Content uploaded by António Amorim
Author content
All content in this area was uploaded by António Amorim
Content may be subject to copyright.
A preview of the PDF is not available
Identification of Species with DNA-Based Technology: Current Progress and Challenges
Abstract and Figures
One of the grand challenges of modern biology is to develop accurate and reliable technologies for a rapid screening of DNA sequence variation. This topic of research is of prime importance for the detection and identification of species in numerous fields of investigation, such as taxonomy, epidemiology, forensics, archaeology or ecology. Molecular identification is also central for the diagnosis, treatment and control of infections caused by different pathogens. In recent years, a variety of DNA-based approaches have been developed for the identification of individuals in a myriad of taxonomic groups. Here, we provide an overview of most commonly used assays, with emphasis on those based on DNA hybridizations, restriction enzymes, random PCR amplifications, species-specific PCR primers and DNA sequencing. A critical evaluation of all methods is presented focusing on their discriminatory power, reproducibility and user-friendliness. Having in mind that the current trend is to develop small-scale devices with a high-throughput capacity, we briefly review recent technological achievements for DNA analysis that offer great potentials for the identification of species.
Figures - uploaded by António Amorim
Author content
All figure content in this area was uploaded by António Amorim
Content may be subject to copyright.
... Modern phylogenetic research relies strongly on molecular data, which, based on a large amount of easily quantifiable information, typically allow more objective and robust inference of phylogeny than with morphological datasets (Suárez-Díaz 2013). Also, DNA sequences are used in alpha-taxonomy, along with morphological and other characters (Pereira et al. 2008). Aside from being monophyletic, there is general agreement that a valid genus should also fulfill additional conditions, including possession of a set of recognizable traits. ...
Analysis of a target enrichment molecular dataset confirms the monophyly of the Neotropical montane butterfly group known as the Pronophila Westwood clade, 1 of 2 major lineages of the satyrine subtribe Pronophilina. The Pronophila clade comprises 18–20 recognized genera and some 125 species. Within this group, the genus Pseudomaniola Röber appears as paraphyletic, and is split here into 3 genera, Pseudomaniola sensu novum with 6 species, including 4 previously considered as subspecies of P. phaselis (Hewitson), the monobasic Fahraeusia Pyrcz n. gen. for Catargynnis asuba Thieme, n. comb., and Boyeriana Pyrcz, Espeland & Willmott n. gen., with 9 species. The adults of all 3 genera can be recognized by their wing color patterns, but the strongest synapomorphies are found in the genitalia, especially those of the male, supporting the above systematic decisions. Notable differences are also found in scale organization and morphology. A divergence time analysis suggests that Fahraeusia diverged from Pseudomaniola + Boyeriana in the mid-Miocene, around 12 Mya, and the subsequent separation of the last 2 genera occurred at the start of the Pliocene at around 5 Mya.
... Upon receiving sequencing data, we visually checked the chromatogram quality and proofread the sequences using the software Geneious Prime, Version 2022.0.1 (Biomatters Ltd). Subsequently, as reported by previous studies on species identification [31][32][33], we used the Basic Local Alignment Search Tool (BLAST) on the NIH sequence database (GenBank) to align sequences with potential matches among the millions of publicly available sequences in this database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). We identified samples by comparing the sequence (query sequence) from our target sample with those (subject sequences) contained in GenBank's comprehensive reference database. ...
... Upon receiving sequencing data, we visually checked the chromatogram quality and proofread the sequences using the software Geneious Prime, Version 2022.0.1 (Biomatters Ltd). Subsequently, as reported by previous studies on species identification [31][32][33], we used the Basic Local Alignment Search Tool (BLAST) on the NIH sequence database (GenBank) to align sequences with potential matches among the millions of publicly available sequences in this database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). We identified samples by comparing the sequence (query sequence) from our target sample with those (subject sequences) contained in GenBank's comprehensive reference database. ...
Renewable energy production and development will drastically affect how we meet global energy demands, while simultaneously reducing the impact of climate change. Although the possible effects of renewable energy production (mainly from solar- and wind-energy facilities) on wildlife have been explored, knowledge gaps still exist, and collecting data from wildlife remains (when negative interactions occur) at energy installations can act as a first step regarding the study of species and communities interacting with facilities. In the case of avian species, samples can be collected relatively easily (as compared to other sampling methods), but may only be able to be identified when morphological characteristics are diagnostic for a species. Therefore, many samples that appear as partial remains, or "feather spots"-known to be of avian origin but not readily assignable to species via morphology-may remain unidentified, reducing the efficiency of sample collection and the accuracy of patterns observed. To obtain data from these samples and ensure their identification and inclusion in subsequent analyses, we applied, for the first time, a DNA barcoding approach that uses mitochondrial genetic data to identify unknown avian samples collected at solar facilities to species. We also verified and compared identifications obtained by our genetic method to traditional morphological identifications using a blind test, and discuss discrepancies observed. Our results suggest that this genetic tool can be used to verify, correct, and supplement identifications made in the field and can produce data that allow accurate comparisons of avian interactions across facilities, locations, or technology types. We recommend implementing this genetic approach to ensure that unknown samples collected are efficiently identified and contribute to a better understanding of wildlife impacts at renewable energy projects.
