Successful PCR Research Articles

Genotyping of the OATP1B1 c. 521 T>C Polymorphism from the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens: an Optimized Protocol.

Organic anion transporting polypeptide (OATP) 1B1 is a liver-specific transport protein that plays an important role in hepatic drug disposition. It transports many drugs from the blood into the liver, including lipid-lowering statins. The c. 521 T>C polymorphism of OATP1B1 has reduced transport activity and is associated with statin-induced myopathy. Formalin-fixed paraffin-embedded (FFPE) liver tissues can be an enriched source for genotyping of this clinically significant OATP1B1 polymorphism in retrospective studies. The successfulness of genotyping using Sanger-sequencing of a PCR product from FFPE tissue relies on a successful PCR amplification using genomic DNA extracted from the FFPE tissues. Such PCR amplification is often limited by the quality of DNA extracted from the FFPE tissue. An optimized method for high-quality DNA extraction and efficient PCR amplification is highly needed in order to genotype polymorphisms such as the c. 521 T>C polymorphism using FFPE tissues. The current study established an optimized and reproducible method for a Sanger-sequencing-based genotyping method using FFPE human liver tissues that is applicable to even small FFPE tissues such as needle-core biopsy specimens.

DNA barcodes from snake venom: a broadly applicable method for extraction of DNA from snake venoms.

DNA barcoding is a simple technique used to develop a large-scale system of classification that is broadly applicable across a wide variety of taxa. DNA-based analysis of snake venoms can provide a system of classification independent of currently accepted taxonomic relationships by generating DNA barcodes specific to each venom sample. DNA purification from dried snake venoms has previously required large amounts of starting material, has resulted in low yields and inconsistent amplification, and was possible with front-fanged snakes only. Here, we present a modified DNA extraction protocol applied to venoms of both front- and rear-fanged snakes that requires significantly less starting material (1mg) and yields sufficient amounts of DNA for successful PCR amplification of regions commonly used for DNA barcoding. [Formula: see text].

The first set of universal nuclear protein-coding loci markers for avian phylogenetic and population genetic studies

Multiple nuclear markers provide genetic polymorphism data for molecular systematics and population genetic studies. They are especially required for the coalescent-based analyses that can be used to accurately estimate species trees and infer population demographic histories. However, in avian evolutionary studies, these powerful coalescent-based methods are hindered by the lack of a sufficient number of markers. In this study, we designed PCR primers to amplify 136 nuclear protein-coding loci (NPCLs) by scanning the published Red Junglefowl (Gallus gallus) and Zebra Finch (Taeniopygia guttata) genomes. To test their utility, we amplified these loci in 41 bird species representing 23 Aves orders. The sixty-three best-performing NPCLs, based on high PCR success rates, were selected which had various mutation rates and were evenly distributed across 17 avian autosomal chromosomes and the Z chromosome. To test phylogenetic resolving power of these markers, we conducted a Neoavian phylogenies analysis using 63 concatenated NPCL markers derived from 48 whole genomes of birds. The resulting phylogenetic topology, to a large extent, is congruence with results resolved by previous whole genome data. To test the level of intraspecific polymorphism in these makers, we examined the genetic diversity in four populations of the Kentish Plover (Charadrius alexandrinus) at 17 of NPCL markers chosen at random. Our results showed that these NPCL markers exhibited a level of polymorphism comparable with mitochondrial loci. Therefore, this set of pan-avian nuclear protein-coding loci has great potential to facilitate studies in avian phylogenetics and population genetics.

A Cohort Study of the Milk Microbiota of Healthy and Inflamed Bovine Mammary Glands From Dryoff Through 150 Days in Milk.

The objective of this longitudinal cohort study was to describe the milk microbiota of dairy cow mammary glands based on inflammation status before and after the dry period. Individual mammary quarters were assigned to cohorts based on culture results and somatic cell count (SCC) at dryoff and twice in the first 2 weeks post-calving. Mammary glands that were microbiologically negative and had low SCC (< 100,000 cells/mL) at all 3 sampling periods were classified as Healthy (n = 80). Microbiologically negative mammary glands that had SCC ≥150,000 cells/mL at dryoff and the first post-calving sample were classified as Chronic Culture-Negative Inflammation (CHRON; n = 17). Quarters that did not have both culture-negative milk and SCC ≥ 150,000 cells/mL at dryoff but were culture-negative with SCC ≥ 150,000 at both post-calving sampling periods were classified as Culture-Negative New Inflammation (NEWINF; n = 6). Mammary glands with bacterial growth and SCC ≥ 150,000 cells/mL at all 3 periods were classified as Positive (POS; n = 3). Milk samples were collected from all enrolled quarters until 150 days in milk and subjected to microbiota analysis. Milk samples underwent total DNA extraction, a 40-cycle PCR to amplify the V4 region of the bacterial 16S rRNA gene, and next-generation sequencing. Healthy quarters had the lowest rate of PCR and sequencing success (53, 67, 83, and 67% for Healthy, CHRON, NEWINF, and POS, respectively). Chao richness was greatest in milk collected from Healthy quarters and Shannon diversity was greater in milk from Healthy and CHRON quarters than in milk collected from glands in the NEWINF or POS cohorts. Regardless of cohort, season was associated with both richness and diversity, but stage of lactation was not. The most prevalent OTUs included typical gut- and skin-associated bacteria such as those in the phylum Bacteroidetes and the genera Enhydrobacter and Corynebacterium. The increased sequencing success in quarters with worse health outcomes, combined with the lack of bacterial growth in most samples and the high PCR cycle number required for amplification of bacterial DNA, suggests that the milk microbiota of culture-negative, healthy mammary glands is less abundant than that of culture-negative glands with a history of inflammation.

DNA barcoding the flowering plants from the tropical coral islands of Xisha (China).

AimDNA barcoding has been widely applied to species diversity assessment in various ecosystems, including temperate forests, subtropical forests, and tropical rain forests. However, tropical coral islands have never been barcoded before due to the difficulties in field exploring. This study aims at barcoding the flowering plants from a unique ecosystem of the tropical coral islands in the Pacific Ocean and supplying valuable evolutionary information for better understanding plant community assembly of those particular islands in the future.LocationXisha Islands, China.MethodsThis study built a DNA barcode database for 155 plant species from the Xisha Islands using three DNA markers (ITS, rbcL, and matK). We applied the sequence similarity method and a phylogenetic‐based method to assess the barcoding resolution.ResultsAll the three DNA barcodes showed high levels of PCR success (96%–99%) and sequencing success (98%–100%). ITS performed the highest rate of species resolution (>95%) among the three markers, while plastid markers delivered a relatively poor species resolution (85%–90%). Our analyses obtained a marginal increase in species resolution when combining the three DNA barcodes.Main conclusionsThis study provides the first plant DNA barcode data for the unique ecosystem of tropical coral islands and considerably supplements the DNA barcode library for the flowering plants on the oceanic islands. Based on the PCR and sequencing success rates, and the discriminatory power of the three DNA regions, we recommend ITS as the most successful DNA barcode to identify the flowering plants from Xisha Islands. Due to its high sequence variation and low fungal contamination, ITS could be a preferable candidate of DNA barcode for plants from other tropical coral islands as well. Our results also shed lights on the importance of biodiversity conservation of tropical coral islands.

Differential Usefulness of Nine Commonly Used Genetic Markers for Identifying Phytophthora Species.

