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A Guide for Mitochondrial DNA Analysis in Non-Human Forensic Investigations
Abstract and Figures
In recent years, mitochondrial DNA (mtDNA) typing has become established as a powerful tool for forensic investigations. The sequencing of mtDNA is now a standard laboratory procedure for the examination of degraded casework samples of human origin, with unique advantages over nuclear DNA profiling systems. Nonetheless, the use of mtDNA in non-human forensic science has only recently gained attention, with applications in the profiling of 'silent witnesses' of crimes, traceability of food products and control of illegal trade of endangered species or drugs, among others. This review briefly describes some important features of mtDNA with relevance for forensic testing in different taxonomic groups and suggests appropriate laboratorial and analytic procedures to avoid erroneous interpreta-tions. It is imperative to merge a solid knowledge of mitochondrial genetics with rigorous methods of error prevention in order to permit the full approval of non-human mtDNA profiling in routine forensic work.
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... Mitochondrial (mt) genomes of multicellular animals (metazoans) are generally closed-circular and double-stranded DNA molecules approximately 14−20 kbp in length [1][2][3]. However, genome size ranges from 6 to 48 kbp [2,4] and linear and fragmented mtDNAs have been found in approximately 9000 animals investigated thus far [5][6][7]. ...
... Mitochondrial (mt) genomes of multicellular animals (metazoans) are generally closed-circular and double-stranded DNA molecules approximately 14−20 kbp in length [1][2][3]. However, genome size ranges from 6 to 48 kbp [2,4] and linear and fragmented mtDNAs have been found in approximately 9000 animals investigated thus far [5][6][7]. In most metazoans, this small organelle genome encodes a typical set of 37 genes: 13 protein genes, involved in the electron transport system of respiration, two rRNA genes (rrns), and 22 tRNA genes (trns). ...
... In most metazoans, this small organelle genome encodes a typical set of 37 genes: 13 protein genes, involved in the electron transport system of respiration, two rRNA genes (rrns), and 22 tRNA genes (trns). In addition, animal mt genomes contain one long non-coding region, harboring several sequence elements related to mt genomic transcription and replication, named the control region (CR), or alternatively the D-loop region [2]. ...
... Mitochondrial (mt) genomes of multicellular animals (metazoans) are generally closed-circular and double-stranded DNA molecules approximately 14-20 kbp in length [1][2][3]. However, genome size ranges from 6 to 48 kbp [2,4], and linear and fragmented mtDNAs have been found in approximately 9000 animals investigated thus far [5][6][7]. ...
... Mitochondrial (mt) genomes of multicellular animals (metazoans) are generally closed-circular and double-stranded DNA molecules approximately 14-20 kbp in length [1][2][3]. However, genome size ranges from 6 to 48 kbp [2,4], and linear and fragmented mtDNAs have been found in approximately 9000 animals investigated thus far [5][6][7]. In most metazoans, this small organelle genome encodes a typical set of 37 genes: 13 protein genes, involved in the electron transport system of respiration, two rRNA genes (rrns), and 22 tRNA genes (trns). ...
... In most metazoans, this small organelle genome encodes a typical set of 37 genes: 13 protein genes, involved in the electron transport system of respiration, two rRNA genes (rrns), and 22 tRNA genes (trns). In addition, animal mt genomes contain one long noncoding region, harboring several sequence elements related to mt genomic transcription and replication, named the control region (CR) or alternatively the D-loop region [2]. ...
The mitochondrial (mt) genome of the bushveld rain frog (Breviceps adspersus, Brevicipitidae, Afrobatrachia) is the largest (28.8 kbp) among the vertebrates investigated to date. The major cause of genome size enlargement in this species is the duplication of multiple genomic regions. To investigate the evolutionary lineage, timing, and process of mt genome enlargement, we sequenced the complete mtDNAs of two congeneric rain frogs, B. mossambicus and B. poweri. The mt genomic organization, gene content, and gene arrangements of these two rain frogs are very similar to each other but differ from those of B. adspersus. The B. mossambicus mt genome (22.5 kbp) does not differ significantly from that of most other afrobatrachians. In contrast, the B. poweri mtDNA (28.1 kbp) is considerably larger: currently the second largest among vertebrates, after B. adspersus. The main causes of genome enlargement differ among Breviceps species. Unusual elongation (12.5 kbp) of the control region (CR), a single major noncoding region of the vertebrate mt genome, is responsible for the extremely large mt genome in B. poweri. Based on the current Breviceps phylogeny and estimated divergence age, it can be concluded that the genome enlargements occurred independently in each species lineage within relatively short periods. Furthermore, a high nucleotide substitution rate and relaxation of selective pressures, which are considered to be involved in changes in genome size, were also detected in afrobatrachian lineages. Our results suggest that these factors were not direct causes but may have indirectly affected mt genome enlargements in Breviceps.