... Now the development of PCR technique makes easy to identify the meat species even from the cooked and spoiled meat in which protein is easily destroyed. Real time PCR is the revolution in this field in which we can identify and monitor the product during its amplification (Filipe et al., 2008). ...
The two main objectives that this study aimed were to determine the effectiveness of
adding chicken breast meat on the chemical and sensory properties of kebab firstly, and to
evaluate the quality of kebab by determining the origin of the animal species in meat
products secondly which is a matter of great concerns such as religious, economical, legal
as well as medical aspects.
To achieve the first objective the standard mixture of kebab considered as control
treatment (20% beef, 5% sheep tail fat and 75% meat of sheep flank area) was treated
with adding different concentration of chicken breast meat (by decreasing the
concentration meat of sheep flank area from 75% to 45%) as T1, T2, T3, T4, T5 in rate of
10%, 15%, 20%, 25%, 30%, respectively. After preparing the samples, sensory evaluation
was conducted for all of the kebab treatments and then chemical analysis were carried out
for raw and grilled samples, which comprised of the following parameters: the percentage
of moisture, protein, fat and ash contents as well as studying the change in the
organoleptic features represented by measuring pH, peroxide value, free fatty acid (Acid
Value), beside cholesterol content and water holding capacity. To achieve the second
objective of the study, DNA was extracted successfully from the kebab treatments. Three
pairs of species-specific primers were selected from previous reports. They depended on
mitochondrial cytochrome b (cyt b) gene for application of PCR to detect the species
involved in the treatments including chicken, beef and sheep.
... If a new approach or genetic marker is being used for the first time, the genetic composition of all taxon species should be established. [5]. Furthermore, studying the genetic structure of insect populations demands gathering a vast number of samples of each species [4]. ...
Polymorphic simple sequence repeats (SSRs), also known as microsatellites are considered as efficient molecular marker for genetic studies. The conventional methods of searching and developing SSR markers are laborious, time consuming, and less efficient. Therefore, in present study, the genome and transcriptome sequences were employed to search for polymorphic SSR using the Basic Sequence Alignment Tool (BLAST). The genomic sequences of Gryllus bimaculatus and transcriptomic sequences of Acheta domesticus were used to search polymorphic SSRs. BLAST allows to search and visualize sequence variation in sequence alignment, which is very imperial to detect insertion or deletion in SSR sequences as gaps in the alignment. The present study focused on a perfect trinucleotide SSRs and their observed variation based on stepwise mutation model. Total 26 polymorphic SSRs from the transcriptome and 25 from the genome were identified by the in silico method. Moreover, their transferability was also checked using transcriptome datasets from different species. The proposed in silico methodology can be used to examine variability and transferability of microsatellites. Moreover, the in silico approach also provides insights into SSRs, that is huge aid for primer design and overall better understanding of SSR sequences containing.
... Pathogenicity, ecological study and industrial importance are the two main characteristics necessary to characterize a microbial isolate beyond species level and determine its subspecies, strain, or even substrain. Strains of microbes are often classified below the species level called typing [59]. Conventional techniques generally employed for microbial identification are based on phenotypic or biochemical characteristics, however these methods are labor-intensive, time-consuming and often inadequate for the differentiation of phenotypically similar species. ...
Microorganisms offer a wide array of useful metabolites and products ranging from environmentally important biofuels
[1,2], industrially important enzymes [3,4], agriculturally important biofertilizers [5,6] and biopesticides [7,8], etc. Apart
from that, microorganisms are a natural source of a plethora of medically significant molecules like antibiotics [9–11],
anticancer agents [12,13], antivirals [14,15], etc. Also, the utilization of microorganisms for commercial production of
industrially important compounds is inherently advantageous. This can be attributed to the literally unending metabolic
diversity of microbes, their shorter life spans, amenability to genetic modification, relatively simpler cultivation requirements,
etc. [16]. Moreover, due to the ever-increasing need for novel therapeutic agents as well as the stress on natural
products, the microbes and fermentation industry are gaining importance day by day [11,17]. Another major force providing
impetus for looking for novel microbial metabolites (especially in the field of medicine), is the ever increasing challenge
of emergence of drug resistance in pathogenic microorganisms which includes drug resistant bacteria, fungi, viruses
as well as protozoa [11,18–20]. Moreover, the recent coronavirus pandemic, caused by severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), has practically caught us unaware with respect to our preparedness for any novel and
emerging infection. So, exploring the microbial metabolic diversity for therapeutic as well as other potential uses is certainly
the need of hour.