The genus Phytophthora is agriculturally and ecologically important. As the number of Phytophthora species continues to grow, identifying isolates in this genus has become increasingly challenging even by DNA sequencing. This study evaluated nine commonly used genetic markers against 154 formally described and 17 provisionally named Phytophthora species. These genetic markers were the cytochrome-c oxidase 1 (cox1), internal transcribed spacer region (ITS), 60S ribosomal protein L10, beta-tubulin (β-tub), elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA, and tigA gene fusion protein (tigA). As indicated by species distance, cox1 had the highest genus-wide resolution, followed by ITS, tigA, and β-tub. Resolution of these four markers also varied with (sub)clade. β-tub alone could readily identify all species in clade 1, cox1 for clade 2, and tigA for clades 7 and 8. Two or more genetic markers were required to identify species in other clades. For PCR consistency, ITS (99% PCR success rate) and β-tub (96%) were easier to amplify than cox1 (75%) and tigA (71%). Accordingly, it is recommended to take a two-step approach: classifying unknown Phytophthora isolates to clade by ITS sequences, as this marker is easy to amplify and its signature sequences are readily available, then identifying to species by one or more of the most informative markers for the respective (sub)clade.

Effect of formalin fixation on pcr amplification of DNA isolated from healthy autopsy tissues

The aim of this study is to investigate the effects of formalin fixation on the degradation of DNA molecules in five different healthy tissues exempted during the autopsy, as well as the selection of the method that is most suitable for the DNA isolation. Heart muscle, liver, brain, lung and kidney tissue obtained from the healthy people who suddenly died from a violent death were used. The parts of tissue were fixed in 10% phosphate-buffered formalin as well as in 4% unbuffered formalin at room temperature. Morphology of tissue was studied using H&E staining. The DNA was isolated 6 h, 1–7 days (every 24 h), 10, 14, 28 days and 2 months after fixation using two different methods: extraction with phenol-chloroform-isoamyl alcohol as well as with PureLink Genomic DNA Kit. Yield and purity of the DNA samples were measured spectrophotometrically at 260 nm and 280 nm. The PCR amplifications of the glycerol-3-phosphate dehydrogenase 1 (GPD1, 150 bp), ß actin (ACTB, 262 bp) and ribosomal protein L4 (RPL4, 407 bp) genes were performed to evaluate the degree of DNA fragmentation. The RPL4 gene was amplified up to 72 h, ACTB gene up to 14 days and GPD1 gene up to 28 days from tissue fixed in phosphate-buffered formalin using phenol-chloroform-isoamylalcohol protocol for DNA isolation. Liver and kidney gave better results of PCR amplification, but statistical significance between tissues was not found. Preserving period, fixative and DNA extracting method are important factors for successful PCR amplification. The healthy tissue, fixed in phosphate-formalin up to 28 days, can be useful source in molecular studies. Tissues fixed in unbuffered formalin are suitable for molecular analysis up to 7 days.

REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS

PCR clamping/wild-type blocking PCR with non-extendable locked nucleic acid (LNA) oligonucleotides is used for sensitive detection of somatic mutations in tumors. Various versions of the technique use different DNA polymerases and LNA oligonucleotides with and without additional phosphorothioate modifications. Here we studied requirements for successful PCR clamping with LNA oligonucleotides and Taq DNA polymerase for analysis of mutations in KRAS and BRAF genes by means of real-time PCR and Sanger sequencing. We found that addition of phosphorothioate linkages at the 5’-end of LNA oligonucleotide to protect from 5’- exonuclease activity of Taq DNA polymerase did not improve clamping. For most target sequences, efficient clamping was observed at melting temperature of LNA oligonucleotide 20‑25°C above annealing/extension temperature of the PCR with a 2-step protocol. Under such conditions, simple and sensitive detection of mutations in KRAS and BRAF genes was feasible using real-time PCR with TaqMan probes or Sanger sequencing.

Enabling large-scale feather mite studies: an Illumina DNA metabarcoding pipeline.

Feather mites are among the most common and diverse ectosymbionts of birds, yet basic questions such as the nature of their relationship remain largely unanswered. One reason for feather mites being understudied is that their morphological identification is often virtually impossible when using female or young individuals. Even for adult male specimens this task is tedious and requires advanced taxonomic expertise, thus hampering large-scale studies. In addition, molecular-based methods are challenging because the low DNA amounts usually obtained from these tiny mites do not reach the levels required for high-throughput sequencing. This work aims to overcome these issues by using a DNA metabarcoding approach to accurately identify and quantify the feather mite species present in a sample. DNA metabarcoding is a widely used molecular technique that takes advantage of high-throughput sequencing methodologies to assign the taxonomic identity to all the organisms present in a complex sample (i.e., a sample made up of multiple specimens that are hard or impossible to individualise). We present a high-throughput method for feather mite identification using a fragment of the COI gene as marker and Illumina Miseq technology. We tested this method by performing two experiments plus a field test over a total of 11,861 individual mites (5360 of which were also morphologically identified). In the first experiment, we tested the probability of detecting a single feather mite in a heterogeneous pool of non-conspecific individuals. In the second experiment, we made 2 × 2 combinations of species and studied the relationship between the proportion of individuals of a given species in a sample and the proportion of sequences retrieved to test whether DNA metabarcoding can reliably quantify the relative abundance of mites in a sample. Here we also tested the efficacy of degenerate primers (i.e., a mixture of similar primers that differ in one or several bases that are designed to increase the chance of annealing) and investigated the relationship between the number of mismatches and PCR success. Finally, we applied our DNA metabarcoding pipeline to a total of 6501 unidentified and unsorted feather mite individuals sampled from 380 European passerine birds belonging to 10 bird species (field test). Our results show that this proposed pipeline is suitable for correct identification and quantitative estimation of the relative abundance of feather mite species in complex samples, especially when dealing with a moderate number (> 30) of individuals per sample.

PCR Amplification Strategies Towards Full-length HIV-1 Genome Sequencing.

The advent of next-generation sequencing has enabled greater resolution of viral diversity and improved feasibility of full viral genome sequencing allowing routine HIV-1 full genome sequencing in both research and diagnostic settings. Regardless of the sequencing platform selected, successful PCR amplification of the HIV-1 genome is essential for sequencing template preparation. As such, full HIV-1 genome amplification is a crucial step in dictating the successful and reliable sequencing downstream. Here we reviewed existing PCR protocols leading to HIV-1 full genome sequencing. In addition to the discussion on basic considerations on relevant PCR design, the advantages as well as the pitfalls of the published protocols were reviewed.

USING DNA BARCODE TO IMPROVE THE IDENTIFICATION OF MARINE FISH LARVAE, CASE STUDY COASTAL WATER NEAR JAKARTA AND BANDA SEA, INDONESIA

The sustainability of the exploitation of the Indonesian fishes depends heavily on many of fish’s basic information including both larvae distribution and dispersal. However, the identification of fish larvae and juvenile to species is very limited. In this study, we employed DNA barcoding techniques to identify marine fish larvae to a species level in Jakarta Bay and Banda Sea by comparing the querries with sequences from adult stage as reference library to contribute on biodiversity information on that particular area. The result revealed that in Jakarta Bay, the molecular marker of a 471 bp region of the mitchondrial cyctochrome c oxidase I gene (COI) has been successfully found to be species-specific, genetic distance within species (0.0 - 1.30 percent). There are total of 8 families, 5 genera and 5 species from a total 15 successful PCR that could be used to calculate the accuracy of larval fish identification in three taxonomic categories. Meanwhile in Banda Sea, for the adult specimens, after some of PCR experiment, we have successfully amplified 27 individuals, only 8 sequences available. There are a total 326 eggs and larvae which been collected from 19 stations, of the 28 successfully amplified PCR samples, 11 sequences were available for DNA analysis and at least 10 species used Banda Sea and surrounding area as their spawning ground. We prove the ability of COI barcodes to identify species level resolution from query sequences and to classify species from distinct geographical origins and determine of how the data retrieved give important information for proposing plans for conserving and managing of fisheries in the sea waters.

Digestive gland inclusion bodies in queen conch (Lobatus gigas) are non-parasitic.