... CO1 gene is maternalistic. CO1 gene has very rapidly mutate or have a high rate of sequence change and showed a sequence divergence in the same species(Herbert ., 2003;Bucklin ., 2003;Pareira ., 2010). CO1 gene has been used to determine the spread of L. in, the results of the indonesian. ...
The aims of this study were to characterize the mitochondrial cytochrome oxidase subunit 1 gene (CO1) of Apis dorsata from Minahasa, North Sulawesi. Apis dorsata Binghami used in this study was obtained from the natural colony in Klabat forest, Airmadi forest and Kombi forest. From each native bee colony, ten workers were taken (n = 30). Femur hind legs used for subsequent DNA extraction were amplified by PCR, underwent electrophoresis and then sequenced. CO1 sequences then were matched with BLAST to obtain sequences from the NCBI that have high levels of homology (>90%). The results showed that the universal primer LCO -1490 and HCO - 2198 success amplify the gene CO1 Apis dorsata of Minahasa. Based on the phylogeny tree, Apis dorsata derived from forest Airmadidi and forest Kombi have the same CO1 sequences but have had differences with Apis dorsata from forests Klabat. BLAST analysis results showed that the partial mitochondrial CO1 sequences Apis dorsata of Minahasa has the highest rate of 94 % similarity with similar sequences recorded in the NCBI gene bank.
... On the other hand, polymerase chain reaction (PCR) with specific primers is a cost-effective way to identify a specific plant (Wallinger et al., 2012). Generally, hypervariable regions of some genes like ycf1, matK,rbcL,ropC1,rpoB, sequences are widely used for plant species identification and phylogenetic analyses (Dong et al., 2015), as the mitochondrial DNA, like that of animal species, is not suitable for plant species identification (Pereira et al., 2010). But the concerning issue is that the design of reliable specific primers from these genes of cpDNA is the most challenging part due to the lower variability between closely related taxa (Wallinger et al., 2012). ...
The phenomenon of incorporating sweet pumpkin pulp into industrially processed mango juice has been observed in various reports from Bangladesh in recent years due to the remarkable resemblance in the matrix and color of both fruits at their matured stage. The increasing prevalence of such incidents has become a growing concern for consumers due to the several potential health implications of sweet pumpkin. However, there is no reliable method available so far for the surveillance of this fraudulent activity. Therefore, the present study highlights a prompt and precise approach for tracing the origin of mango and pumpkin species in commercial mango juices based on a polymerase chain reaction. This approach involves the use of newly designed primer sets that target the chloroplast ycf1 genes and can produce the specific detectable amplicons of 206 and 70 bp from ≥10^(−4) and ≥10^(−3) ng/µl of total DNA for mango and pumpkin, respectively. Analyzing six samples of industrially produced mango juice, only four were traceable to their respective origins, and neither mango nor sweet pumpkin were detected in the two remaining samples. However, the non-reactive samples also did not succeed during performance analysis with a plant-specific universal primer set, indicating that the non-reactive samples may contain target unamplifiable or amplifiable DNA along with PCR inhibitors or non-target DNA without PCR inhibitors. Therefore, based on these consistent findings with the plant-specific universal primer set, the newly designed primer sets can be applied to pumpkin adulterant detection in commercial mango juice items.
... Mitochondrial DNA is a useful tool in forensic sciences because it helps to establish the source of a biological sample by means of linage identification [1]. The advantage of mitochondrial-based DNA analyses derives from the fact that there are several mitochondria per cell and many mitochondrial DNA molecules within each mitochondrion, increasing the possibility of obtaining results from critical samples [2]. ...
... Our previous works have targeted the mitochondrial DNA (mtDNA) of animals, taking advantage of its relatively high mutation rate [11][12][13]. However, the mtDNA of plants is not suitable for species identification procedures since it is usually slowly evolving, resulting in the absence of inter-specific variation, has high intra-molecular recombination and pseudogenes [14][15][16]. Therefore, researches have used the chloroplast DNA (cpDNA) for identification of plant species [17][18][19]. ...
The correct identification of species in the highly divergent group of plants is crucial for several forensic investigations. Previous works had difficulties in the establishment of a rapid and robust method for the identification of plants. For instance, DNA barcoding requires the analysis of two or three different genomic regions to attain reasonable levels of discrimination. Therefore, new methods for the molecular identification of plants are clearly needed. Here we tested the utility of variable-length sequences in the chloroplast DNA (cpDNA) as a way to identify plant species. The SPInDel (Species Identification by Insertions/Deletions) approach targets hypervariable genomic regions that contain multiple insertions/deletions (indels) and length variability, which are found interspersed with highly conserved regions. The combination of fragment lengths defines a unique numeric profile for each species, allowing its identification. We analysed more than 44,000 sequences retrieved from public databases belonging to 206 different plant families. Four target regions were identified as suitable for the SPInDel concept: atpF-atpH, psbA-trnH, trnL CD and trnL GH. When considered alone, the discrimination power of each region was low, varying from 5.18% (trnL GH) to 42.54% (trnL CD). However, the discrimination power reached more than 90% when the length of some of these regions is combined. We also observed low diversity in intraspecific data sets for all target regions, suggesting they can be used for identification purposes. Our results demonstrate the utility of the SPInDel concept for the identification of plants.
