RESUMO
As one of the most vital organelles within biological cells, mitochondria hold an irreplaceable status and play crucial roles in various diseases. Research and therapies targeting mitochondria have achieved significant progress in numerous conditions. Throughout an organism's lifespan, mitochondrial dynamics persist continuously, and due to their inherent characteristics and various external factors, mitochondria are highly susceptible to damage. This susceptibility is particularly evident during aging, where the decline in biological function is closely intertwined with mitochondrial dysfunction. Despite being an ancient and enigmatic organelle, much remains unknown about mitochondria. Here, we will explore the past and present knowledge of mitochondria, providing a comprehensive review of their intrinsic properties and interactions with nuclear DNA, as well as the challenges and impacts they face during the aging process.
RESUMO
The inference of body fluids and tissues is critical in reconstructing crime scenes and inferring criminal behaviors. Nevertheless, present methods are incompatible with conventional DNA genotyping, and additional testing might result in excessive consumption of forensic scene materials. This study aims to investigate the feasibility of distinguishing common body fluids/tissues through the difference in mitochondrial DNA copy number (mtDNAcn). Four types of body fluids/tissues were analyzed in this study - hair, saliva, semen, and skeletal muscle. MtDNAcn was estimated by dividing the read counts of mitochondrial DNA to that of nuclear DNA (RRmt/nu). Results indicated that there were significant differences in RRmt/nu between different body fluids/tissues. Specifically, hair samples exhibited the highest RRmt/nu (log10RRmt/nu: 4.3 ± 0.28), while semen samples showed the lowest RRmt/nu (log10RRmt/nu: -0.1 ± 0.28). RRmt/nu values for DNA samples without extraction were notably higher (approximately 2.9 times) than those obtained after extraction. However, no significant difference in RRmt/nu was observed between various age and gender groups. Hierarchical clustering and Kmeans clustering analyses showed that body fluids/tissues of the same type clustered closely to each other and could be inferred with high accuracy. In conclusion, this study demonstrated that the simultaneous detection of nuclear and mitochondrial DNA made it possible to perform conventional DNA analyses and body fluid/tissue inference at the same time, thus killing two birds with one stone. Furthermore, mtDNAcn has the potential to serve as a novel and promising biomarker for the identification of body fluids/tissues.
RESUMO
Most of Switzerland is inhabited by the nominotypical subspecies of the barred grass snake (Natrixhelveticahelvetica), which is characterized by mitochondrial DNA lineage E. Only in the northeast of the country, the common grass snake (N.natrix) occurs and hybridizes with N.h.helvetica in a narrow contact zone. However, we discovered that in southern and western Switzerland barred grass snakes representing another mtDNA lineage (lineage C) are widely distributed. Lineage C is typical for Alpine populations of the southern subspecies N.h.sicula. Our microsatellite analyses of the Swiss samples revealed differences between the two subspecies and also a substructure with two clusters in each subspecies. Furthermore, we discovered a contact and hybrid zone of N.h.helvetica and N.h.sicula along the northern shore of Lake Geneva and also confirm that interbreeding with alien common grass snakes (N.n.moreotica, mtDNA lineage 7) occurs there. This finding is of concern for nature conservation and measures should be taken to prevent further genetic pollution. Using morphometrics, we found no differences between the two subspecies of N.helvetica, while N.natrix was slightly distinct from N.helvetica.
RESUMO
OBJECTIVE: Identify the genotype and clinical characteristics of mitochondrial epilepsy caused by nDNA mutations in Chinese children and explore the treatment and prognosis of the condition. STUDY DESIGN: This is a retrospective cohort study conducted at a single center, including patients diagnosed with an established nDNA mutation-associated primary mitochondrial disease between October 2012 and March 2023 who also met the practical clinical definition of epilepsy published by the ILAE in 2014. RESULTS: Of the 58 patients identified, 74.1% had an onset before the age of 1 year and 63.8% had seizures as their initial symptom. Developmental and epileptic encephalopathy (DEE) (31%) are the most common phenotypes. The most frequently observed MRI abnormalities include abnormal signal asymmetry in the bilateral basal ganglia and/or brainstem (34.7%), as well as brain atrophy, myelin sheath dysplasia, and corpus callosum dysplasia (32.7%). Of the 40 patients followed, seizure treatment was effective in 18 of the cases, while it was ineffective in 22. The mitochondrial DNA depletion syndrome (MDS) was found to be more difficult to control seizures than other phenotypes (P < 0.05). Additionally, the MDS was associated with a significantly higher mortality rate compared to alternative phenotypes (P < 0.05). CONCLUSIONS: The onset of mitochondrial epilepsy due to nDNA mutations is early and seizures are the most common initial symptom. DEE is the most common phenotype. Characteristic MRI abnormalities in the brain may be helpful in the diagnosis of primary mitochondrial disease. People with MDS typically face challenges in seizure control and have a poor prognosis.
RESUMO
Sesamum indicum L. (Pedaliaceae) is one of the most economically important oil crops in the world, thanks to the high oil content of its seeds and its nutritional value. It is cultivated all over the world, mainly in Asia and Africa. Well adapted to arid environments, sesame offers a good opportunity as an alternative subsistence crop for farmers in Africa, particularly Niger, to cope with climate change. For the first time, the variation in genome size among 75 accessions of the Nigerien germplasm was studied. The sample was collected throughout Niger, revealing various morphological, biochemical and phenological traits. For comparison, an additional accession from Thailand was evaluated as an available Asian representative. In the Niger sample, the 2C DNA value ranged from 0.77 to 1 pg (753 to 978 Mbp), with an average of 0.85 ± 0.037 pg (831 Mbp). Statistical analysis showed a significant difference in 2C DNA values among 58 pairs of Niger accessions (p-value < 0.05). This significant variation indicates the likely genetic diversity of sesame germplasm, offering valuable insights into its possible potential for climate-resilient agriculture. Our results therefore raise a fundamental question: is intraspecific variability in the genome size of Nigerien sesame correlated with specific morphological and physiological traits?
Assuntos
Tamanho do Genoma , Genoma de Planta , Sesamum , Sesamum/genética , Níger , Variação Genética , Sementes/genéticaRESUMO
The Africanized honey bee, a hybrid of Apis mellifera scutellata from Africa with European subspecies, has been considered an invasive species and a problem for beekeeping. Africanized bees arrived in Mexico in 1986, 30 years after their accidental release in Brazil. Although government programs were implemented for its eradication, Africanized populations persist in Mexico, but precise information on the patterns of genetic introgression and racial ancestry is scarce. We determined maternal and parental racial ancestry of managed and feral honey bees across the five beekeeping regions of Mexico, using mitochondrial (mtDNA, COI-COII intergenic region) and nuclear markers (94 ancestrally informative SNPs), to assess the relationship between beekeeping management, beekeeping region, altitude, and latitude with the distribution of maternal and parental racial ancestry. Results revealed a predominantly African ancestry in the Mexican honey bees, but the proportion varied according to management, beekeeping regions, and latitude. The Mexican honey bees showed 31 haplotypes of four evolutionary lineages (A, M, C, and O). Managed honey bees had mitochondrial and nuclear higher proportions of European ancestry than feral honey bees, which had a higher proportion of African ancestry. Beekeeping regions of lower latitudes had higher proportions of African nuclear ancestry. Managed and feral honey bees showed differences in the proportion of maternal and nuclear racial ancestry. Managed honey bees from the Yucatan Peninsula and feral honey bees had a higher mtDNA than nuclear proportions of African ancestry. Managed honey bees, except those on the Yucatan Peninsula, had a higher nuclear than mtDNA proportion of African ancestry. Our study demonstrates that Africanized honey bee populations are genetically diverse and well established in Mexico, which highlights the limitations of management and government programs to contain the Africanization process and demands the incorporation of this lineage in any breeding program for sustainable beekeeping.
RESUMO
Introduction: Outcome-prediction in patients with sepsis is challenging and currently relies on the serial measurement of many parameters. Standard diagnostic tools, such as serum creatinine (SCr), lack sensitivity and specificity for acute kidney injury (AKI). Circulating cell-free DNA (cfDNA), which can be obtained from liquid biopsies, can potentially contribute to the quantification of tissue damage and the prediction of sepsis mortality and sepsis-associated AKI (SA-AKI). Methods: We investigated the clinical significance of cfDNA levels as a predictor of 28-day mortality, the occurrence of SA-AKI and the initiation of renal replacement therapy (RRT) in patients with sepsis. Furthermore, we investigated the long-term course of cfDNA levels in sepsis survivors at 6 and 12 months after sepsis onset. Specifically, we measured mitochondrial DNA (mitochondrially encoded NADH-ubiquinone oxidoreductase chain 1, mt-ND1, and mitochondrially encoded cytochrome C oxidase subunit III, mt-CO3) and nuclear DNA (nuclear ribosomal protein S18, n-Rps18) in 81 healthy controls and all available samples of 150 intensive care unit patients with sepsis obtained at 3 ± 1 days, 7 ± 1 days, 6 ± 2 months and 12 ± 2 months after sepsis onset. Results: Our analysis revealed that, at day 3, patients with sepsis had elevated levels of cfDNA (mt-ND1, and n-Rps18, all p<0.001) which decreased after the acute phase of sepsis. 28-day non-survivors of sepsis (16%) had higher levels of cfDNA (all p<0.05) compared with 28-day survivors (84%). Patients with SA-AKI had higher levels of cfDNA compared to patients without AKI (all p<0.05). Cell-free DNA was also significantly increased in patients requiring RRT (all p<0.05). All parameters improved the AUC for SCr in predicting RRT (AUC=0.88) as well as APACHE II in predicting mortality (AUC=0.86). Conclusion: In summary, cfDNA could potentially improve risk prediction models for mortality, SA-AKI and RRT in patients with sepsis. The predictive value of cfDNA, even with a single measurement at the onset of sepsis, could offer a significant advantage over conventional diagnostic methods that require repeated measurements or a baseline value for risk assessment. Considering that our data show that cfDNA levels decrease after the first insult, future studies could investigate cfDNA as a "memoryless" marker and thus bring further innovation to the complex field of SA-AKI diagnostics.
Assuntos
Injúria Renal Aguda , Biomarcadores , Ácidos Nucleicos Livres , Sepse , Humanos , Sepse/mortalidade , Sepse/sangue , Sepse/complicações , Ácidos Nucleicos Livres/sangue , Masculino , Injúria Renal Aguda/mortalidade , Injúria Renal Aguda/sangue , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/etiologia , Feminino , Pessoa de Meia-Idade , Idoso , Biomarcadores/sangue , Prognóstico , DNA Mitocondrial/sangue , Terapia de Substituição RenalRESUMO
In this protocol paper, we review a set of methods developed in recent years for analyzing nuclear reads obtained from genome skimming. As the cost of sequencing drops, genome skimming (low-coverage shotgun sequencing of a sample) becomes increasingly a cost-effective method of measuring biodiversity at high resolution. While most practitioners only use assembled over-represented organelle reads from a genome skim, the vast majority of the reads are nuclear. Using assembly-free and alignment-free methods described in this protocol, we can compare samples to each other and reference genomes to compute distances, characterize underlying genomes, and infer evolutionary relationships.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Genômica/métodos , Genoma/genética , Software , Núcleo Celular/genética , Biologia Computacional/métodos , HumanosRESUMO
Introduction: Rare disorders that are genetically and clinically heterogeneous, such as mitochondrial diseases (MDs), have a challenging diagnosis. Nuclear genes codify most proteins involved in mitochondrial biogenesis, despite all mitochondria having their own DNA. The development of next-generation sequencing (NGS) technologies has revolutionized the understanding of many genes involved in the pathogenesis of MDs. In this new genetic era, using the NGS approach, we aimed to identify the genetic etiology for a suspected MD in a cohort of 450 Portuguese patients. Methods: We examined 450 patients using a combined NGS strategy, starting with the analysis of a targeted mitochondrial panel of 213 nuclear genes, and then proceeding to analyze the whole mitochondrial DNA. Results and Discussion: In this study, we identified disease-related variants in 134 (30%) analyzed patients, 88 with nuclear DNA (nDNA) and 46 with mitochondrial DNA (mtDNA) variants, most of them being pediatric patients (66%), of which 77% were identified in nDNA and 23% in mtDNA. The molecular analysis of this cohort revealed 72 already described pathogenic and 20 novel, probably pathogenic, variants, as well as 62 variants of unknown significance. For this cohort of patients with suspected MDs, the use of a customized gene panel provided a molecular diagnosis in a timely and cost-effective manner. Patients who cannot be diagnosed after this initial approach will be further selected for whole-exome sequencing. Conclusion: As a national laboratory for the study and research of MDs, we demonstrated the power of NGS to achieve a molecular etiology, expanding the mutational spectrum and proposing accurate genetic counseling in this group of heterogeneous diseases without therapeutic options.
RESUMO
Major strides have been made in the development of FLASH radiotherapy (FLASH RT) in the last ten years, but there are still many obstacles to overcome for transfer to the clinic to become a reality. Although preclinical and first-in-human clinical evidence suggests that ultra-high dose rates (UHDRs) induce a sparing effect in normal tissue without modifying the therapeutic effect on the tumor, successful clinical translation of FLASH-RT depends on a better understanding of the biological mechanisms underpinning the sparing effect. Suitable in vitro studies are required to fully understand the radiobiological mechanisms associated with UHDRs. From a technical point of view, it is also crucial to develop optimal technologies in terms of beam irradiation parameters for producing FLASH conditions. This review provides an overview of the research progress of FLASH RT and discusses the potential challenges to be faced before its clinical application. We critically summarize the preclinical evidence and in vitro studies on DNA damage following UHDR irradiation. We also highlight the ongoing developments of technologies for delivering FLASH-compliant beams, with a focus on laser-driven plasma accelerators suitable for performing basic radiobiological research on the UHDR effects.
Assuntos
Dano ao DNA , Motivação , Humanos , Cabeça , Plasma , RadiobiologiaRESUMO
During a 1-year study, Trichuris adults were obtained after necropsy of Arabian camels (Camelus dromedarius) from a slaughterhouse in Kuwait. Morphological and molecular identification was performed to confirm the identity of the Trichuris specimens obtained from C. dromedarius. Fifteen male Trichuris specimens were selected, and molecular identification was performed using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA, 16S ribosomal RNA genes and the nuclear internal transcribed spacer 2 (ITS2) region. Through phylogenetic analysis, 2 distinct groups were obtained using the mitochondrial genes, where group 1 showed a close relationship to Trichuris globulosa while group 2 showed a close relationship to Trichuris ovis, providing molecular evidence of a possible T. globulosa species complex. Additionally, the nuclear ITS2 region did not provide enough resolution to distinguish between the 2 groups of Trichuris specimens. Observation of morphological characters revealed variations in the shape of the male spicule sheath, where specimens present either a globular posteriorly truncated swelling or the absence of posteriorly truncated swelling. Moreover, the variations in male spicule sheath does not corroborate with the results of molecular data, suggesting the limited use of this character for identification of T. globulosa. In conclusion, molecular analysis suggests a possible species complex in T. globulosa, with the mitochondrial genetic markers successfully differentiating between the 2 groups. The limited use of the male spicule sheath as a diagnostic character for identification of T. globulosa is suggested.
RESUMO
Angiosperms, which inhabit diverse environments across all continents, exhibit significant variation in genome sizes, making them an excellent model system for examining hypotheses about the global distribution of genome size. These include the previously proposed large genome constraint, mutational hazard, polyploidy-mediated, and climate-mediated hypotheses. We compiled the largest genome size dataset to date, encompassing 16 017 (> 5% of known) angiosperm species, and analyzed genome size distribution using a comprehensive geographic distribution dataset for all angiosperms. We observed that angiosperms with large range sizes generally had small genomes, supporting the large genome constraint hypothesis. Climate was shown to exert a strong influence on genome size distribution along the global latitudinal gradient, while the frequency of polyploidy and the type of growth form had negligible effects. In contrast to the unimodal patterns along the global latitudinal gradient shown by plant size traits and polyploid proportions, the increase in angiosperm genome size from the equator to 40-50°N/S is probably mediated by different (mostly climatic) mechanisms than the decrease in genome sizes observed from 40 to 50°N northward. Our analysis suggests that the global distribution of genome sizes in angiosperms is mainly shaped by climatically mediated purifying selection, genetic drift, relaxed selection, and environmental filtering.
Assuntos
Magnoliopsida , Magnoliopsida/genética , Tamanho do Genoma , Genoma de Planta , Poliploidia , Plantas/genética , FilogeniaRESUMO
Astronauts in space are subject to continuous exposure to ionizing radiation. There is concern about the acute and late-occurring adverse health effects that astronauts could incur following a protracted exposure to the space radiation environment. Therefore, it is vital to consider the current tools and models used to describe and study the organic consequences of ionizing radiation exposure. It is equally important to see where these models could be improved. Historically, radiobiological models focused on how radiation damages nuclear deoxyribonucleic acid (DNA) and the role DNA repair mechanisms play in resulting biological effects, building on the hypotheses of Crowther and Lea from the 1940s and 1960s, and they neglected other subcellular targets outside of nuclear DNA. The development of these models and the current state of knowledge about radiation effects impacting astronauts in orbit, as well as how the radiation environment and cellular microenvironment are incorporated into these radiobiological models, aid our understanding of the influence space travel may have on astronaut health. It is vital to consider the current tools and models used to describe the organic consequences of ionizing radiation exposure and identify where they can be further improved.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Exposição à Radiação , Lesões por Radiação , Humanos , Astronautas , Microambiente Celular , DNARESUMO
Objective:To optimize the existing protocols for the detection of sperm nuclear DNA integrity and to explore their application value in assisted reproductive technology.Methods:194 couples intending to undergo in vitro fertilization-embryo transfer(IVF-ET)treatment at the Affiliated Reproductive Hospital of Jiangxi University of Traditional Chinese Medicine from January 1,2021,to December 8,2022,were selected as the study subjects.The sperm samples from the male partners were collected as the control group,and the same semen,processed after optimization using a double-layer density gradient centrifugation method,was used as the observation group.According to the DNA fragmentation index(DFI)results,the control group and the observation group were divided into three subgroups,control group A and observation group A:DFI<15%;control group B and observation group B:DFI 15%~30%;control group C and observation group C:DFI ≥30%.Then the DFI values of the observation group and control group were compared.The conditions of assisted pregnancy and pregnancy were analyzed among the subgroups.Results:①The sperm DNA fragmentation index(DFI)of the observation group was signifi-cantly lower than that of the control group[(13.55±10.17)%vs.(18.56±11.54)%,P<0.05].②There was no significant difference in fertilization rate,cleavage rate and high-quality embryo rate among the six subgroups(P>0.05).③There were significant differences in pregnancy rate and implantation rate among the six subgroups(P<0.05);The clinical pregnancy rate(all above 65.00%)and implantation rate(all above 50.00%)were com-pared among four groups:control group A,control group B,observation group A and observation group B.There was no significant difference among the four groups(P>0.05),but they were all higher than those of control group C(43.24%,31.67%)and observation group C(13.64%,8.82%)(P<0.05).The clinical pregnancy rate and implantation rate in the control group were significantly higher than those in the observation group C(P<0.05).Conclusions:The DNA integrity of sperm nucleus can be improved obviously after the sperm was opti-mized.Both of the two methods have good application value in assisted reproductive technology,but the DFI≥30%of semen after optimal treatment has a better predictive value for adverse pregnancy outcomes in ART.
RESUMO
Toxoplasma gondii is a zoonotic protist pathogen that infects up to one third of the human population. This apicomplexan parasite contains three genome sequences: nuclear (65 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear integrants of mitochondrial DNA) and NUPTs (nuclear integrants of plastid DNA) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome-the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma ~28 mya, revealed that the movement and fixation of five NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb), and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together, these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.
Assuntos
Toxoplasma , Humanos , Toxoplasma/genética , Genoma , DNA Mitocondrial/genética , Mitocôndrias/genética , Evolução Molecular , Núcleo Celular/genética , Análise de Sequência de DNARESUMO
Bite mark analysis is among the most interesting research fields in forensic odontology; however, it is limited by its dependence on the employed method as well as assessor subjectivity, particularly when using morphological analysis or DNA profiling. These limitations are due to differences in DNA collected from saliva adhering to a living or inanimate body, as well as differences in exocrine fluid secretion and deposition amount among individuals. This study aimed to analyze the effectiveness of DNA profiling when there are differences in the amount of saliva adhering to a living body and when time has elapsed since deposition. Most allele peaks could be identified in 1 µl of saliva, even 9 h after saliva deposition and examination. Consistent results were obtained following saliva deposition in an individual who had engaged in up to 9 h of free activity. The results of this study demonstrate the validity and reliability of DNA profiling for bite mark analysis and are extremely important as they can demonstrate the usefulness of the little information left by a suspect on a victim's body.
