Target DNA Fragments Research Articles

Quantification of eDNA to Map the Distribution of Cold-Water Coral Reefs

An effective management of vulnerable marine ecosystems is dependent on thorough knowledge of their location. Multibeam echo-sounders and remotely operated vehicles (ROVs) are currently used to map areas impacted by industrial activities when sensitive and vulnerable species are expected, however surveying large areas using ROVs is slow and expensive. Here, we developed a species-specific eDNA assay targeting a 178 bp fragment in the control region of the mitochondrial DNA of the cold-water coral (CWC) Lophelia pertusa. The aim was to test if concentrations of L. pertusa eDNA in seawater, determined using droplet digital PCR (ddPCR) technology, could be used to rapidly assess the distribution of CWC reefs in a region. Our assay successfully amplified L. pertusa DNA from seawater. In laboratory we documented an exponential decay rate of the targeted DNA fragment and a linear correlation between coral biomass and eDNA concentrations in flow through microcosms. The ability of the method to detect CWC reefs in situ was tested in the fjords south of Bergen, Norway, where such reefs are common. We tested 5 sites with, and 5 sites without, reefs. Lophelia pertusa eDNA was detected in all 10 deep sites examined. However, concentrations were elevated by 5 to 10 times in water sampled off the two large reefs growing on vertical surfaces. Water sampled 10 m above CWC reefs growing on the flat seabed did not produce an equally clear eDNA signal, nor did single CWC colonies growing on vertical surfaces. Our dispersal model used experimentally derived values for L. pertusa eDNA decay and shedding rates in realistic temperature and salinity. Treating the eDNA as a passive particle with no active vertical or horizontal movement, we successfully modelled the dispersal of eDNA from the known CWC reefs in the region and achieved a good fit with measured eDNA concentrations. In all, our study demonstrated a great potential for eDNA measurements as a cost-efficient tool for a rapid screening of the distribution of CWC reefs growing on vertical surfaces (so called wall reefs) that are challenging to detect using traditional mapping techniques.

A Facile and Sensitive DNA Sensing of Harmful Algal Blooms Based on Graphene Oxide Nanosheets.

Gene detection has important applications in biology, biomedical engineering, clinical, environmental, and marine fields. Rapid, sensitive, and selective recognition of specific genes is essential in practical applications. In this study, we describe a facile and sensitive DNA sensing platform for specific and quantitative detection of Heterosigma akashiwo, which is one of the causative agents of red tides. Fast and sensitive detection is achieved by using chemically synthesized graphene oxide (GO) nanosheets. Probe DNA is designed according to the specific DNA fragments of harmful algae and labeled with fluorescent molecules FAM (fluorescein-based dye). GO nanosheet solution is made, in which the strong interaction between FAM-labeled probe and GO nanosheets keeps them in close proximity, facilitating the fluorescence quenching of the fluorophore by GO nanosheets. In the presence of a complementary target DNA, the FAM-labeled DNA probe and the target DNA hybridize and desorb from the surface of GO nanosheets, resulting in restoration of fluorophore fluorescence. The concentration of target DNA fragments is analyzed by the fluorescence intensity at ~ 520nm with emission wavelength of 480nm. The sensitive detecting platform achieved stable measurement of 1pM specific genes from Heterosigma akashiwo. Our GO nanosheet-based DNA-sensing platform performs fast and sensitive detection of trace amount of DNA, and enables quantitative recognition of harmful algae, which has promising applications in real-time monitoring in the marine environment of red tide generative dynamics, allowing effective control, particularly in relation to marine aquaculture.

