the most effective way of learning, and DNA barcoding method allows to merge necessary-to-study experimental techniques such as DNA isolation and purification, PCR, and basic BLAST search into a two- or three-week inquiry-based student project. It also provides a research-based experience to the students, who, when organized in groups, can design their own DNA-barcoding project if they wish. Here, we describe how DNA barcoding can be offered in an undergraduate college or advanced high school settings. This chapter is intended to help college and high school instructors to include DNA barcoding in their classes.

Soft interface confined DNA walker for sensitive and specific detection of SARS-CoV-2 variants.

Nucleic acid detection is conducive to preventing the spread of COVID-19 pandemic. In this work, we successfully designed a soft interface confined DNA walker by anchoring hairpin reporter probes on cell membranes for the detection of SARS-CoV-2 variants. In the presence of target RNA, the cyclic self-assembly reaction occurred between hairpin probes H1 and H2, and the continuous walking of target RNA on cell membranes led to the gradual amplification of fluorescence signal. The enrichment of H1 on membranes and the unique fluidity of membranes promoted the collision efficiency between DNA strands in the reaction process, endowing this method with high sensitivity. In addition, the double-blind test of synthetic RNA in 5% normal human serum demonstrated the good stability and anti-interference in complex environment of this method, which exhibited great potential in clinical diagnostics.

Development of decellularization protocols for female cat reproductive organs.

Decellularization is an innovative method to create natural scaffolds by removing all cellular materials while preserving the composition and three-dimensional ultrastructure of the extracellular matrix (ECM). The obtention of decellularized reproductive organs in cats might facilitate the development of assisted reproductive techniques not only in this species but also in other felids. The aim was to compare the efficiency of three decellularization protocols on reproductive organs (ovary, oviduct, and uterine horn) in domestic cats. The decellularization protocol involved 0.1% sodium dodecyl sulfate and 1%Triton X-100. Protocol 1 (P1) entailed 2-cycles of decellularization using these detergents. Protocol 2 (P2) was like P1 but included 3-cycles. Protocol 3 (P3) was similar to P2, with the addition of deoxyribonuclease incubation. Reproductive organs from nine cats were separated into two sides. One side served as the control (non-decellularized organ) while the contralateral side was the treated group (decellularized organ). The treated organs were subdivided into 3 groups (n = 3 per group) for each protocol. Both control and treated samples were analyzed for DNA content, histology (nuclear and ECM (collagen, elastin, and glycosaminoglycans (GAGs)) density), ultrastructure by electron microscopy, and cytotoxicity. The results of the study showed that P3 was the only protocol that displayed no nucleus residue and significantly reduced DNA content in decellularized samples (in all the studied organs) compared to the control (P < 0.05). The ECM content in the ovaries remained similar across all protocols compared with controls (P > 0.05). However, elastic fibers and GAGs decreased in decellularized oviducts (P < 0.05), while collagen levels remained unchanged (P > 0.05). Regarding the uterus, the ECM content decreased in decellularized uterine horns from P3 (P < 0.05). Electron microscopy revealed that the microarchitecture of the decellularized samples was maintained compared to controls. The decellularized tissues, upon being washed for 24 h, showed cytocompatibility following co-incubation with sperm. In conclusion, when comparing different decellularization methods, P3 proved to be the most efficient in removing nuclear material from reproductive organs compared to P1 and P2. P3 demonstrated its success in decellularizing ovarian samples by significantly decreasing DNA content while maintaining ECM components and tissue microarchitecture. However, P3 was less effective in maintaining ECM contents in decellularized oviducts and uterine horns.

Fecal DNA metabarcoding helps characterize the Canada jay's diet and confirms its reliance on stored food for winter survival and breeding.

