Successful PCR Amplification Research Articles

A mini-primer set in a multiplex PCR fashion covering the MTDNA control region from submerged skeletal remains

When DNA is recovered in a small quantity and low quality, typing methods based on mtDNA should be considered. Mini-primer sets multiplex with reduced amplicons has been successful in amplifying the entire CR. This study developed a mini-primer set PCR multiplex assay to amplify the entire CR of mtDNA from fragments of human skeletal remains submerged in water up to 30, 60 and 90days. So far, the new mini-primer set for PCR multiplex assay was tested on four samples, three of them submerged in river water for a period of 30 months. This approach gave successful PCR amplifications for the three samples submerged and the control. The following sequencing analysis brought partial mtDNA control region profiles to the samples and a complete to the control.

Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples.

Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next‐generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high‐quality DNA extraction procedures for obtaining the minute quantities of short‐fragmented food DNA. Automatic high‐throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole‐body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high‐throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor‐intensive, low‐throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost‐efficient and innovative methodology at low contamination risk also in trophic ecology.

Mini-DNA barcode in identification of the ornamental fish: A case study from Northeast India

The ornamental fishes were exported under the trade names or generic names, thus creating problems in species identification. In this regard, DNA barcoding could effectively elucidate the actual species status. However, the problem arises if the specimen is having taxonomic disputes, falsified by trade/generic names, etc., On the other hand, barcoding the archival museum specimens would be of greater benefit to address such issues as it would create firm, error-free reference database for rapid identification of any species. This can be achieved only by generating short sequences as DNA from chemically preserved are mostly degraded. Here we aimed to identify a short stretch of informative sites within the full-length barcode segment, capable of delineating diverse group of ornamental fish species, commonly traded from NE India. We analyzed 287 full-length barcode sequences from the major fish orders and compared the interspecific K2P distance with nucleotide substitutions patterns and found a strong correlation of interspecies distance with transversions (0.95, p<0.001). We, therefore, proposed a short stretch of 171bp (transversion rich) segment as mini-barcode. The proposed segment was compared with the full-length barcodes and found to delineate the species effectively. Successful PCR amplification and sequencing of the 171bp segment using designed primers for different orders validated it as mini-barcodes for ornamental fishes. Thus, our findings would be helpful in strengthening the global database with the sequence of archived fish species as well as an effective identification tool of the traded ornamental fish species, as a less time consuming, cost effective field-based application.

OPTIMIZED DNA ISOLATION PROTOCOL ENABLES AN INSIGHT INTO MOLECULAR GENETIC BACKGROUND OF ENDEMIC Moltkia petraea (Tratt.) Griseb. FROM BOSNIA AND HERZEGOVINA

The Dinaric endemic plant species Moltkia petraea (Tratt.) Griseb. is often called a "living fossil" of ancient Tertiary flora, with great importance for Bosnia and Herzegovina’s biodiversity. Considering its narrow and limited distribution range, insufficient data on the molecular background of this species is given so far. Due to the presence of various secondary metabolites that interfere with the DNA, isolation of nucleic acids from plant cells is known to be challenging. Even in closely related species it is necessary to optimize DNA isolation protocol in order to obtain high quality PCR amplifiable DNA. We collected 91 samples from five populations in Herzegovina. Doyle and Doyle (1987) CTAB protocol was modified by adding vitamin C (ascorbic acid) to the cell lysis buffer to improve DNA yield and quality. trnL(UAA) intron and nrDNA (ITS1, ITS2) molecular markers were applied to demonstrate amplifiability of isolated DNA and elucidate the intra- and interpopulation genetic diversity. Our results suggest a successful PCR amplification for 81% of the analyzed samples. PCR-RFLP analysis of trnL(UAA) revealed that all individuals in five populations have the same haplotype based on the obtained enzymatic profile for three enzymes (TaqI, HinfI, HindII). Alignment and comparison of ITS sequences didn’t reveal any hypervariable portion that could be informative in elucidating the genetic diversity of M. petraea populations. Further studies with additional application of microsatellite loci, RAPD and AFLP methods are necessary in an attempt to get insights into the genetic diversity of M. petraea.

Design of character-based DNA barcode motif for species identification: A computational approach and its validation in fishes.