Loop mediated isothermal amplification (LAMP) of Toxoplasma DNA from dried blood spots

IntroductionThe cosmopolitan protozoan Toxoplasma gondii is a major parasite of warm-blooded animals including man. Early and accurate diagnosis is a must for proper treatment that prevents life threatening sequels. Loop-mediated isothermal amplification (LAMP) is a novel technique that can amplify DNA with high sensitivity and specificity under isothermal conditions. Aim of the studyTo validate a LAMP-specific protocol for detection of Toxoplasma DNA using dried blood spots (DBS) from mice experimentally infected with the cystogenic Toxoplasma ME-49 strain. MethodsIn this study, the target DNA fragment was the Toxoplasma 529-bp repeat element that exists in 200–300 copies per T. gondii genome. The sensitivity of both LAMP and conventional PCR techniques was estimated in DBS samples from experimental mice at 1-week and 8-weeks post-infection. ResultsOut of 20 blood samples gathered on Whatman filter paper from mice at 1-week post-infection, 18 and 16 were positive by LAMP and conventional PCR, respectively. Neither techniques detected parasite DNA in blood at 8th week of infection. ConclusionDried blood spots are easy source of material for molecular studies. LAMP assay proved higher sensitivity than the conventional PCR in detecting parasitemia in early infection with the cystogenic Toxoplasma strain.

PCR-RFLP as a detection method of allelic diversity seahorse Hippocampus comes (Cantor, 1849) from Bintan waters, Riau Island

Seahorse (Hippocampus comes) is a unique species which reproductive system pregnancy occur in male organisms. According to IUCN and CITES, seahorses are noted as endangered or vulnerable and Appendix II species. The aimed of this study was to identify the allelic diversity based on Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP). Samples were collected from Tanjung Berakit, Bintan waters, Riau islands. Total of 11 individuals of the species were analyzed two types of restriction enzyme, EcoRI and AluI. This study showed that there were two types of DNA fragmentations based on restriction enzyme. EcoRI enzyme was unable to cut the target DNA fragments. AluI enzyme was capable of bypassing the target DNA fragment so it could be visualized by two alleles. The genetic diversity values of less than one showed that the genetic diversity of the population of seahorse species of H. comes in Bintan waters was low.

Sequence Variations and Molecular Phylogeny of some Red Sea Parrotfishes (Scaridae) Using Mitochondrial Gene Sequences

Targeted DNA fragments were isolated from four scarids species; Scarus collana Ruppell 1835; Scarus frenatus Lacepede 1802; Scarus (Chlorurus) sordidus Forsskal 1775 and Scarus niger Forsskal 1775 using mitochondrial 16S rRNA gene-specific primers. Analysis of obtained partial 16S rRNA gene nucleotide sequences showed a high level of nucleotide identity in the studied regions. This reflects a close genetic relationship and shared ancestry among studied parrotfishes. Nucleotide compositions of partial 16S rRNA gene nucleotide sequence biased towards adenine and similar preference towards thymine, cytocine, and guanine. Also, base constitutions revealed preference towards higher DNA conservations. Phylogenetic analysis displayed patterns of assembly for studied species, and other included related taxa, which reflect their similar genetic makeup and their tendency to have similar niches. The phylogenetic trees revealed two evolutionary lineages splitting Scaridae and Wrasses which assumed that Scaridae should maintain the family status. Obtained data could be beneficial for parootfishes classification, conservation, and their needed environments. Therefore, the acquisition of nucleotide sequences from other parrotfishes using the developed mt16S rRNA gene-specific primers utilized here would contribute in the future to the phylogenetic

Ultrasensitive Monolayer MoS2 Field-Effect Transistor Based DNA Sensors for Screening of Down Syndrome.

Field-effect transistor (FET) biosensors based on low-dimensional materials present the advantages of low cost, high speed, small size, and excellent compatibility with integrated circuits (ICs). In this work, we fabricated highly sensitive FET-based DNA biosensors based on chemical vapor deposition (CVD)-grown monolayer MoS2 films in batches and explored their application in noninvasive prenatal testing (NIPT) for trisomy 21 syndrome. Specifically, MoS2 was functionalized with gold nanoparticles (Au NPs) of an optimized size and at an ideal density, and then, probe DNAs for the specific capture of target DNAs were immobilized on the nanoparticles. The fabricated FET biosensors are able to reliably detect target DNA fragments (chromosome 21 or 13) with a detection limit below 100 aM, a high response up to 240%, and a high specificity, which satisfy the requirement for the screening of Down syndrome. In addition, a real-time test was conducted to show that the biosensor clearly responds to the target DNA at concentrations as low as 1 fM. Our approach shows the potential for detecting the over-expression of chromosome 21 in the peripheral blood of pregnant women and achieving Down syndrome screening.

Electrochemical DNA Biosensing via Electrochemically Controlled Reversible Addition-Fragmentation Chain Transfer Polymerization.

Sensitive and selective sensing of biological molecules is fundamental to disease diagnosis and infectious disease surveillance. Herein, an ultrasensitive and highly selective electrochemical DNA biosensor is described by exploiting the electrochemically controlled reversible addition-fragmentation chain-transfer (eRAFT) polymerization as a signal amplification strategy and the peptide nucleic acid (PNA) probes as the recognition elements. Specifically, the PNA probes with a thiol at their 5'-terminals are anchored to a gold electrode surface (via gold-sulfur self-assembly) for sequence-specific recognition of target DNA (tDNA) fragments, of which the phosphate sites serve as the anchorages for the targeted labeling (via the well-established phosphate-Zr4+-carboxylate chemistry) of the carboxyl-group-containing chain-transfer agents (CTAs) for the succedent eRAFT polymerization, wherein the initiating radicals are generated through electrochemical reduction of aryl diazonium salts under a potentiostatic condition. In the presence of ferrocenylmethyl methacrylate (FcCH═CH2) as the monomer, the grafting of polymer chains from the CTA-anchored sites as a result of the eRAFT polymerization brings numerous electroactive Fc tags to the electrode surface, outputting a high electrochemical sensing signal even in the presence of trace amounts of tDNA fragments. Under the optimized conditions, the linear range of the described electrochemical DNA biosensor spans from 10 aM to 10 pM ( R2 = 0.998), with an attomolar detection limit (4.1 aM) being achieved. Moreover, the described electrochemical DNA biosensor is highly selective and applicable to the sensing of tDNA fragments in complex serum samples. Given its high efficiency, easy operation, and low cost, this biosensor shows great promise in real applications.

Phage serine integrase-mediated genome engineering for efficient expression of chemical biosynthetic pathway in gas-fermenting Clostridium ljungdahlii

The real value of gas-fermenting clostridia, capable of using CO and CO2, resides in their potential of being developed into cell factories to produce various bulk chemicals and fuels. This process requires rapid chromosomal integration of heterologous chemical biosynthetic pathways, which is impeded by the absence of genetic tools competent for efficient genome engineering in these anaerobes. Here, we developed a phage serine integrase-mediated site-specific genome engineering technique in Clostridium ljungdahlii, one of the major acetogenic gas-fermenting microbes. Two heterologous phage attachment/integration (Att/Int) systems (from Clostridium difficile and Streptomyces) were introduced into C. ljungdahlii and proven to be highly active, achieving efficient chromosomal integration of a whole donor vector via single-crossover recombination. Based on this, we further realized markerless chromosomal integration of target DNA fragments through a “dual integrase cassette exchange” (DICE) strategy with the assistance of the CRISPR-Cas9 editing system. As a proof of concept, a butyric acid production pathway from Clostridium acetobutylicum was integrated into the C. ljungdahlii genome without the introduction of extra markers, enabling stable expression of the pathway genes. The resulting engineered strain produced 1.01 g/L of butyric acid within 3 days by fermenting synthesis gas (CO2/CO). More importantly, the engineered strain showed good genetic stability and maintained butyric acid production ability after continuous subculturing. The system developed in this study overcomes the deficiencies of currently available genetic tools in the chromosomal integration of large DNA fragments (rapid, markerless and stable) in C. ljungdahlii, and may be extended to other Clostridium species.

ICatch: a new strategy for capturing large DNA fragments using homing endonucleases.

Natural genetic materials contain many biosynthetic gene clusters encoding potentially valuable natural products, many of which can be used directly without codon optimization or other manipulations. With the development of synthetic biology, several DNA assembly standards have been proposed, conveniently facilitating the reuse of natural materials. Among these standards, the iBrick assembly standard was developed by our laboratory to manipulate large DNA fragments, employing two homing endonucleases. Considering the difficulty of cloning large iBrick parts using conventional endonuclease-mediated restriction and ligation methods, we herein present a new method, known as iCatch, which readily captures biosynthetic gene clusters. As the clusters cloned by iCatch have the prefix and suffix of the iBrick standard, they serve as new iBrick parts and are therefore conducive to further editing and assembly with the iBrick standard. iCatch employs the natural homologous recombination system to flank the region of interest with I-SceI and PI-PspI recognition sites, after which the genome is digested with I-SceI or PI-PspI and the fragments are then self-ligated to clone the target DNA fragments. We used this method to successfully capture the actinorhodin biosynthetic cluster from Streptomyces coelicolor and then heterologously expressed this cluster in a thermophilic Streptomyces strain. We propose that iCatch can be used for the cloning of DNA sequences that are dozens of kilobases in length, facilitating the heterologous expression of microbial natural products. Moreover, this cloning methodology can be a complementary tool for the iBrick standard, especially in applications requiring the manipulation of large DNA fragments.

Surface-Initiated-Reversible-Addition-Fragmentation-Chain-Transfer Polymerization for Electrochemical DNA Biosensing.

Sensitive detection of biomolecules is integral for biomarker screening and early diagnosis. Herein, surface-initiated reversible-addition-fragmentation-chain-transfer (SI-RAFT) polymerization is exploited as a novel amplification strategy for highly sensitive electrochemical biosensing of DNA. Briefly, thiol-terminated peptide nucleic acid (PNA) probes are first self-assembled onto a gold electrode for the specific capture of target-DNA fragments; the carboxyl-group-containing dithiobenzoate 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPAD) is then tethered to the hybridized PNA-DNA heteroduplexes by means of the well-established carboxylate-Zr4+-phosphate chemistry and serves as the chain-transfer agent (CTAs) for subsequent SI-RAFT polymerization, which is thermally initiated in the presence of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) as the free-radical initiator and ferrocenylmethyl methacrylate (FcMMA) as the monomer. Through SI-RAFT polymerization, one target-DNA fragment can be labeled by a large number of electroactive Fc tags, giving rise to significant amplification of the electrochemical signal. The SI-RAFT-polymerization-based strategy does not involve the use of natural enzymes or complex nanomaterials, offering the benefits of low cost and easy operation. Under optimal conditions, the electrochemical signal is linearly related to the logarithm of the concentration of target DNA over the range from 10 aM to 10 pM ( R2 = 0.997), with a detection limit down to 3.2 aM, which is much lower than those of other amplification-by-polymerization-based methods. By virtue of its easy operation, low cost, and high efficiency, the SI-RAFT-polymerization-based amplification strategy is believed to have great application prospects in the sensitive detection of biomolecules.

A multiplex PCR amplification strategy coupled with microchip electrophoresis for simultaneous and sensitive detection of three foodborne bacteria

Foodborne bacteria are some of the most important human pathogens and can cause many diseases. In this study, multiplex PCR amplification combined with microchip electrophoresis (MCE) was studied to simultaneously and sensitively detect Staphylococcus aureus, Proteus mirabilis, and Enterobacter sakazakii. In order to simultaneously and accurately detect the aim bacteria, three pairs of primers were specially designed for the multiplex PCR amplification of the target genes of three bacteria, which were the specific genes corresponding to these bacteria respectively. After the DNA fragments of three bacteria were simultaneously extracted, the multiplex PCR amplification was performed by adding the three pairs of specific primers in the mixed DNA fragments solution. The multiplex PCR products of the three food-borne pathogens were analyzed by MCE and the limits of detection of target DNA fragments were 1.2–2.2ngμL−1, (S/N=3). The limits of detection of the aim bacteria were calculated as 53CFUmL−1 for Enterobacter sakazakii, 32CFUmL−1 for Proteus mirabilis, 28CFUmL−1 for Staphylococcus aureus, respectively. Satisfactory results were obtained when this method was applied to detect the three foodborne bacteria in milk samples. The experimental results show that this method has the advantages of quickness, less sample consumption, high selectivity and high sensitivity.