Unusual inclusion bodies occur within the epithelial cells of the digestive gland of queen conch, Lobatus gigas, and have previously been described as apicomplexan parasites. The aim of this study was to investigate the parasitic features of these inclusion bodies in queen conch. L. gigas from St. Kitts (Caribbean Sea) consistently (100% of n = 61) showed large numbers of ovoid to tri-bulbous dark brown inclusion bodies (15 × 30 µm) within vacuolar cells. Histochemical stains demonstrated iron, melanin, and glycoprotein and/or mucopolysaccharide within the inclusion bodies. Microscopic features indicative of a host response to injury were lacking in every case, as were consistent morphological forms to indicate distinct parasitic stages. Transmission electron microscopy failed to reveal cellular organelles of parasitic organisms and DNA extractions of purified inclusion bodies did not yield sufficient concentrations for successful PCR amplification. Scanning electron microscopy with energy dispersive X-ray analysis revealed a number of elements, particularly iron, within the inclusion bodies. We conclude that the inclusion bodies are not an infectious agent, and hypothesize that they represent a storage form for iron, and potentially other elements, within a protein matrix. Similar structures have been described in the digestive glands of other invertebrates, including prosobranchs.

Engineering, Cloning and Expression of DNA Sequence Coding of OMP31 Epitope of Brucella melitensis linked to IL-2 in Escherichia coli

: Brucellosis is a zoonetic disease that is caused by Brucella melitensis, which affects human health and animal husbandry industry. Omp31 is the most exposed outer membrane proteins (OMPs) and is a dominant antigen in smooth strains of B. melitensis, thus it is considered a desirable protein in subunit vaccine against brucellosis disease. Besides, IL-2 acts as a growth and differentiation stimulator of T-cells in cellular immune responses, therefore, it can serve as an adjuvant in subunit vaccines against intracellular pathogens. It seems that chimera scaffold, consisting of some copies of OMP31 experimental epitope region and IL-2, can provide a good immunization. In this regard, these tow fragments are linked together through using rigid linker and splicing overlap extension (SOE) PCR technique. Successful PCR products were TA cloned and then sub-cloned into pET-22b (+) vector as an expression vector. Recombinant protein was expressed in Escherichia coli BL21 (DE3) as an expressing lineage bacterium and then confirmed by SDS-PAGE and Western-blotting analysis. Secondary and tertiary structure prediction of recombinant protein and its immunity characterization were evaluated using in silico process. Further studies on the immunogenicity properties of this protein are expected to be carried out in the future to confirm the results in vivo.

Molecular Cloning and Structure–Function Analysis of a Trypsin Inhibitor from Tartary Buckwheat and Its Application in Combating Phytopathogenic Fungi

Host plant protease inhibitors offer resistance to proteases from invading pathogens. Trypsin inhibitors (TIs), in particular, serve as protective agents against insect and pathogen attacks. In this study, we designed a pair of degenerate primers based on highly conserved motifs at the N- and C-termini of the TI from tartary buckwheat (Fagopyrum tataricum; Ft) to clone the central portion. Genomic walking was performed to isolate the 5′ and 3′ flanking regions of FtTI. We demonstrated the successful PCR amplification of a 644 bp portion of FtTI. The full-length DNA of FtTI contains a complete open reading frame of 264 bp, encoding 87 amino acids with a mass of approximately 9.5 kDa. The FtTI protein sequence was 49% identical and 67% similar to potato protease inhibitors. Site-directed mutagenesis identified the residues, Asp67 and Arg68, as crucial for the inhibitory activity of the FtTI. Recombinant and mutant FtTI inhibited both the hyphal growth and spore germination of Alternaria solani. The calculated 50% inhibitory concentrations of FtTI ranged from 5–100 μg mL−1 for spore germination and 1–50 μg mL−1 for fungal growth. Thus, recombinant FtTI may function in host resistance against a variety of fungal plant pathogens.