... Finding these quantitative characteristic loci opens the door to genetic advancement and makes poultry breeding operations easier. In the chicken industry, the use of genetic selection techniques has boosted growth rate and carcass quality (Pereira et al., 2008). Over the past few decades, advances in DNA technology and molecular genetics have coincided with remarkable developments in animal breeding and genetic improvement. ...
This study examined the growth characteristics, nucleotide sequence variations, and economic evaluation of two duck breeds. For this investigation, 150 each of 300 male Muscovy and Pekin ducklings aged one week were used. Body weight gain (BWG) as well as the initial and final live body weights were noted. Feed conversion ratios (FCR) and average feed intake (AFI) were also calculated. The percentages of the giblets and eviscerated carcass to live body weight were recorded after the giblets and carcass were weighed. For the purpose of extracting DNA, blood samples from ducks of each breed were taken and placed in tubes with disodium EDTA as an anticoagulant. For real-time analysis, RNA was taken from the liver and breast muscle of each breed. In terms of economic criteria, each breed's total variable costs (TVC), total costs (TC), total return (TR), net return (NR), values for the difference in net profit, and economic efficiency percent were assessed. In comparison to Pekin breed, the Muscovy breed exhibited higher ultimate BW, BWG, dressing percentages, and liver percentages. However, the Muscovy breed experienced a considerable fall in AFI and FCR. PCR-DNA sequencing of IGFBP3, FGF5, MSTN, PGAM2, ApoVLDL-II, ACACA, SCD, FASN and LPL genes revealed nucleotide sequence variations in the form of single nucleotide polymorphisms (SNPs) between Muscovy and Pekin breeds. Muscovy had a much higher gene expression profile for growth markers than Pekin breed. Muscovy breed displayed greater TR, NR, net profit difference%, and economic efficiency% compared to Pekin ducks. According to this study, Muscovy ducks showed greater growth features than Pekin ducks. Identified SNPs and the gene expression profile of growth promoters, together with economic factors, may serve as proxy indicators for selection the best breed of ducks.
... DNA barcoding is essential for identification, evolutionary relationship, and taxonomy. DNA barcoding technology has been adopted as a good approach for species identification (Pereira et al., 2008). This genetic variation and divergence may be due to habitat effects, spatial variations (Doherty, 1991), and physical variables which have a great influence on the fish distribution and other aquatic organisms (Lasram et al., 2010). ...
Molecular appraoch for identification of unknown species by using Cytochrome b gene is an effective and reliable as compared with morphological based identification. For DNA barcoding universal molecular genes were used to identify the species. Cytochrome b is a specific gene used for identification purpose. DNA barcoding is a reliable and effective method compared to the different traditional morphological methods of specie identification. So,in the present study which was conducted to identify the species, a total of 50 fish samples were collected from five different sites. DNA was extracted by using the Phenol Chloroform method from muscle tissue. Five sequences were sequenced (one from each site), analyzed, and identified specific species as Pangasius pangasius. Identified sequences were variable in length from 369 bp (Site 1), 364 bp (Site 2), 364 bp (Site 3), 352 bp (Site 4), and 334 bp (Site 5). Identity matches on the NCBI database confirmed the specific specie as P. pangasius. A distancing tree was drawn to show maximum likelihood among the same and different species. Yet, in many cases fishes on diverse development stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative tool for species identification and phylogenetic study. This work intends to provide an updated and extensive overview on the DNA based methods for fish species identification by using Cytochrome b gene as targeted markers for identification purpose.
Phetchabun Basin is located in the lower northern part of Thailand and presents many
interesting geological features, including Cenozoic columnar basalt, national geopark,
petroleum, and other geological resources. Along the national highway number 225 area, the outcrop appeared by a new road cut. A distinctive point of the outcrop is the large porphyritic texture of basalt. This work aims to identify rock names and magma series as well as classify phenocrysts of basaltic rocks. The methodology comprises petrography, geochemistry analyzed by Handheld ED-XRF, and mineralogy of phenocrysts studied by SEM-EDS. Petrography classifies the rocks as porphyritic olivine tholeiite basalt, consisting of plagioclase (andesine), olivine, and clinopyroxene with a small amount of orthopyroxene opaque minerals. According to the geochemistry of phenocrysts, the plagioclases are classified as andesine-bytownite (An33-70), and olivine is hyalosiderite (Fo57). Major oxides and trace elements classify magma series as sub-alkali, including tholeiite and calc-alkali. So, the results show that these basaltic andesites were generated from transitional tholeiitic magma and erupted in the continental rifting process of the Phetchabun Basin.