Accurately determining the diet of wild animals can be challenging if food items are small, visible only briefly, or rendered visually unidentifiable in the digestive system. In some food caching species, an additional challenge is determining whether consumed diet items have been previously stored or are fresh. The Canada jay (Perisoreus canadensis) is a generalist resident of North American boreal and subalpine forests with anatomical and behavioural adaptations allowing it to make thousands of arboreal food caches in summer and fall that are presumably responsible for its high winter survival and late winter/early spring breeding. We used DNA fecal metabarcoding to obtain novel information on nestling diets and compiled a dataset of 662 published and unpublished direct observations or stomach contents identifications of natural foods consumed by Canada jays throughout the year. We then used detailed natural history information to make informed decisions on whether each item identified to species in the diets of winter adults and nestlings was best characterized as 'likely cached', 'likely fresh' (i.e., was available as a non-cached item when it appeared in a jay's feces or stomach), or 'either possible'. Of the 87 food items consumed by adults in the winter, 39% were classified as 'likely cached' and 6% were deemed to be 'likely fresh'. For nestlings, 29% of 125 food items identified to species were 'likely cached' and 38% were 'likely fresh'. Our results support both the indispensability of cached food for Canada jay winter survival and previous suggestions that cached food is important for late winter/early spring breeding. Our work highlights the value of combining metabarcoding, stomach contents analysis, and direct observations to determine the cached vs. non-cached origins of consumed food items and the identity of food caches, some of which could be especially vulnerable to degradation through climate change.

A fluorescence-electrochemical dual-mode aptasensor based on novel DNA-dependent PBNFs@PtPd for highly selective and sensitive detection of procymidone through hybridization chain reaction.

Herein, a study for the first application of a hybridization chain reaction, a 1,8-naphthalimides-DNA (NDs) intercalator, and DNA-dependent Prussian blue nanoflowers@PtPd materials (PBNFs@PtPd) in the development of a fluorescence-electrochemical (FL-EC) aptasensor. This construction establishes an efficient sensing platform for the detection of procymidone (PCM). In the context of the described experiment, dual-mode detection is achieved through the generation of FL signals by an aptamer labeled with a Cy5 moiety and the formation of DPV signals by the modification of a thionine-appended 1,8-naphthalimide (Thi-NDs). In the presence of PCM, specific recognition occurs, followed by the utilization of magnetic separation technology to release DNA1 (S1) and aptamer-Cy5 (Apt-Cy5), subsequently introducing them onto both fluorescence and EC platforms. The presence of S1 effectively activates hybridization chain reaction (HCR) for the electrode surface, thereby significantly increasing the binding sites for Thi-NDs and consequently greatly amplifying the response signal of differential pulse voltammetry (DPV). The developed FL-EC dual-mode sensing platform demonstrates high sensitivity in the detection of PCM, with the detection limits of 0.173 µg·ml-1 (within the detection range of 500 pg·ml-1 to 500 ng·ml-1) and 0.074 ng·ml-1 (within the detection range of 100 pg·ml-1 to 100 ng·ml-1), respectively. The designed dual-mode sensor exhibits notable characteristics, including high selectivity, reproducibility, synergy, and reliable monitoring/capability for PCM in real samples.

Tunable nanofluidic device for digital nucleic acid analysis.

Nano/microfluidic-based nucleic acid tests have been proposed as a rapid and reliable diagnostic technology. Two key steps for many of these tests are target nucleic acid (NA) immobilization followed by an enzymatic reaction on the captured NAs to detect the presence of a disease-associated sequence. NA capture within a geometrically confined volume is an attractive alternative to NA surface immobilization that eliminates the need for sample pre-treatment (e.g. label-based methods such as lateral flow assays) or use of external actuators (e.g. dielectrophoresis) that are required for most nano/microfluidic-based NA tests. However, geometrically confined spaces hinder sample loading while making it challenging to capture, subsequently, retain and simultaneously expose target NAs to required enzymes. Here, using a nanofluidic device that features real-time confinement control via pneumatic actuation of a thin membrane lid, we demonstrate the loading of digital nanocavities by target NAs and exposure of target NAs to required enzymes/co-factors while the NAs are retained. In particular, as proof of principle, we amplified single-stranded DNAs (M13mp18 plasmid vector) in an array of nanocavities via two isothermal amplification approaches (loop-mediated isothermal amplification and rolling circle amplification).

PCR in Forensic Science: A Critical Review.

The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The introduction of PCR into forensic science marked the beginning of a new era of DNA profiling. This era has pushed PCR to its limits and allowed genetic data to be generated from trace DNA. Trace samples contain very small amounts of degraded DNA associated with inhibitory compounds and ions. Despite significant development in the PCR process since it was first introduced, the challenges of profiling inhibited and degraded samples remain. This review examines the evolution of the PCR from its inception in the 1980s, through to its current application in forensic science. The driving factors behind PCR evolution for DNA profiling are discussed along with a critical comparison of cycling conditions used in commercial PCR kits. Newer PCR methods that are currently used in forensic practice and beyond are examined, and possible future directions of PCR for DNA profiling are evaluated.