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Two new efficient, fast and low-cost metagenomic DNA extraction methods were developed for different Persian oak tissues (leaf, stem, root, and rhizospheric) and soil samples. The new “omics” studies on the genus Quercus are of importance to help finding efficient strategies for overcoming environmental challenges, and to do this, presence of efficient DNA extraction protocols for different Quercus species are very critical. The objective of the present study was to develop new efficient methods for extraction of metagenomic DNA (mDNA) from of Persian oak (Quercus brantii Lindl.) tissues. The efficiency of two newly developed mDNA extraction methods, including indirect SDS-based (ISB or concentrate method) and one spin column-based method (SCB) were compared to that of two classical direct methods, including CTAB-based and SDS-based methods, and two commercial mDNA extraction kits. The maximum average yield of mDNA for all samples (leaf, stem, root, bulk, and rhizospheric soils) was obtained by SCB (258 ng/μl) and ISB (189 ng/μl) methods, respectively. Successful PCR amplification for 16SrRNA and ITS sequence was consistently observed for ISB, SCB, and kit-extracted mDNAs, which confirmed the high purity of mDNA extracted by these methods. The new methods showed more than 96% quantitative PCR efficiency, and partial restriction digestion and metagenomic library construction confirmed the high efficiency of the newly developed methods. It could be concluded that two new protocols enhanced efficiency (yield, purity, and cost) of mDNA extraction from different tissues of Persian oak.

Molecular identification of temperate Cricetidae and Muridae rodent species using fecal samples collected in a natural habitat

Molecular species identification from biological material collected at field sites has become an established ecological tool. However, extracting and amplifying DNA from degraded field samples, such as prey remains and feces that have been exposed to the elements, remains a challenge and costly. We collected 115 fecal samples of unknown small mammals, resembling fecal droppings of voles and mice (i.e., Cricetidae and Muridae), from a salt marsh in The Netherlands. We modified a previously published protocol into a relatively low-cost method with a PCR success of 95%. We demonstrate that species identification is possible for both Cricetidae and Muridae species using fecal samples of unknown age deposited in the field. For 90 samples, sequences of the variable control region in the mitochondrial genome were obtained and compared to published DNA sequences of small mammals occurring in north European salt marshes. A single sample, probably environmentally contaminated, appeared as Sus scrofa (n = 1). We positively identified house mouse Mus musculus, being the positive control (n = 1), and common vole Microtus arvalis (n = 88). In 81 sequences of 251 nt without ambiguous bases, ten haplotypes were present. These haplotypes, representing the central lineage of the western subspecies M. arvalis arvalis, were separated by 20 mutations from published control region haplotypes of the western European lineages sampled in France. Unlike earlier studies of cytochrome b variation in coastal European populations, we did not find indications of recent purging of genetic variation in our study area.

PCR Amplification of Base-Modified DNA.

An efficient PCR amplification of various templates (short 57-mer, random 67- and 82-mer, and long DNA) with base-modified nucleoside triphosphates is presented here. Using 5-substituted pyrimidine and 7-substituted-7-deaza- or 8-substituted purine nucleoside triphosphates as substrates for thermostable DNA polymerases [Taq and Vent (exo- )], successful PCR amplification of partially or entirely modified DNA libraries and long DNA constructs (up to 1.5kb) is achieved. Visualization of double-stranded PCR product formation is improved through the use of primers with different fluorescent labels. This allows one to monitor the efficiency of modified substrate incorporation and the enzymatic recognition of the modified template during PCR. The redesigned fully base-modified DNA (denoted 'DZA') can be utilized for the straightforward production of diverse libraries for in vitro selection of aptamer and catalytic nucleic acids as well as for the synthesis of artificial genetic templates, replicons, or complex vectors. © 2018 by John Wiley & Sons, Inc.

Simultaneous amplification of exons 18 to 21 of the EGFR gene using 5′ tailed primers and a two-stage protocol

ABSTRACTObjective: Reduction of non-specific amplification and achievement of efficient amplification of multiple gene fragments under the same reaction condition is the basic goal of PCR diagnosis; however, this is often difficult. This study was conducted to establish a highly specific and effective amplification of the epidermal growth factor receptor (EGFR) gene's exons, 18–21, simultaneously. Methods: The 5′-tailed primers were synthesized by adding 10 to 20 bp of a non-specific sequence to the 5′-terminus of sequence-specific primers (tailless primers). The two-stage protocol consisted of 5–10 cycles of a conventional 3-step cycling, which was then followed by 30–35 cycles of two-step cycling. The exons 18–21 of EGFR gene were amplified in 28 non-small cell lung cancer (NSCLC) patients using an optimized PCR that combined 5′ tailed primers with a two-stage protocol. Results: The 5′ tailed primers exhibited a wider range of suitable annealing temperatures, similar range of primer concentration, similar sensitivity, specificity, and reproducibility, as well as a reduced, non-specific amplification compared with the corresponding tailless primers. The amplification of exons 18–21 of EGFR gene in NSCLC patients revealed that a combination of 5′ tailed primers with two-stage protocol (optimized PCR) had a similar PCR success rate (P = 0.873) but had significantly reduced non-specific amplification (P <0.001) compared to conventional PCR. Conclusion: 5′ tailed primers exhibited a wider range of suitable annealing temperatures and improved specificity compared with conventional PCR primers. An optimized PCR was established with 5′ tailed primers and a two-stage protocol to amplify exons 18–21 of the EGFR gene in NSCLC patients.

A simple, efficient and universal method for the extraction of genomic DNA from bacteria, yeasts, molds and microalgae suitable for PCR-based applications

The extraction of genomic DNA from microbial cells plays a significant role in PCR-based applications such as molecular diagnosis, microbial taxonomy, screening of genetically engineered microorganisms, and other such PCR-based applications. Currently, many methods for extraction of genomic DNA from microorganisms have been developed. However, these methods either require hazardous chemicals or consist of time-consuming steps for effective execution. In this study, we have established a simple and universal genomic DNA extraction method for different microorganisms including bacteria, yeasts, molds, and microalgae. Our method does not require harmful reagents such as phenol and chloroform for the extraction process to minimize the generation of hazardous wastes. The obtained genomic DNA products displayed high concentrations and represented a good purity level with the average 260 nm/280 nm absorbance ratios (A260/280) that range from 1.6 to 2.0. The DNA molecules further remained considerably intact when analyzed on agarose gels. More importantly, these DNA products were qualified through successful PCR amplifications of 16S rRNA gene, rDNA internal transcribed spacer (ITS), or 18S rRNA gene from genomes of bacteria, fungi, and microalgae respectively. Furthermore, with the extracted genomic DNA products, the processes of the identification of the haploid and diploid states of the Saccharomyces yeast strains or detection of putative strains of Aspergillus oryzae and Aspergillus flavus that have been isolated from infected food materials through PCR analyses are facilitated. The genomic DNA extraction method established in this study is easy to manage, time saving and cost-effective, and environmentally friendly.

DNA Barcoding for Species Identification of Insect Skins: A Test on Chironomidae (Diptera) Pupal Exuviae

Chironomidae (Diptera) pupal exuviae samples are commonly used for biological monitoring of aquatic habitats. DNA barcoding has proved useful for species identification of chironomid life stages containing cellular tissue, but the barcoding success of chironomid pupal exuviae is unknown. We assessed whether standard DNA barcoding could be efficiently used for species identification of chironomid pupal exuviae when compared with morphological techniques and if there were differences in performance between temperate and tropical ecosystems, subfamilies, and tribes. PCR, sequence, and identification success differed significantly between geographic regions and taxonomic groups. For Norway, 27 out of 190 (14.2%) of pupal exuviae resulted in high-quality chironomid sequences that match species. For Costa Rica, 69 out of 190 (36.3%) Costa Rican pupal exuviae resulted in high-quality sequences, but none matched known species. Standard DNA barcoding of chironomid pupal exuviae had limited success in species identification of unknown specimens due to contaminations and lack of matching references in available barcode libraries, especially from Costa Rica. Therefore, we recommend future biodiversity studies that focus their efforts on understudied regions, to simultaneously use morphological and molecular identification techniques to identify all life stages of chironomids and populate the barcode reference library with identified sequences.