The use of deoxyribonucleic acid, i.e. DNA, for the testing in criminal justice explains the term forensic DNA analysis in simple words. It was first introduced in 1981. The term forensis which is a Latin word has given birth to the forensic science where forensic means pertaining to; thus, the term forensic sciences means the use of various applications for the resolution of criminal disputes either criminal or civil. The DNA analysis has become an indispensable part of the modern forensic science; with the use of PCR techniques, it has become a major tool for the analysis of the biological material. The method of DNA analysis used in forensic science is also known by the popular term DNA profiling [1]. The main focus of this science is on the use of the genetic material in case of the criminal justice for solving the cases and answering the concerns related to the cases. It has become a very significant source for establishment and expansion of the databases of DNA collected from the suspected criminals. The technique of DNA analysis involves the use of genetic material in this process; the sample in the form of hair, skin and blood is collected from the suspects that are linked to a particular or various crime scenes [2]. Then the large numbers of isolated DNA sample are interrogated with the DNA databases in order to match the profiles of DNA at the scene of the crime. A DNA database comprises of the profiles of different DNA that are used for the analysis of genetic diseases, genetic fingerprinting or genealogy purposes.
In the event of fires, natural disasters, and other events associated with high temperature, bones and teeth are the only source of genetic material for identifying human or animal carcasses. To obtain reliable final results of identification tests, the use of appropriate nucleic acid extraction methods is crucial. Therefore, the main objective of this research was to evaluate the effectiveness of selected methods of DNA isolation from animal burnt bones and teeth. In addition, the effect of the duration of high temperature on the stability of nuclear and mitochondrial DNA in these tissues was determined, as well as the possibility of using the genetic material obtained for species identification of remains of unknown origin. Bones and teeth collected during necropsy of dogs were burnt in a laboratory oven at 400 °C (752 °F; 673.15 K) for 5, 10, 15, 30, 45 and 60 min. DNA was isolated according to four different protocols, using three commercial kits, i.e. the PrepFiler® Forensic DNA Extraction Kit from Applied Biosystems, the QIAamp® DNA Investigator Kit from QIAGEN, and the DNA Mini Kit from Syngen, as well as a classic organic method. The effectiveness of these methods was compared by assessing the amount of isolated DNA using Real-Time PCR and its purity using a NanoDrop™ spectrophotometer. Each isolate was also subjected to PCR with primers designed to amplify fragments of dog mitochondrial DNA. The effectiveness of species identification was assessed for the method showing the best DNA recovery and for the organic method, considered the gold standard for analysis of difficult material. The QIAamp® DNA Investigator Kit showed the highest efficiency of DNA isolation from bones and teeth burnt for 15 min (the longest burning time for which DNA could still be recovered from bones and teeth). The results of the experiment clearly indicate that DNA stability in hard tissues depends on how long they burn. In the case of exposure to 400 °C, reliable genetic testing, including species identification, is possible when the burning time does not exceed 15 min. Among the hard tissues examined, bones proved more suitable than teeth for identification purposes. It was also concluded that identification of bone remains with extreme heat damage should be based on mitochondrial DNA analysis.
MTCYB gene produces a protein which is a component of respiratory chain complex III. This gene is also important in phylogenetic relationships between and among species. Presently many hereditary carcinomas are also associated with the alterations of this gene. The aim of the current study is to develop primers for amplification and sequencing of complete human mitochondrial cytochrome b (CYTB) gene and use of the FTA card for isolation and preservation of mitochondrial DNA. DNA from 6 blood samples were isolated by the phenol chloroform method as well as by the FTA card method which was further used for the PCR amplification and sequencing process. Novel human mitochondrial cytochrome b (CYTB) gene specific primers (two sets) were designed and examined for amplification and sequencing. DNA trapped on FTA cards and DNA isolated by the phenol chloroform method produced a brighter clear band of approximately 1320 bp with both designed primers. During sequencing reverse and forward primers of both primer sets were produced good quality DNA sequence and prepared consensus sequences covers complete cytochrome b (CYTB) gene. Whatman FTA Classic card was successfully used for storage and amplification of Human mitochondrial DNA as well as, two sets of Human mitochondrial cytochrome b (cyt b) gene specific novel primers namely MVM-MT-Cyb-01 and MVM-MT-Cyb-02. Keywords: FTA classic card, cytochrome b gene, PCR amplification, sequencing, human mitochondria.
Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
The assumption that human mitochondrial DNA is inherited from one parent only and therefore does not recombine is questionable.
Linkage disequilibrium in human and chimpanzee mitochondrial DNA declines as a function of the distance between sites. This
pattern can be attributed to one mechanism only: recombination.
Shark DNA extracted from products used in trade (e.g. soup and dried fins) was amplified using PCR. A strategy is described that permits the identification of amplified material to species (for mtDNA Cytb and NADH2 loci) and the species-specific amplification of basking shark DNA from mixed templates (for the Cytb locus). These methods will be useful for forensic applications to govern trade in these heavily exploited species.
The ability to discriminate between species using barcoding loci has proved more difficult in plants than animals, raising the possibility that plant species boundaries are less well defined. Here, we review a selection of published barcoding data sets to compare species discrimination in plants vs. animals. Although the use of different genetic markers, analytical methods and depths of taxon sampling may complicate comparisons, our results using common metrics demonstrate that the number of species supported as monophyletic using barcoding markers is higher in animals (> 90%) than plants (~70%), even after controlling for the amount of parsimony-informative information per species. This suggests that more than a simple lack of variability limits species discrimination in plants. Both animal and plant species pairs have variable size gaps between intra- and interspecific genetic distances, but animal species tend to have larger gaps than plants, even in relatively densely sampled genera. An analysis of 12 plant genera suggests that hybridization contributes significantly to variation in genetic discontinuity in plants. Barcoding success may be improved in some plant groups by careful choice of markers and appropriate sampling; however, overall fine-scale species discrimination in plants relative to animals may be inherently more difficult because of greater levels of gene-tree paraphyly.
The ability to extract DNA from ivory provides the basis for genetically tracking the origin of poached ivory and thus has important implications for elephant conservation and management. We describe a method to isolate and amplify both genomic and mitochondrial DNA from African elephant ivory that requires very small amounts of ivory taken from any location on the tusk. We pulverized ivory and isolated DNA with a modified QIAamp kit. Ivory as old as 10 to 20 years, stored at ambient conditions, was amenable to DNA isolation with this method. The isolated DNA was robustly amplified at 16 elephant microsatellite loci and two mitochondrial DNA loci. This method has important applications for the forensic analysis of poached African elephant ivory. It enables determination of where stronger antipoaching efforts are needed and provides the basis for monitoring the extent of the trade as well as the consequences of future international trade decisions.
With the discovery of the polymerase chain reaction (PCR) in the mid-1980's, the last in a series of critical molecular biology techniques (to include the isolation of DNA from human and non-human biological material, and primary sequence analysis of DNA) had been developed to rapidly analyze minute quantities of mitochondrial DNA (mtDNA). This was especially true for mtDNA isolated from challenged sources, such as ancient or aged skeletal material and hair shafts. One of the beneficiaries of this work has been the forensic community. Over the last decade, a significant amount of research has been conducted to develop PCR-based sequencing assays for the mtDNA control region (CR), which have subsequently been used to further characterize the CR. As a result, the reliability of these assays has been investigated, the limitations of the procedures have been determined, and critical aspects of the analysis process have been identified, so that careful control and monitoring will provide the basis for reliable testing. With the application of these assays to forensic identification casework, mtDNA sequence analysis has been properly validated, and is a reliable procedure for the examination of biological evidence encountered in forensic criminalistic cases.
Copyright © 1999 Central Police University.
The mitochondrial oxidative phosphorylation (OXPHOS) system is the final
biochemical pathway in the production of ATP. The OXPHOS system consists of
five multiprotein complexes, the individual subunits of which are encoded
either by the mitochondrial or by the nuclear genome. Defects in the OXPHOS
system result in devastating, mainly multisystem, diseases, and recent years
have seen the description of the underlying genetic mutations in mitochondrial
and nuclear genes. Advances in this arena have profited from progress in various
genome projects, as well as improvements in our ability to create relevant
animal models.
We here describe a multiplex–PCR method to generate six overlapping short amplicons (100–130 bp) in two separate PCR reactions of non-overlapping fragments for full sequencing of the whole hypervariable region I (HV1). The performance of this multiplex system has been evaluated not only on ancient bone remains (5000–4000 BP) but also on different forensic samples with highly degraded DNA (bone remains, hair shafts, . . .) that yielded negative PCR results with the mtDNA amplification strategies usually employed in forensic genetics. The multiplex–PCR methodology described in this study also illustrates a potential high throughput strategy to reconstruct the sequence of both coding and non-coding regions of the mtDNA genome from ancient DNA.