The DNA barcodes are generally interpreted using distance-based and character-based methods. The former uses clustering of comparable groups, based on the relative genetic distance, while the latter is based on the presence or absence of discrete nucleotide substitutions. The distance-based approach has a limitation in defining a universal species boundary across the taxa as the rate of mtDNA evolution is not constant throughout the taxa. However, character-based approach more accurately defines this using a unique set of nucleotide characters. The character-based analysis of full-length barcode has some inherent limitations, like sequencing of the full-length barcode, use of a sparse-data matrix and lack of a uniform diagnostic position for each group. A short continuous stretch of a fragment can be used to resolve the limitations. Here, we observe that a 154-bp fragment, from the transversion-rich domain of 1367 COI barcode sequences can successfully delimit species in the three most diverse orders of freshwater fishes. This fragment is used to design species-specific barcode motifs for 109 species by the character-based method, which successfully identifies the correct species using a pattern-matching program. The motifs also correctly identify geographically isolated population of the Cypriniformes species. Further, this region is validated as a species-specific mini-barcode for freshwater fishes by successful PCR amplification and sequencing of the motif (154bp) using the designed primers. We anticipate that use of such motifs will enhance the diagnostic power of DNA barcode, and the mini-barcode approach will greatly benefit the field-based system of rapid species identification.

DNA Barcoding and Morphological Identification of Benthic Nematodes Assemblages of Estuarine Intertidal Sediments: Advances in Molecular Tools for Biodiversity Assessment

Concerns regarding the status of marine ecosystems have increased in part due to traditional and emerging human activities in marine waters, driving a demand for approaches with high sample throughput capability to improve ecosystem monitoring. Nematodes are already used as indicator species in biodiversity assessments and biomonitoring of terrestrial and marine systems, with molecular approaches offering the opportunity to utilize these organisms further in large scale ecological surveys and environmental assessments. Based on an available nematode dataset for estuarine sediments of the Mira estuary (SW coast, Portugal), we evaluated the diversity of the nematode community of this system, using the molecular markers 18S rRNA and COI genes. These approaches were compared to voucher specimens from a morphological characterization of the same samples allowing validation and comparison between nematode communities. The spatial and temporal variability of the density and diversity of the nematode assemblages was analyzed based on morphological characterization to allow the validation and efficiency of the genetic characterization. A PCO ordination plot showed a distinct separation of the assemblages between sampling occasions confirmed by PERMANOVA analysis, which showed significant differences, although no significant differences were detected between sampling sites. The morphological characterization identified 50 genera of which only 26 and 25 distinct 18S rRNA and COI DNA barcodes, respectively, were obtained. 90.2% of the morphologically identified specimens representing eleven different genera, successfully generated DNA barcodes for both 18S rRNA and COI genes. This study confirmed that the success of the 18S rRNA gene PCR amplification is higher than of COI gene with 43 species amplified against 34. The study highlights a limitation of available sequences for both targets in databases when compared to the known diversity of marine nematodes. The gene sequences of this study enriched the databases, contributing gene sequences from 7 and 16 new genera for the 18S rRNA and COI genes, respectively. A robust database of gene sequences is a prerequisite for the development of robust high sample throughput techniques to be applied in marine assessing and monitoring programs.

DNA Barcoding for Efficient Species- and Pathovar-Level Identification of the Quarantine Plant Pathogen Xanthomonas.

Genus Xanthomonas comprises many economically important plant pathogens that affect a wide range of hosts. Indeed, fourteen Xanthomonas species/pathovars have been regarded as official quarantine bacteria for imports in China. To date, however, a rapid and accurate method capable of identifying all of the quarantine species/pathovars has yet to be developed. In this study, we therefore evaluated the capacity of DNA barcoding as a digital identification method for discriminating quarantine species/pathovars of Xanthomonas. For these analyses, 327 isolates, representing 45 Xanthomonas species/pathovars, as well as five additional species/pathovars from GenBank (50 species/pathovars total), were utilized to test the efficacy of four DNA barcode candidate genes (16S rRNA gene, cpn60, gyrB, and avrBs2). Of these candidate genes, cpn60 displayed the highest rate of PCR amplification and sequencing success. The tree-building (Neighbor-joining), ‘best close match’, and barcode gap methods were subsequently employed to assess the species- and pathovar-level resolution of each gene. Notably, all isolates of each quarantine species/pathovars formed a monophyletic group in the neighbor-joining tree constructed using the cpn60 sequences. Moreover, cpn60 also demonstrated the most satisfactory results in both barcoding gap analysis and the ‘best close match’ test. Thus, compared with the other markers tested, cpn60 proved to be a powerful DNA barcode, providing a reliable and effective means for the species- and pathovar-level identification of the quarantine plant pathogen Xanthomonas.

Sex determination of baleen whale artefacts: Implications for ancient DNA use in zooarchaeology

Methods to determine the sex from tissue samples of mammals include the amplification of Y chromosome specific regions, which should only amplify from males, or amplification of homologous regions of the X and Y chromosome containing XY specific SNPs. A disadvantage of the first approach is that PCR failure can be misinterpreted as the identification of a female. The latter approach is proposed to identify PCR failure through non-amplification of the X homologue, which should be present in both sexes. This method is therefore potentially more suitable for molecular sexing of degraded DNA with a high probability of PCR failure, such as for example, ancient DNA samples. Here, we investigate the validity of this assumption regarding the use of XY homologue PCR assays for molecular sexing of ancient DNA. We tested a primer set targeting the ZFX/ZFY alleles using ancient DNA extracts from 100 to 4500years old bowhead whale samples, and for comparison on dilution series from modern bowhead whales of known sex. DNA sequencing of PCR products obtained from the ancient material confirmed a higher proportion of successful PCR amplifications of the X homologue over the Y homologue. This potentially biased sex determination was further assessed by testing highly diluted DNA extracts of modern samples, for which a consistently higher success rate of PCR amplification and lower PCR cycle threshold was found for the X homologue from females than either homologue from males. This is most likely due to the higher copy number of the X homologue in females, although other yet unknown attributes of the protocol may also cause the observed bias. The current case study provides a valuable example of a potential pitfall in molecular sex determination of ancient mammal DNA in zooarchaeology. High-throughput sequencing methods, in which sufficiently large numbers of reads can be unambiguously mapped to X and Y regions, should overcome such biases and be the most robust approach for molecular sex determination using degraded DNA.

Assessment of mangroves from Goa, west coast India using DNA barcode.

Mangroves are salt-tolerant forest ecosystems of tropical and subtropical intertidal regions. They are among most productive, diverse, biologically important ecosystem and inclined toward threatened system. Identification of mangrove species is of critical importance in conserving and utilizing biodiversity, which apparently hindered by a lack of taxonomic expertise. In recent years, DNA barcoding using plastid markers rbcL and matK has been suggested as an effective method to enrich traditional taxonomic expertise for rapid species identification and biodiversity inventories. In the present study, we performed assessment of available 14 mangrove species of Goa, west coast India based on core DNA barcode markers, rbcL and matK. PCR amplification success rate, intra- and inter-specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in rbcL (97.7 %) and matK (95.5 %) region. The two candidate chloroplast barcoding regions (rbcL, matK) yielded barcode gaps. Our results clearly demonstrated that matK locus assigned highest correct identification rates (72.09 %) based on TaxonDNA Best Match criteria. The concatenated rbcL + matK loci were able to adequately discriminate all mangrove genera and species to some extent except those in Rhizophora, Sonneratia and Avicennia. Our study provides the first endorsement of the species resolution among mangroves using plastid genes with few exceptions. Our future work will be focused on evaluation of other barcode markers to delineate complete resolution of mangrove species and identification of putative hybrids.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3191-4) contains supplementary material, which is available to authorized users.

Applying DNA Barcodes to Identify Closely Related Species of Ferns: A Case Study of the Chinese Adiantum (Pteridaceae)

DNA barcoding is a fast-developing technique to identify species by using short and standard DNA sequences. Universal selection of DNA barcodes in ferns remains unresolved. In this study, five plastid regions (rbcL, matK, trnH-psbA, trnL-F and rps4-trnS) and eight nuclear regions (ITS, pgiC, gapC, LEAFY, ITS2, IBR3_2, DET1, and SQD1_1) were screened and evaluated in the fern genus Adiantum from China and neighboring areas. Due to low primer universality (matK) and/or the existence of multiple copies (ITS), the commonly used barcodes matK and ITS were not appropriate for Adiantum. The PCR amplification rate was extremely low in all nuclear genes except for IBR3_2. rbcL had the highest PCR amplification rate (94.33%) and sequencing success rate (90.78%), while trnH-psbA had the highest species identification rate (75%). With the consideration of discriminatory power, cost-efficiency and effort, the two-barcode combination of rbcL+ trnH-psbA seems to be the best choice for barcoding Adiantum, and perhaps basal polypod ferns in general. The nuclear IBR3_2 showed 100% PCR amplification success rate in Adiantum, however, it seemed that only diploid species could acquire clean sequences without cloning. With cloning, IBR3_2 can successfully distinguish cryptic species and hybrid species from their related species. Because hybridization and allopolyploidy are common in ferns, we argue for including a selected group of nuclear loci as barcodes, especially via the next-generation sequencing, as it is much more efficient to obtain single-copy nuclear loci without the cloning procedure.