The objective of this review is to summarize numerous studies on the use of the random amplified polymorphic DNA (RAPD) technique on rice, corn, wheat, sorghum, barley, rye, and oats to examine its feasibility and alidity for assessment of genetic variation, population genetics, mapping, linkage and marker assisted selection, phylogenetic analysis, and the detection of somaclonal variation. Also we discuss the advantages and limitations of RAPD. Molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The simplicity of the RAPD technique made it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics.
The recent explosion of interest in DNA-based tools for species identification has prompted widespread speculation on the
future availability of inexpensive, rapid, and accurate means of identifying specimens and assessing biodiversity. One applied
field that may benefit dramatically from the development of such technologies is the detection, identification, and monitoring
of invasive species. Recent studies have demonstrated the feasibility of DNA-based tools for such important tasks as confirmation
of specimen identity and targeted screening for known or anticipated invaders. However, significant technological hurdles
must be overcome before more ambitious applications, including estimation of propagule pressure and comprehensive surveys
of complex environmental samples, are to be realized. Here we review existing methods, examine the technical difficulties
associated with development of more sophisticated tools, and consider the potential utility of these DNA-based technologies
for various applications relevant to invasive species monitoring.
A convenient DNA-based identification system is described for testing the species origin of meat samples. Probes are generated
by PCR with primers binding to species-specific satellite DNA and hybridized to DNA purified from meat. This method is more
robust and versatile than methods based on oligonucleotide hybridization. With the exception of a slight cross-reaction of
mutton and beef, each probe only recognized the species from which it was derived. Purifying the DNA with a DNA-binding resin
improved the sensitivity. Admixtures of 0.1–0.5% can be detected in raw meat and 0.5–5% in autoclaved meat samples. The method
can be adapted to detect any eukaryotic species for which species-specific DNA sequences are available. This method has proven
its value in the routine inspection of meat samples by revealing more cases of deliberate or accidental species substitution
and admixture than conventional techniques.
Single-nucleotide polymorphisms (SNPs) are the most abundant type of human genetic variation. These variable sites are present at high density in the genome, making them powerful tools for mapping and diagnosing disease-related alleles. We have developed a sensitive and rapid flow cytometry-based assay for the multiplexed analysis of SNPs based on polymerase-mediated primer extension, or minisequencing, using microspheres as solid supports. The new method involves subnanomolar concentrations of sample in small volumes (∼10 μl) which can be analyzed at rates of one sample per minute or faster, without a wash step. Further, genomic analysis using multiplexing microsphere arrays (GAMMArrays), enables the simultaneous analysis of dozens, and potentially hundreds of SNPs per sample. We have tested the new method by genotyping the Glu69 variant from the HLA DPB1 locus, a SNP associated with chronic beryllium disease, as well as HLA DPA1 alleles using the multiplexed method. The results demonstrate the sensitivity and accuracy of flow cytometry-based minisequencing, a powerful new tool for genome- and global-scale SNP analysis.
Bicyclic nucleoside analogues with a fixed N-type conformation, 2′-O,4′-C-methyleneuridine and -cytidine, were incorporated into oligonucleotides, and the binding efficiency of the modified oligonucleotides to the complementary DNA and RNA as well as the CD spectra of the modified DNA-DNA and modified DNA-RNA duplexes were studied.
Recent trends and challenges in the electrochemical methods for the detection of DNA hybridization are reviewed. Electrochemistry has superior properties over the other existing measurement systems, because electrochemical biosensors can provide rapid, simple and low-cost on-field detection. Electrochemical measurement protocols are also suitable for mass fabrication of miniaturized devices. Electrochemical detection of hybridization is mainly based on the differences in the electrochemical behaviour of the labels towards the hybridization reaction on the electrode surface or in the solution. Basic criteria for electrochemical DNA biosensor technology, and already commercialized products, are also introduced. Future prospects towards PCR-free DNA chips are discussed.
Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of
any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters
are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter
and restriction site sequence, with additional nucleotides at the 3′-end, are used as selective agents to amplify a subset
of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide
gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for
the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes
for determining genetic relationships and for epidemiological typing. This review describes the AFLP procedure, and recent,
novel applications in the molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms.