Amplification Of Nuclear DNA Research Articles

Optimization of Molecular Genetic Method for Identification of Dairy Raw Materials

Introduction: This article addresses the pressing issue of ensuring the quality and safety of dairy products by combating adulteration, a challenge that continues to plague the dairy industry. Adulteration, often driven by cost-cutting motives, involves altering the composition, quality, or origin of dairy products, even in the face of tightened control measures and improved monitoring systems. Specifically, this study hones in on the problem of species-specific adulteration, particularly the substitution of goat milk with cow milk due to the significant cost differential. Detecting and identifying such adulteration is crucial, and while traditional methods like electrophoresis and chromatography have been used, they are often expensive and labor-intensive. The article explores the use of molecular genetic methods, particularly polymerase chain reaction (PCR), as a more efficient and accurate means of identifying species-specific milk adulteration. Methods like PCR offer high specificity, sensitivity, speed, and the ability to perform quantitative and multiplex analyses.Purpose: The objective of this study is to optimise a method for species identification of dairy products by PCR-based screening using DNA isolated from cow’s and goat’s milk comatic cells.Materials and Methods: This research conducted at the Central Laboratory of Microbiology, All-Russian Research Institute of Dairy Industry, focuses on milk species identification of cattle and small ruminants using a specific set of primers targeting the polymorphic amylogenin gene. The study involves PCR screening with DNA extracted from cow’s and goat’s raw milk utilizing a MiniAmp instrument for the analysis. Qualitative and quantitative assessments of DNA preparations were performed, measuring DNA concentration with a Qubit 4 fluorimeter and Qubit dsDNA BR Assay Kit. PCR analysis was carried out in 25 μl reactions, including key components such as 5xScreen Mix, specific primers, DNA samples, and mQ H2O. The amplification program comprised denaturation, annealing, and elongation steps over a series of cycles.Results: The experiment was aimed at assessing the suitability of primers SE47 and SE48 for amplification of nuclear DNA of milk somatic cells of cattle (Bos taurus) and goats (Capra hircus). Initially, a calculated annealing temperature of 56 °C was used in the PCR reaction, yielding in numerous nonspecific fragments appeared on the electropherogram. To solve this problem, we gradually increased the annealing temperature, which resulted in a significant decrease in the nonspecific fragments number and their complete absence at annealing temperature of 70°C.Conclusion: We succeeded in optimizing a PCR-based detection system for the milk species identification of cattle and small ruminants. The results obtained confirm the possibility of using genomic DNA of milk somatic cells for the successful amplification of species-specific nuclear markers, but there remains a need for further research to determine the sensitivity of the PCR system and the possibility of its use in the analysis of milk processing products.

DNA from mollusc shell: a valuable and underutilised substrate for genetic analyses.

Mollusc shells are an abundant resource that have been long used to predict the structures of ancient ecological communities, examine evolutionary processes, reconstruct paleoenvironmental conditions, track and predict responses to climatic change, and explore the movement of hominids across the globe. Despite the ubiquity of mollusc shell in many environments, it remains relatively unexplored as a substrate for molecular genetic analysis. Here we undertook a series of experiments using the New Zealand endemic greenshell mussel, Perna canaliculus, to explore the utility of fresh, aged, beach-cast and cooked mollusc shell for molecular genetic analyses. We find that reasonable quantities of DNA (0.002–21.48 ng/mg shell) can be derived from aged, beach-cast and cooked mussel shell and that this can routinely provide enough material to undertake PCR analyses of mitochondrial and nuclear gene fragments. Mitochondrial PCR amplification had an average success rate of 96.5% from shell tissue extracted thirteen months after the animal’s death. A success rate of 93.75% was obtained for cooked shells. Amplification of nuclear DNA (chitin synthase gene) was less successful (80% success from fresh shells, decreasing to 10% with time, and 75% from cooked shells). Our results demonstrate the promise of mollusc shell as a substrate for genetic analyses targeting both mitochondrial and nuclear genes.

Lost at sea: A pilot study investigating DNA recovery from teeth in a South African natural marine environment

Research investigating DNA recovery from samples exposed to seawater is lacking and limited to artificial or semi-realistic environments. This pilot study assessed DNA recovery from pig (Sus scrofa) teeth (n = 28) submerged in-situ in a natural marine environment of South Africa. Amplification of nuclear DNA was successful in 60% (17/28) of samples for a 96 bp fragment, and in 46% (13/28) for 200 bp fragment. By comparison, mitochondrial DNA was detected in 57% (16/28) for a 486 bp fragment. DNA was more consistently amplified in teeth submerged during winter, suggesting colder seawater temperatures contributed towards DNA preservation. Non-specific amplification was linked to a marine microbe origin. The hypothesis being marine bacteria may contribute towards DNA degradation. The study demonstrates the complexity in understanding the effect seawater has on DNA recovery. Future research on human samples is required to improve success rates of identifying marine wash-ups.

Rapid and inexpensive NaOH based direct PCR for amplification of nuclear and organelle DNA from ramie (Boehmeria nivea), a bast fibre crop containing complex polysaccharides

Most of the PCR based approaches in plant science rely on lengthy and expensive DNA isolation protocols, which often involve use of hazardous chemicals. Direct PCR methods save time and cost of experiments and also increase efficiency of PCR. We have compared three rapid DNA extraction processes for direct PCR in ramie (Boehmeria nivea), a fibre crop species containing high amount of gummy complex polysaccharides and developed modified protocols for direct PCR using NaOH as extraction buffer and Tris/Tris–HCl/Tris–EDTA as dilution buffer. These protocols were successful in amplification of nuclear DNA from leaf and stem tissues using ISSR and SSR markers and also chloroplast DNA amplification using primers for rbcL gene. Our results also show that nature and quantity of dilution buffer are important for increasing efficiency of direct PCR. The NaOH based methods are simpler, cheaper and economical compared to other direct PCR methods and work very well for tissues containing high amount of complex polysaccharides. The protocols are suitable for batch processing and high throughput genotyping within a short time period, which will have many applications in plant genomic researches.

High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species

Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent—the Harvest mouse Micromys minutus—as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.

The Effects of Different Maceration Techniques on Nuclear DNA Amplification Using Human Bone

Forensic anthropologists routinely macerate human bone for the purposes of identity and trauma analysis, but the heat and chemical treatments used can destroy genetic evidence. As a follow-up to a previous study on nuclear DNA recovery that used pig ribs, this study utilizes human skeletal remains treated with various bone maceration techniques for nuclear DNA amplification using the standard Combined DNA Index System (CODIS) markers. DNA was extracted from 18 samples of human lower leg bones subjected to nine chemical and heat maceration techniques. Genotyping was carried out using the AmpFlSTR COfiler and AmpFlSTR Profiler Plus ID kits. Results showed that heat treatments via microwave or Biz/Na(2)CO(3) in sub-boiling water efficiently macerate bone and produce amplifiable nuclear DNA for genetic analysis. Long-term use of chemicals such as hydrogen peroxide is discouraged as it results in poor bone quality and has deleterious effects on DNA amplification.

Remotely plucked hair genotyping: a reliable and non-invasive method for censusing pine marten (Martes martes, L. 1758) populations

We investigated the feasibility of using genetic techniques to census pine marten (Martes martes) populations by genotyping non-invasively collected samples (plucked hair and scats), with particular reference to the genetically depauperate Irish population. Novel real-time polymerase chain reaction methods were developed for species and sex identification, targeting short DNA sequences. Background genetic variation at 17 microsatellite loci was very low in the Irish population, with an average of 2.29 alleles per locus and expected heterozygosity of 0.35. Despite such low polymorphism, a panel of eight loci with a sibling probability of identity of 0.011 reliably identified individual pine marten and their gender, as determined by reference to genotypes of live trapped individuals. With high nuclear DNA amplification success rates (93.8%) and low genotyping error rates (1.8%), plucked hairs may represent a more reliable and cost-effective DNA source than scats for monitoring populations of this elusive carnivore, and similar taxa such as the sympatric stone marten Martes foina.

Human mitochondrial DNA complete amplification and sequencing: A new validated primer set that prevents nuclear DNA sequences of mitochondrial origin co‐amplification

To date, there are no published primers to amplify the entire mitochondrial DNA (mtDNA) that completely prevent the amplification of nuclear DNA (nDNA) sequences of mitochondrial origin. The main goal of this work was to design, validate and describe a set of primers, to specifically amplify and sequence the complete human mtDNA, allowing the correct interpretation of mtDNA heteroplasmy in healthy and pathological samples. Validation was performed using two different approaches: (i) Basic Local Alignment Search Tool and (ii) amplification using isolated nDNA obtained from sperm cells by differential lyses. During the validation process, two mtDNA regions, with high similarity with nDNA, represent the major problematic areas for primer design. One of these could represent a non-published nuclear DNA sequence of mitochondrial origin. For two of the initially designed fragments, the amplification results reveal PCR artifacts that can be attributed to the poor quality of the DNA. After the validation, nine overlapping primer pairs to perform mtDNA amplification and 22 additional internal primers for mtDNA sequencing were obtained. These primers could be a useful tool in future projects that deal with mtDNA complete sequencing and heteroplasmy detection, since they represent a set of primers that have been tested for the non-amplification of nDNA.

Germline mutation identified after amplification of nuclear DNA from citrated plasma

Germline mutation identified after amplification of nuclear DNA from citrated plasma

Blastocysts produced by nuclear transfer between chicken blastodermal cells and rabbit oocytes.

Interspecies nuclear transfer (INT) has been used as an invaluable tool for studying nucleus-cytoplasm interactions; and it may also be a method for rescuing endangered species whose oocytes are difficult to obtain. In the present study, we investigated interaction of the chicken genome with the rabbit oocyte cytoplasm. When chicken blastodermal cells were transferred into the perivitelline space of rabbit oocytes, 79.3% of the couplets were fused and 9.7% of the fused embryos developed to the blastocyst stage. Both M199 and SOF medium were used for culturing chicken-rabbit cloned embryos; embryo development was arrested at the 8-cell stage obtained in SOF medium, while the rates of morulae and blastocysts were 12.1 and 9.7%, respectively, in M199 medium. Polymerase chain reaction (PCR) amplification of nuclear DNA and karyotype analyses confirmed that genetic material of morulae and blastocysts was derived from the chicken donor cells. Analysis mitochondrial constitution of the chicken-rabbit cloned embryos found that mitochondria, from both donor cells and enucleated oocytes, co-existed. Our results suggest that: (1) chicken genome can coordinate with rabbit oocyte cytoplasm in early embryo development; (2) there may be an 8- to 16-cell stage block for the chicken-rabbit cloned embryos when cultured in vitro; (3) mitochondrial DNA from the chicken donor cells was not eliminated until the blastocyst stage in the chicken-rabbit cloned embryos; (4) factors existing in ooplasm for somatic nucleus reprogramming may be highly conservative.

Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts.

While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region of rrn operon of Frankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS1 region of the nuclear rrn operon of Alnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.

An evaluation of long-term preservation methods for brown bear (Ursus arctos) faecal DNA samples

Relatively few large-scale faecal DNA studieshave been initiated due to difficulties inamplifying low quality and quantity DNAtemplate. To improve brown bear faecal DNA PCRamplification success rates and to determinepost collection sample longevity, fivepreservation methods were evaluated: 90%ethanol, DETs buffer, silica-dried, oven-driedstored at room temperature, and oven-driedstored at −20 °C. Preservationeffectiveness was evaluated for 50 faecalsamples by PCR amplification of a mitochondrialDNA (mtDNA) locus (∼146 bp) and a nuclear DNA(nDNA) locus (∼200 bp) at time points of oneweek, one month, three months and six months. Preservation method and storage timesignificantly impacted mtDNA and nDNAamplification success rates. For mtDNA, allpreservation methods had ≥ 75% success atone week, but storage time had a significantimpact on the effectiveness of the silicapreservation method. Ethanol preserved sampleshad the highest success rates for both mtDNA(86.5%) and nDNA (84%). Nuclear DNAamplification success rates ranged from 26–88%, and storage time had a significant impacton all methods but ethanol. Preservationmethod and storage time should be importantconsiderations for researchers planningprojects utilizing faecal DNA. We recommendpreservation of faecal samples in 90% ethanolwhen feasible, although when collecting inremote field conditions or for both DNA andhormone assays a dry collection method may beadvantageous.

Characterization of the switch of kinetoplast DNA minicircle dominance during development and reversion of drug resistance in Leishmania

To characterize the differences between kDNA minicircles of drug-resistant Leishmania mexicana amazonensis variants that show nuclear DNA amplification and minicircles of variants without nuclear DNA amplification, we sequenced minicircles from repeatedly cloned parasites. The dominant minicircles from arsenite- and tunicamycin-resistant parasites with DNA amplification were found to preexist as minor conserved divergent classes in parental wild-type cells. These classes shared very limited similarity with the predominant wild-type minicircle sequences or sequences from drug resistant parasites without amplification. These minor classes were preferentially selected to replicate in variants with DNA amplification and subsequently became the dominant sequences in these variants. Kinetic studies of the correlation between amplification and deamplification of the nuclear DNA and the switch in kDNA minicircle dominance indicated that factor(s) other than the amplified chromosomal DNA itself caused the minicircles to switch. Treating the kDNA networks isolated from cells at the switch transition period with single cutter endonucleases specific for either wild-type or variant-specific minicircles resulted in structural modifications consistent with both minicircle sequence classes being present simultaneously in the same network. This establishes the ‘ trans’ nature of the switch.

Nuclear DNA amplification in cultured cells of Oryza sativa L.

Highly repeated nuclear DNA sequences from suspension cultured cells of Oryza sativa L. cv. 'Roncarolo' have been cloned in pBR322. Ten clones with specific digestion patterns have been randomly selected. Nine sequences appear to be organized in a clustered tandem array while one is interpersed in the rice genome. The clones have been used to gather information on: (a) their modulation in cultured cells as compared to whole plant and (b) their distribution in different rice cultivars belonging to the Japonica or Indica subspecies of Oryza sativa L. Hybridization with nuclear DNA isolated either from suspension or from seedlings of the 'Roncarolo' cultivar revealed extensive quantitative variations, with most cloned sequences showing amplification (up to 75-fold) in cultured cells. Hybridization with nuclear DNA isolated from seedlings or suspension cultured cells from different cultivars belonging to the Japonica or to the Indica sub-species of O. sativa have shown that (a) amplification also occurs in a similar pattern in the case of DNA from the other tested suspension cultured cell types but not in the case of DNA from seedlings; (b) in some cases the tested sequences show minor but significant variations in different rice accessions.