English

Genomic DNA isolation is fundamental step to study genetic diversity, phylogeny of species and cloning of desired genes. The preferable protocol needs to be efficient, less time consuming, economic and non-destructive particularly for the endangered species. The current study was aimed at economic isolation of considerable quantity of DNA manually from a particular tissue of the fish without any potential damage to the animal. For this purpose fin tissue was preferably selected over scale due to its universal occurrence in fish. A total of 180 samples of fin tissues belonging to twenty nine species of fresh water fish preserved in different preservatives, were used for DNA isolation. DNA could not be isolated from the fin tissues preserved in formalin, alcohol, air dried and ice preserved fins more than one week by any of two previously described methods including salt extraction and urea treatment. However, introducing some modifications in salt extraction method resulted in large quantity of high quality DNA from those fin tissues that were isolated from the living animal and immediately preserved in absolute ethanol. The modifications include use of low quantity of proteinase K and slight increase in incubation period due to hard nature of fin tissue. The extracted DNA by the modified salt extraction method was very pure, of high quality and resulted in successful PCR amplification by Random Amplified Polymorphic DNA primer and cytochrome oxidase subunit 1 gene specific primers. This modified procedure results in highest yield of isolated DNA reported so far. The method is particularly suited for DNA isolation from endangered and sparse species as it requires minute quantity of fin tissue, without any detrimental effect on fish.

Creation of reference DNA barcode library and authentication of medicinal plant raw drugs used in Ayurvedic medicine.

BackgroundAyurveda is a system of traditional medicine that originated in ancient India, and it is still in practice. Medicinal plants are the backbone of Ayurveda, which heavily relies on the plant-derived therapeutics. While Ayurveda is becoming more popular in several countries throughout the World, lack of authenticated medicinal plant raw drugs is a growing concern. Our aim was to DNA barcode the medicinal plants that are listed in the Ayurvedic Pharmacopoeia of India (API) to create a reference DNA barcode library, and to use the same to authenticate the raw drugs that are sold in markets.MethodsWe have DNA barcoded 347 medicinal plants using rbcL marker, and curated rbcL DNA barcodes for 27 medicinal plants from public databases. These sequences were used to create Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL). This library was used to authenticate 100 medicinal plant raw drugs, which were in the form of powders (82) and seeds (18).ResultsAyurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL) was created with high quality and authentic rbcL barcodes for 374 out of the 395 medicinal plants that are included in the API. The rbcL DNA barcode differentiated 319 species (85 %) with the pairwise divergence ranging between 0.2 and 29.9 %. PCR amplification and DNA sequencing success rate of rbcL marker was 100 % even for the poorly preserved medicinal plant raw drugs that were collected from local markets. DNA barcoding revealed that only 79 % raw drugs were authentic, and the remaining 21 % samples were adulterated. Further, adulteration was found to be much higher with powders (ca. 25 %) when compared to seeds (ca. 5 %).ConclusionsThe present study demonstrated the utility of DNA barcoding in authenticating medicinal plant raw drugs, and found that approximately one fifth of the market samples were adulterated. Powdered raw drugs, which are very difficult to be identified by taxonomists as well as common people, seem to be the easy target for adulteration. Developing a quality control protocol for medicinal plant raw drugs by incorporating DNA barcoding as a component is essential to ensure safety to the consumers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1086-0) contains supplementary material, which is available to authorized users.

