PCR Amplification Success Rate Research Articles

Identification and diversity assessment of Ziziphus species using DNA barcoding

The genus Ziziphus, belonging to the family Rhamnaceae, is of great economic and medicinal importance worldwide due to the value of its fruits in household income and food security. However, many species within this genus are phenotypically identical, leading to imprecise identification and hindering their conservation, variation in the gene pool, and exploitation. To address this issue, we collected 26 individuals from different regions of Khyber Pakhtunkhwa, Pakistan and Shanxi province, China, to assess the efficiency of DNA barcoding regions in identifying Ziziphus species. We tested the ability of five DNA barcodes regions, namely ITS, matK, rbcL, trnH-psbA, and trnL-UAA, to distinguish between Ziziphus species as an alternative approach for species identification and the phylogenetic reconstruction. The results of the study, as the success rate of PCR amplification of matK, trnL-UAA, and ITS, provided the highest species resolution with 100 % accuracy, except for trnH-psbA (90.78 %). ITS and matK were identified as the most effective single barcode, with a species identification success rate of 87.4 %±0.98 % and 84.89 %±0.66 %. These results suggest that matK, trnL-UAA, and ITS are universal barcode regions for identification of all Ziziphus species identification and their discrimination power to differentiate. In contrast, the combination of trnL-UAA+ITS region displayed the highest species identification rate (92.1 %), the five barcodes region combination, and superior to any barcodes regions combination that included rbcL+trnH-psbA. Additionally, the study found high haplotypes and nucleotide diversity in Pakistani Ziziphus species, mainly in the ITS barcode, with values of 0.84 and 0.0086, respectively. In conclusion, the study establishes that the DNA fragments ITS, trnL-UAA, and matK are effective in identifying and distinguishing Ziziphus species. The approach can be considered as an accurate tool for identifying these species and will be used as a universal barcode's region for Ziziphus species. DNA barcoding is a useful approach and provides a reliable and effective means for Ziziphus discrimination, and in combination with morphology-based taxonomy will be a robust approach for tackling taxonomically complex species. Additionally, this study provides valuable insights into the genomic architecture of Ziziphus species, which has implications for their classification, conservation, and germplasm resource improvement. This research provides a novel approach to the problem of incorrectly identifying economically and medicinally important species, particularly those in the genus Ziziphus.

Comprehensive analysis using DNA metabarcoding, SCAR marker based PCR assay, and HPLC unveils the adulteration in Brahmi herbal products.

Brahmi is one of the important nootropic botanicals, widely sold in the market, with the name "Brahmi'' being used to describe both Bacopa monnieri and Centella asiatica species. The Brahmi herbal products market is expanding; hence, economically motivated adulteration is highly prevalent. This study aimed to develop DNA-based methods, including SCAR marker-based PCR and metabarcoding, to authenticate Brahmi herbal products and compare these methods with HPLC. These methods have been validated using mock controls (in-house blended formulations). All targeted plant species in mock controls were detected successfully with all three methods, whereas, in market samples, only 22.2%, 55.6%, and 50.0% were found positive for Brahmi by PCR assay, DNA metabarcoding, and HPLC, respectively. Metabarcoding can detect the presence of non-labeled plants together with targeted species, which is an advantage over PCR assay or HPLC. SCAR marker-based PCR is a rapid and cost-effective method for detecting the presence of B. monnieri and C. asiatica. However, in this study, the success rate of PCR amplification was relatively low because the primers targeted either RAPD or ITS-based SCAR markers. HPLC assay, although an alternative, was unable to detect the presence of other botanicals, just like the SCAR marker-based PCR assay. On the other hand, metabarcoding can be utilized to identify the target plants, even in very small quantities, while also providing simulated identification of other botanicals. This study successfully addressed the need for quality control of Brahmi herbal products and provided the first-time report of DNA metabarcoding for such products.