RESUMO
The genome size variation is an important attribute in evolutionary and species characterization. Musa L. is regarded as one of the taxonomically complicated genera within the order Zingiberales, with more than 75 species from wild seeded to seedless cultivars that may be diploid, triploid or tetraploid. The knowledge of total nuclear DNA content in terms of genome size and ploidy level in wild species of Musa is absolutely important in evolutionary and genomic studies. METHODS: In this paper, chromosome spreading was performed via protoplast isolation and a fast air-dry dropping method and flow cytometry were used with Raphanus sativus L. (Brassicaceae) as a standard for ploidy and genome size estimation. RESULTS: The results showed that genome size (2C) varied amongst Musa species, based on the ratio of G1 peak positions. The lowest genome size (2C) was found in M. balbisiana var. andamanica (1.051 ± 0.060 pg) and the highest genome size (2C) was recorded for Musa ABB.cv. Meitei-hei (1.812 ± 0.108 pg) for the section Eumusa. Among the species belonging to the section Rhodochlamys, M. rosae had the lowest 2C content of 1.194 ± 0.033 pg whereas the highest nuclear DNA content (2C) was observed in M. velutina (1.488 ± 0.203 pg). Cytogenetic analysis revealed that the chromosome number of 14 wild Musa species was 2n = 22, while 1 species-Ensete glaucum-showed a chromosome number of 2n = 18 (diploid), and for 3 species, the chromosome number was 2n = 33 (triploids). An association study based on the Pearson correlation coefficient showed 2C nuclear DNA content was significant and positively correlated with ploidy level (R = 0.9) and chromosome number (R = 0.84). CONCLUSIONS: The present study provides reliable information on the genome size and ploidy level of wild Musa species from the Indian region through flow cytometric analysis, which could be further utilized in taxonomic and crop improvement programs. For the first time, the nuclear DNA content of eight wild diploid and three triploid Indian species were estimated and reported. Genome size could be an effective indicator in identification of species and evolutionary studies in Musa with varying ploidy levels and morphological similarities.
RESUMO
The accurate quantification of DNA in forensic samples is of utmost importance. These samples are often present in limited amounts; therefore, it is indicated to use the appropriate analysis route with the optimum DNA amount (when possible). Also, DNA quantification can inform about the degradation stage and therefore support the decision on which downstream genotyping method to use. Consequently, DNA quantification aids in getting the best possible results from a forensic sample, considering both its DNA quantity and quality limitations. Here, we introduce NuMY, a new quantitative real-time PCR (qPCR) method for the parallel quantification of human nuclear (n) and mitochondrial (mt) DNA, assessing the male portion in mixtures of both sexes and testing for possible PCR inhibition. NuMY is based on previous work and follows the MIQE guidelines whenever applicable. Although quantification of nuclear (n)DNA by simultaneously analyzing autosomal and male-specific targets is available in commercial qPCR kits, tools that include the quantification of mtDNA are sparse. The quantification of mtDNA has proven relevant for samples with low nDNA content when conventional DNA fingerprinting techniques cannot be followed. Furthermore, the development and use of new massively parallel sequencing assays that combine multiple marker types, i.e., autosomal, Y-chromosomal, and mtDNA, can be optimized when precisely knowing the amount of each DNA component present in the input sample. For high-quality DNA extracts, NuMY provided nDNA results comparable to those of another quantification technique and has also proven to be a reliable tool for challenging, forensically relevant samples such as mixtures, inhibited, and naturally degraded samples.
Assuntos
DNA Mitocondrial , Mitocôndrias , Feminino , Humanos , Masculino , DNA Mitocondrial/genética , Cromossomos Humanos Y/genética , Bioensaio , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Cocoa (Theobroma cacao L.) is an international commodity used as an ingredient in the manufacturing of chocolate making its authentication a key issue in the cocoa chain. Various molecular techniques have been increasingly applied for quality requirements. These issues highlight the need for techniques that allow the extraction and detection of cocoa DNA from highly processed cocoa products and chocolate. The applicability of real-time PCR to highly processed cocoa-derived products for authentication purposes depends on the possibility of extracting high-quality and amplifiable DNA and further developing efficient PCR tests. This methodology herein describes the use of a classical CTAB method providing DNA suitable for TaqMan real-time PCR amplification. Real-time PCR is a simple and fast method, with a high potential application in a wide range of food products. The main features of this technique are focused on two DNA targets, one located in the nuclear genome (vicilin-li PCR test) and a second one based on chloroplast DNA (lipids PCR test), which successfully passed the performance criteria considering the specificity, sensitivity, efficiency of amplification, robustness, and applicability in processed cocoa-derived products and chocolate.