Is DNA barcoding child's play? Science education and the utility of DNA barcoding for the discrimination of UK tree species

We present the findings of a DNA barcoding study of the UK tree flora, implemented as part of an innovative, research-based science education programme called ‘Tree School’. The UK tree flora comprises native and introduced species, and is a taxonomically diverse study group for the exploration of the potential and limitations of DNA barcoding. The children participating in the project collected voucher specimens and generated DNA barcode sequences from trees and shrubs found in the grounds and surrounding woodlands of a residential field centre in Dorset, UK. We assessed the potential of rbcL and matK markers for amplification and DNA sequencing success and for species discrimination among the 67 tree and shrub species included in this study. Although we achieved 100% PCR amplification and sequencing success for rbcL and matK, mononucleotide repeats affected sequence quality in matK for some taxonomic groups (e.g. Rosaceae). Species discrimination success ranged from 65% to 71% using tree-based methods to 86% using BLASTN. The occurrence of known hybrids (diploid and polyploid) and their progenitors on the study site reduced the overall species discrimination success for both loci. This study demonstrates that, even in a floristic context, rbcL and matK alone are insufficient for the discrimination of UK tree species, especially where taxonomically complex groups are present. From a science education perspective, DNA barcoding represents a compelling and accessible platform for the engagement of non-experts in ongoing research, providing an opportunity for them to contribute authentic scientific data to an international research campaign.

Characterization of Novel Microsatellite Loci for Primula poissonii (Primulaceae) Using High-Throughput Sequencing Technology

Primula poissonii (Primulaceae) is a perennial herb, widely distributed in the Hengduan Mountain region of Southwest China. In this study, Roche 454 pyrosequencing was used to isolate microsatellite markers. A total of 4528 unique sequences were identified from 68,070 unique reads. Of these, eighty-seven microsatellite loci were screened for utility using two criteria: successful PCR amplification and variation of these loci within three wild P. poissonii populations. Twenty loci were successfully amplified and exhibited polymorphic alleles. The number of observed alleles ranged from 1 to 9 with an average of 3.5. The observed and expected heterozygosities ranged from 0.087 to 1.000 and from 0.124 to 0.828, respectively. Among these SSR loci, only the P69 locus could not be cross-amplified successfully in two closely related species P. wilsonii and P. anisodora. The microsatellite loci developed in this study will be useful for studying genetic diversity and speciation events between P. poissonii and closely related Primula species.

Comparison of methods to preserve Rheum palmatum (Polygonaceae) for efficient DNA extraction and PCR amplification.

In this study, we compared the quality of DNA extracted using the modified CTAB method, from Rheum palmatum leaves preserved using fourteen different methods, including ones used commonly in other species: under ultra-cold (-80°C) temperatures, after drying with an absorbent paper, desiccating using a silica gel, drying at 60°C, in 70% ethanol, absolute ethanol, 70% ethanol supplemented with 50 mM EDTA, SDS-DNA extracting solution, nuclear separation buffer, improved NaCl-CTAB solution, TE-buffer, I-solution, or II-solution. DNA extracted from fresh leaves was used as the control. The quality of extracted DNA was evaluated based on the success of PCR amplification of the ITS2 region and a microsatellite marker. DNA was not extracted from samples preserved in the nuclear separation buffer and II-solution. The purities of DNA extracted from leaves preserved in ultra-cold temperatures, 70% ethanol, and 70% ethanol with 50 mM EDTA, and after desiccating using a silica gel and drying were higher, and comparable to the purity of DNA extracted from fresh leaves, than those of leaves preserved using other methods. In the present study, combined with the PCR amplifications, the preservation using ultra-cold temperatures, silica gel desiccation, or drying, and PCR amplification of the extracted DNA can be used for further molecular studies in R. palmatum.

Applications of Multiple Nuclear Genes to the Molecular Phylogeny, Population Genetics and Hybrid Identification in the Mangrove Genus Rhizophora.

The genus Rhizophora is one of the most important components of mangrove forests. It is an ideal system for studying biogeography, molecular evolution, population genetics, hybridization and conservation genetics of mangroves. However, there are no sufficient molecular markers to address these topics. Here, we developed 77 pairs of nuclear gene primers, which showed successful PCR amplifications across all five Rhizophora species and sequencing in R. apiculata. Here, we present three tentative applications using a subset of the developed nuclear genes to (I) reconstruct the phylogeny, (II) examine the genetic structure and (III) identify natural hybridization in Rhizophora. Phylogenetic analyses support the hypothesis that Rhizophora had disappeared in the Atlantic-East Pacific (AEP) region and was re-colonized from the IWP region approximately 12.7 Mya. Population genetics analyses in four natural populations of R. apiculata in Hainan, China, revealed extremely low genetic diversity, strong population differentiation and extensive admixture, suggesting that the Pleistocene glaciations, particularly the last glacial maximum, greatly influenced the population dynamics of R. apiculata in Hainan. We also verified the hybrid status of a morphologically intermediate individual between R. apiculata and R. stylosa in Hainan. Based on the sequences of five nuclear genes and one chloroplast intergenic spacer, this individual is likely to be an F1 hybrid, with R. stylosa as its maternal parent. The nuclear gene markers developed in this study should be of great value for characterizing the hybridization and introgression patterns in other cases of this genus and testing the role of natural selection using population genomics approaches.

Noninvasive and nondestructive sampling for avian microsatellite genotyping: a case study on the vulnerable Chinese Egret (Egretta eulophotes)

Noninvasive and nondestructive DNA sampling techniques are becoming more important in genetic studies because they can provide genetic material from wild animals with less or even without disturbance, which is particularly useful for the study of endangered species, i.e., birds. However, nondestructively and noninvasively sampled DNA may, in some cases, be inadequate in the amount and quality of the material collected, which can lead to low amplification success rates and high genotyping errors. In this study, noninvasive (eggshell swab, shed feather and feces), nondestructive (plucked feather and buccal swab) and invasive (blood) DNA samples were collected from the vulnerable Chinese Egret (Egretta eulophotes). DNA concentrations, PCR amplification success and microsatellite genotyping errors of different sample types were evaluated and compared to determine whether noninvasive and nondestructive samples performed as well as invasive samples in our experimental procedures. A total of 159 samples were collected in the field. Among the different sample types, the highest DNA concentrations (154.0–385.5 ng/μL) were obtained from blood. Those extracted from fecal samples were the lowest, ranging from 1.25 to 27.5 ng/μL. Almost all of the DNA samples, i.e., 95.59 %, were successfully amplified for mtDNA (n = 152) and 92.76 % of mtDNA samples were successfully genotyped for at least five of the nine microsatellite loci tested (n = 141). Blood samples and buccal swabs produced reliable genotypes with no genotyping errors, but in feces, allelic dropouts and false alleles occurred in all nine loci, with error rates ranging from 6.67 to 38.10 % for the dropouts and from 6.06 to 15.15 % for the false alleles. These results indicate that both nondestructive and noninvasive samplings are suitable for avian microsatellite genotyping, save for fecal DNA. However, we should remain cautious of the appearance of genotyping errors, especially when using noninvasive material.

A simple method to overcome the inhibitory effect of heparin on DNA amplification.

Genetic material from large patient cohorts is increasingly central to translational genetic research. However, patient blood samples are a finite resource and their supply and storage are often dictated by clinical and not research protocols. Our experience supports difficulty in amplifying DNA from blood stored in herparin; a scenario that other researchers may have or will encounter. This technical note describes a number of simple steps that enable successful PCR amplification. DNA was extracted using the Illustra Nucleon Genomic DNA Extraction Kit. PCR amplification was attempted using a number of commercially available PCR mastermixes. PCR DNA amplification failed using ReddyMix™ PCR Master Mix, Thermo-Start® (Thermo Scientific Inc. US) and ZymoTaq™ (Zymo research, US) PCR mastermixes, as demonstrated absence of products on gel electrophoresis. However, using the Invitrogen™ (Thermo Scientific Inc., US) Platinum® Taq DNA Polymerase, PCR products were identified on a 1% agarose gel for all samples. PCR products were cleaned with ExoSAP-IT® (Affymetrix Inc., US) and a sequencing reaction undertaken using a standard Big Dye protocol. Subsequent genotyping was successful for all samples for alleles at the CDH1 locus. From our experience a standard phenol/chloroform purification and using the Invitrogen™ Platinum® Taq has enabled the amplification of whole blood samples taken into lithium heparin and stored frozen for up to a month. This simple method may enable investigators to utilise blood taken in lithium heparin for DNA extraction and amplification.

To beat or not to beat a tick: comparison of DNA extraction methods for ticks (Ixodes scapularis).

Background. Blacklegged ticks (Ixodes scapularis) are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield.Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis—nymph and adult—that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods, we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material.Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification Kit resulted in the highest DNA yields and the most consistent PCR amplification when combined with either cutting or bead beating with select matrices across life stages. DNA isolation methods using ammonium hydroxide as well as the MoBio PowerSoil kit also produced strong and successful PCR amplification, but only for females.Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.

Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.