Species identification of Ligustrum lucidum

Ligustrum lucidum is a woody perennial plant of genus Ligustrum in family Oleaceae. Its dried fruit has high medicinal value. In this study, the authors evaluated the variability and species identification efficiency of three specific DAN barcodes(rbcL-accD, ycf1a, ycf1b) and four general DAN barcodes(matK, rbcL, trnH-psbA, ITS2) for a rapid and accurate molecular identification of Ligustrum species. The results revealed that matK, rbcL, trnH-psbA, ITS2 and ycf1a were inefficient for identifying the Ligustrum species, and a large number of insertions and deletions were observed in rbcL-accD sequence, which was thus unsuitable for development as specific barcode. The ycf1b-2 barcode had DNA barcoding gap and high success rate of PCR amplification and DNA sequencing, which was the most suitable DNA barcode for L. lucidum identification and achieved an accurate result. In addition, to optimize the DNA extraction experiment, the authors extracted and analyzed the DNA of the exocarp, mesocarp, endocarp and seed of L. lucidum fruit. It was found that seed was the most effective part for DNA extraction, where DNAs of high concentration and quality were obtained, meeting the needs of species identification. In this study, the experimental method for DNA extraction of L. lucidum was optimized, and the seed was determined as the optimal part for DNA extraction and ycf1b-2 was the specific DNA barcode for L. lucidum identification. This study laid a foundation for the market regulation of L. lucidum.

Molecular identification and phylogenetic relationships of Colletotrichum isolates pathogenic on cultivated plants in Bulgaria

Fungi of the genus Colletotrichum are causal agents of plant diseases with constantly growing economic importance. Accurate pathogen identification is a significant prerequisite for effective disease control. The aim of the present investigation was to clarify the species affiliation of Colletotrichum isolates obtained from different hosts in Bulgaria and to determine the phylogenetic relationships between them by applying DNA barcoding. Thirty-five fungal isolates obtained from five botanical families (Solanaceae, Rosaceae, Musaceae, Cucurbitaceae and Caryophyllaceae) were morphologically characterized and subjected to molecular analysis based on four fungal barcode markers – the primary ITS barcode and the secondary marker regions ACT, EF-1a and TUB2 (Fig. 1). Three of the barcodes (ITS, ACT and TUB2) showed complete success rate of PCR amplification and sequencing and proved efficient for reliable identification at species level. BLAST analyses identified eleven Colletotrichum species assigned to five different complexes – C. coccodes, C. acutatum, C. gloeosporioides, C. dematium and C. spaethianum. The resolution power of ITS region was not sufficient to discriminate interspecies variations within C. coccodes, C. dematium and C. spaethianum complexes confirming the requirement for secondary barcodes in order to resolve the genetic variability of the Colletotrichum isolates. DNA barcoding analyses revealed that the highest species variation was observed among the isolates from pepper (Capsicum annuum). Interestingly, an isolate from the same host identified as C. truncatum on the basis of morphological characters appeared to be C. circinans when applying DNA barcode markers. According to our knowledge, this species has not been reported as a causal agent of pepper anthracnose. Data obtained in this study improve our understanding of the genetic diversity within the Colletotrichum population pathogenic on cultivated plants in Bulgaria.

Technical note: Polyvinylpyrrolidone (PVP) and proteinase-K improve the efficiency of DNA extraction from Japanese larch wood and PCR success rate

Illegal distribution of timber disrupts the timber market and depletes forest resources. DNA markers are used to verify the legal distribution of wood. However, it is difficult to obtain the quantity and quality of DNA suitable for genetic analysis because of the physicochemical properties of wood; therefore, an efficient wood DNA extraction method is required. In this study, to prepare an efficient DNA extraction method from Japanese larch (Larix kaempferi) wood, we investigated the ability of polyvinylpyrrolidone (PVP) and proteinase-K to improve DNA extraction efficiency and PCR success rate. It was found that the addition of PVP resulted in a significant increase in the DNA concentration of the treatment group compared to that of the control group, while the purity (A260/A280) showed no difference. Moreover, the addition of proteinase-K significantly increased both the DNA concentration and purity of the treatment group compared to those of the control group. Further analysis showed that the PCR success rate of psbC (approximately 350 bp) was higher than 90% in the control, PVP treatment, and proteinase-K treatment groups. However, in the PCR success rate of rbcL (approximately 1.3 kb) was higher in the proteinase-K and PVP treatment groups than in the control group. The addition of PVP and proteinase-K increased the success rate of PCR amplification for long regions by preventing DNA damage caused by phenolic compounds and proteins in the wood. The results of this study can thus develop DNA extraction methods to identify the species and origin of woods.

Isolation and characterization of DNA barcodes from distinctive and rare terrestrial animals in China using universal COI and 16S primers

Background: Accurate taxonomic identification is the cornerstone for monitoring, conservation and management of ecological resources. China has the highest biodiversities and the richest species assemblages in the world, but is lacking in sufficient assessment to the abundant genetic variability. DNA barcoding is a proven tool employing sequence information for rapid and unambiguous species delineation. However, the ability of barcodes to distinguish species that are archaic and distinctive evolutionary lines remains largely untested.Methods: In order to investigate the resources of terrestrial animals in China, regions from mitochondrial COI and 16S are barcoded for 395 specimens belonging to 54 selected species, many of which are indigenous representatives in danger. High success rate of PCR amplification is achieved by using universal COI and 16S primers with many numts pseudogenes co-amplified from mammalian samples.Results: Application of barcodes to flag species is generally straightforward since no COI or 16S haplotypes are shared between closely related species. Barcoding gap, species resolution and phylogenetic relationships relying on our barcode libraries are further compared using distance and tree based approaches.Conclusion: Results show that the discriminatory power of the two barcode markers could differentiate on a case-by-case basis, and also suggest a careful consideration of the nuclear numts for barcoding studies as they might provide a new understanding for evolution.

DNA Barcoding Mushroom Spawn Using EF-1α Barcodes: A Case Study in Oyster Mushrooms (Pleurotus).

Oyster mushrooms (genus Pleurotus) are widespread and comprise the most commonly cultivated edible mushrooms in the world. Species identification of oyster mushroom spawn based on cultural, morphological, and cultivated characteristics is time consuming and can be extraordinarily difficult, which has impeded mushroom breeding and caused economic loss for mushroom growers. To explore a precise and concise approach for species identification, the nuclear ribosomal internal transcribed spacer (ITS), 28S rDNA, and the widely used protein-coding marker translation elongation factor 1α (EF-1α) gene were evaluated as candidate DNA barcode markers to investigate their feasibility in identifying 13 oyster mushroom species. A total of 160 sequences of the candidate loci were analyzed. Intra- and interspecific divergences and the ease of nucleotide sequence acquisition were the criteria used to evaluate the candidate genes. EF-1α showed the best intra- and interspecific variation among the candidate markers and discriminated 84.6% of the species tested, only being unable to distinguish two closely related species Pleurotus citrinopileatus and Pleurotus cornucopiae. Furthermore, EF-1α was more likely to be acquired than ITS or 28S rDNA, with an 84% success rate of PCR amplification and sequencing. For ITS and 28S rDNA, the intraspecific differences of several species were distinctly larger than the interspecific differences, and the species identification efficiency of the two candidate markers was worse (61.5 and 46.2%, respectively). In addition, these markers had some sequencing problems, with 55 and 76% success rates of sequencing, respectively. Hence, we propose EF-1α as a possible DNA barcode marker for oyster mushroom spawn.

Species identification in complex groups of medicinal plants based on DNA barcoding: a case study on Astragalus spp. (Fabaceae) from southwest China

Species from the genus Astragalus L. (Fabaceae) are distributed throughout the world, with southwest China being one of the distribution centers of this genus. Many Astragalus species are used by local Chinese people as traditional medicines for enhancing immunity. However, the complexity of morphological characters in many Astragalus species makes it difficult to correctly identify them. DNA barcoding is a useful tool for species identification based on standardized DNA markers. In this study, internal transcribed spacer (ITS) region and three chloroplast regions (matK, rbcL, and trnH–psbA) were used for the identification of Astragalus species from SW China. The ITS region showed a high success rate of PCR amplification and bidirectional sequencing, as well as a high discriminatory ability (72%). The matK region also possessed relatively perfect university and had a high discriminatory ability (61%); while the rbcL region, the most popular cpDNA marker, showed good university but low discriminatory ability (44%). We found that the trnH–psbA region was not suitable for identifying Astragalus species because of its low success rate of DNA sequencing. In the studied species, combination of multiple DNA barcodes could improve the discriminatory ability. The ITS region was able to identify frequently used Astragalus species in SW China, such as A. yunnanensis, A. acaulis, A. bhotanensis, and A. camptodontoides, and we concluded that this region could be listed as the core barcode for species identification of medicinal Astragalus species from SW China.

The origin identification method for crude drugs derived from arthropods and annelids using molecular biological techniques.

We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots.

Detection of adulteration in black gram-based food products using DNA barcoding

DNA barcoding is gaining importance in food authenticity studies due to its sensitivity, reliability and accuracy in identification of adulterant species from pure food commodity. In the present study, three barcoding loci viz., rbcL, trnH-psbA and ITS were explored to test their potential in detection of adulteration of black gram flour and its products with refined wheat flour (maida) and white pea flour. All three loci exhibited 100% success rate of PCR amplification and sequencing. Amplicons of band size 600 bp, 380 bp and 680 bp were obtained for rbcL, trnH-psbA and ITS, respectively. The method was validated on simulated samples of black gram flour (variety TPU-4) adulterated with 5% each of refined wheat flour and white pea flour. Sequence analysis and BLAST searches of the model blends revealed the presence of these two potential adulterants, thereby making the method sensitive enough to detect adulteration at 5% level. Among the 3 loci studied, loci rbcL and trnH-psbA, served as ideal candidates for detection of refined wheat flour adulteration in black gram flour. Eleven market samples of black gram products such as flour, papad, instant medu vada mix and papad atta of different brands were analysed using DNA barcoding. The method could successfully detect the presence of refined wheat flour in one of the papad samples. Therefore, molecular identification of black gram flour products and its adulterants can be done using the developed DNA barcoding method based on rbcL and trnH-psbA sequences.

Application of the ITS2 Region for Barcoding Plants of the Genus Triticum L. and Aegilops L.

Molecular taxonomic studies have been performed in the past in order to identify different wheat species and construct a molecular phylogeny. These were based on universal but sufficiently divergent sequences from both the nuclear and chloroplastic genomes of wheat. They included two short plastid sequences from the plastid genes rbcL and matK which have been proposed as the core “barcode” sequences by the “barcoding” guidelines for general plant identification. Historically, in molecular plant taxonomy, plastidic sequences had been favored over nuclear sequences, due to their uniparental inheritance and consequently lower intra-molecular recombination. However recently, the short nuclear sequence from the internal transcribed spacer 2 (ITS2) has been used successfully for the accurate identification of many medicinal and other plant species. Herein, we have used the plastidic matK, rbcL trnL, and the nuclear ITS2 region for the identification of different wheat species of Triticum L. and goatgrass species of Aegilops L. We have successfully discriminated all species that were examined from both genera, thus, validating the ITS2 region as a ‘barcode tool’ for accurate distinction of plants in the genus Triticum L. and Aegilops L. The success rate of PCR amplification and sequencing of the ITS2 region was 100%. We report also that matK, rbcL and trnL regions could not discriminate all species in contrast to the ITS2 region which demonstrated full discriminatory capacity.

A simple and efficient total genomic DNA extraction method for individual zooplankton.

Molecular approaches are widely applied in species identification and taxonomic studies of minute zooplankton. One of the most focused zooplankton nowadays is from Subclass Copepoda. Accurate species identification of all life stages of the generally small sized copepods through molecular analysis is important, especially in taxonomic and systematic assessment of harpacticoid copepod populations and to understand their dynamics within the marine community. However, total genomic DNA (TGDNA) extraction from individual harpacticoid copepods can be problematic due to their small size and epibenthic behavior. In this research, six TGDNA extraction methods done on individual harpacticoid copepods were compared. The first new simple, feasible, efficient and consistent TGDNA extraction method was designed and compared with the commercial kit and modified available TGDNA extraction methods. The newly described TGDNA extraction method, “Incubation in PCR buffer” method, yielded good and consistent results based on the high success rate of PCR amplification (82%) compared to other methods. Coupled with its relatively consistent and economical method the “Incubation in PCR buffer” method is highly recommended in the TGDNA extraction of other minute zooplankton species.

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.

MOLECULAR IDENTIFICATION OF ORIGINAL PLANTS OF FRITILLARIAE CIRRHOSAE BULBUS, A TRADTIONAL CHINESE MEDICINE (TCM) USING PLANT DNA BARCODING.

Background:DNA barcoding is a widely used tool that enables rapid and accurate identification of species based on standardized DNA regions.Materials and Methods:In this study, potential DNA barcodes, namely three plastid regions (rbcL, trnH-psbA and matK) and one nuclear ribosomal internal transcribed spacer (ITS) were adopted for species identification of original plants of Fritillariae Cirrhosae Bulbus.Results:The rbcL and trnH-psbA regions showed better success rate of PCR amplification and DNA sequencing, as well as superior discriminatory ability. On the contrary, ITS region did not possess effective genetic variation and matK was faced with low success rate of sequencing. Combination of multi-loci sequences could improve identification ability of DNA barcoding. The trnH-psbA + rbcL could discriminate 25% - 100% species based on the Blast, Tree-Building and Distance methods.Conclusion:The potential DNA barcodes could not completely solving species identification of botanic origins of Fritillariae Cirrhosae Bulbus. In future, we should pay more attention to super-barcoding or specific barcode that enhance ability to discriminate the closely related plants.

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.

Copy Number Variation Analysis of Circulating Tumor Cells at a Single Cell Level Based on Hydrogel Encapsulation

Circulating tumor cells (CTCs) are defined as tumor cells circulating in peripheral blood of patients with metastatic cancer. CTCs have potential as a biomarker for cancer diagnosis or drug discovery. Recently, much attention has been paid to genomic analyses such as copy number variation (CNV) analysis of CTCs, because it will provide important information related to treatment for personalized therapy. These analyses should be performed at a single cell level because of the heterogeneity of cancer cells. However, CTCs are extremely rare cell in blood (1 in 109blood cells), so highly efficient technique for CTC recovery and single-cell isolation is required. Our group has developed “microcavity array system” on which rare CTCs can be efficiently captured from whole blood on the basis of differences in the size and deformability between tumor and blood cells. Furthermore, a novel technique for a single cell manipulation using a photo-polymerized hydrogel, “hydrogel-based cell encapsulation” has been demonstrated. In this study, to evaluate the performance of “microcavity array system” and “hydrogel-based cell encapsulation”, whole genome amplification (WGA) was performed using single-CTCs isolated by our proposed method. Furthermore, the utility of the proposed method in CNV analysis were evaluated. The microcavity array (φ= 8 μm, 4×103 cavities) was created by electroforming technique as microfabricated filter. The microcavity array was fabricated with PDMS structure equipped with a vacuum microchannel to apply negative pressure for cell entrapment. A549 cells (lung cancer cell line), SKBR-3 cells (breast cancer cell line) or MCF-7 cells (breast cancer cell line) were used as model CTCs. These cells were stained with fluorescent dyes, and suspended in PBS or healthy whole blood. These cell suspensions were introduced into the microcavity array at a flow rate of 150 µl/min. After cell recovery process, the number of cancer cells captured on the microcavity array was counted under a fluorescence microscope. As a results, more than 95% of cancer cells was successfully recovered on the microcavity array from PBS and whole blood. Then, poly (ethylene glycol) diacrylate (PEGDA) hydrogel was introduced onto the MCA. An excitation light (λ=365 nm) was irradiated to the targeted cancer cells for 30 seconds, leading to cross-linking reaction of PEGDA hydrogel and encapsulation of a single-cell. Solidified PEGDA hydrogel can be easily recovered from microcavity array by tweezers. Fluorescent microscopic analysis revealed that targeted single-cells were successfully encapsulated with PEGDA hydrogel. The hydrogel-based cell encapsulation process for 20 single-cells was completed within 10 min. To evaluate the effect of hydrogel on genome amplification, hydrogels with single-cells were subjected to the WGA, and the yields of WGA products were measured by PicoGreen assay. Genome amplification was also successfully carried out in all single-cells encapsulated on the hydrogel, and the yields of WGA products were the same with those obtained by using the micromanipulation as a conventional method. The amplification bias of WGA was evaluated based on the success rate of PCR amplification using 9 different primer sets (multiple loci). The PCR amplification of 9 fragments revealed that no significant bias was observed between WGA samples from single-cells isolated by hydrogel encapsulation and micromanipulation. Based on these results, we concluded that our single cell isolation method could be utilized for WGA at single cell level. Furthermore, quantitative PCR was performed to evaluate CNV analysis using SKBR-3 and MCF-7 cells, which have different HER2 status. As a result, the level of HER2 copy number in WGA products obtained from single-cells was similar with that from bulk analysis (103cells).Determination and monitoring of HER2 status in genome is well known to serve as a useful biomarker for diagnosis of breast cancer. Therefore, our proposed method for CNV analysis of CTCs is expected to be widely utilized towards real-time cancer diagnosis without repeating surgical biopsy.

DNA Barcoding of Highly Threatened Sacred Plant - Aegle marmelos L.

DNA barcoding is a useful tool to define operational taxonomic units based on standardized DNA regions. In this study, the matK chloroplast gene was tested for species identification of highly threatened sacred plant Aegle marmelos L., to document their reliable identification, discrimination, similarities and evolutionary trend with their related taxa for future use. DNA isolation from leaf sample of Aegle marmelos was carried out by using a modified Cetyl Trimethyl Ammonium Bromide method (CTAB). Gradient PCR amplification was performed for the isolated DNA using matK gene and the primers, matK472F and matK1248R. Pairwise sequence alignments were made with BLAST and multiple sequence alignments were also made with ClustalW. Based on the sequence alignments, dendrograms were constructed using the software, Mega 5 and Neighbor Joining tree method was used to study the phylogenetic aspects of the species studied and with their related taxa. The success rate of PCR amplification was 90-95% and the success rate of DNA sequence was 95%. The maximum identity of the present study sample was 100% with Aegle marmelos and the similarity of other related species, Severinia buxifolia, Citrus trifoliata and Citrus reticulata was 97% respectively.

植物の法科学的検査に適したPCRキットの比較

Recently, plant DNA has been utilized to discriminate or identify forensic botanical samples. In a forensic examination of a plant, various species and plant parts are considered as evidence. PCR amplification has sometimes failed because the samples contain some PCR inhibitors. In this study, to increase the success rate of PCR amplification of plant materials, four kinds of commercially available PCR kits, which showed resistance to PCR inhibitors, were evaluated by two experiments. One experiment was PCR amplification with the addition of chemicals to PCR solution; the other one was PCR amplification using non-purified DNA extracted from plants. The concentrations of chemicals, which inhibited PCR amplification, were different between four kits. Humic acid showed a high inhibitory power, and the concentration of it inhibiting PCR amplification was smaller than the other chemicals by one or two orders of magnitude. PCR amplification from non-purified DNA was succeeded by using a PCR kit, but there was no kit that enabled us to amplify all plant samples. Two kits could amplify more plant samples than the other kits and previous methods. The results indicate that these two PCR kits, which are designed for PCR of plant-derived DNA, are more suitable for forensic examinations of plants.

Identification and persistence of Pinus pollen DNA on cotton fabrics: A forensic application

Advances in plant genomics have had an impact on the field of forensic botany. However, the use of pollen DNA profiling in forensic investigations has yet to be applied. Five volunteers wore a jacket with Pinus echinata pollen-containing cotton swatches for a 14-day period. Pollen decay was evaluated at days 0, 3, 6, 9 and 14 by microscopy. Pollen grains were then transferred to slides using a portable forensic vacuum handle. Ten single grains per swatch were isolated for DNA analysis. DNA was extracted using a high throughput extraction method. A nine-locus short tandem repeat (STR) multiplex system, including previously published primers from Pinus taeda, was developed. DNA was amplified by PCR using fluorescent dyes and analyzed by capillary electrophoresis. Pollen counts from cotton swatches in a 14-day period exhibited an exponential decay from 100% to 17%. The success rate of PCR amplification was 81.2%. Complete and partial STR profiles were generated from 250 pollen grains analyzed (44% and 37%, respectively). Due to the limited amount of DNA, drop-in events were observed (1.87%). However, the rate of contamination with pollen from other pine individuals originating from environmental sources was 4.4%. In conclusion, this study has shown that pollen can be a stable source of forensic DNA evidence, as a proof-of-principle, and that may persist on cotton clothing for at least 14days of wear. This method can be applied in forensic cases where pollen grains larger than 10μm (e.g., from herbs or trees) may be transferred to clothing (worn by suspect or victim) by primary contact.