Detection and identification of Mycoplasma species by multiplex PCR in cats

Mycoplasma (M.) felis and M. canis is related with pneumonia or conjunctivitis in domestic cats and several diseases in a variety of other animals, including lower respiratory tract disease or pleuritis. Polymerase chain reaction (PCR) assays has been reported as an easy and useful method. It could be conducted even on nasal swab samples as a non-invasive rapid testing tools for large numbers of Mycoplasma species. However, PCR assays have to conduct multiple assays because of a lot of Mycoplasma species. Therefore, it need to perform several tests and reveal time consuming procedures. In this study, we developed a sensitive and specific multiplex polymerase chain reaction (PCR) assay that detects simultaneously two species like as M. felis and M. canis. The simultaneous detection of M. felis and M. canis primers were used to differentiate two Mycoplasma species. The target DNA fragments were specifically amplified M. felis and M. canis PCR with 16S ribosomal DNA primers. Single and mixed Mycoplasma species DNA templates were submitted to validate the specificity of the multiplex PCR. The corresponding specific DNA products were amplified for each pathogen. The detection limit of the developed multiplex PCR is 102 pg with M. felis and M. canis DNA. Furthermore, the developed multiplex PCR detected successfully M. felis in feline nasal specimens. The multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of M. felis and M. canis in cats.

An Optical Biosensor-Based Quantification of the Microcystin Synthetase A Gene: Early Warning of Toxic Cyanobacterial Blooming.

The monitoring and control of toxic cyanobacterial strains, which can produce microcystins, is critical to protect human and ecological health. We herein reported an optical-biosensor-based quantification of the microcystin synthetase A (mcyA) gene so as to discriminate microcystin-producing strains from nonproducing strains. In this assay, the mcyA-specific ssDNA probes were designed in silico with an on-line tool and then synthesized to be covalently immobilized on an optical-fiber surface. Production of fluorescently modified target DNA fragment amplicons was accomplished through the use of Cy5-tagged deoxycytidine triphosphates (dCTPs) in the polymerase chain reaction (PCR) method, which resulted in copies with internally labeled multiple sites per DNA molecule and delivered great sensitivity. With a facile surface-based hybridization process, the PCR amplicons were captured on the optical-fiber surface and were induced by an evanescent-wave field into fluorescence emission. Under the optimum conditions, the detection limit was found to be 10 pM (S/N ratio = 3) and equaled 103 gene copies/mL. The assay was triumphantly demonstrated for PCR amplicons of mcyA detection and showed satisfactory stability and reproducibility. Moreover, the sensing system exhibited excellent selectivity with quantitative spike recoveries from 87 to 102% for M. aeruginosa species in the mixed samples. There results confirmed that the method would serve as an accurate, cost-effective, and rapid technique for in-field testing of toxic Microcystis sp. in water, giving early information for water quality monitoring against microcystin-producing cyanobacteria.

Multifragment melting analysis of yeast species isolated from spoiled fruits.

In this study, we developed a novel approach, multifragment melting analysis (MFMA), based on the simultaneous analysis of the melting characteristics of multiple DNA fragments for the differentiation and identification of yeast species isolated from spoiled fruits. In total, 183 yeast isolates recovered from spoiled fruit samples were differentiated and grouped using MFMA. Six different DNA fragments of the 26S rRNA gene showing a high interspecific heterogeneity were amplified, and the PCR products were individually subjected to MFMA. An excellent discrimination and classification were obtained when the melting characteristics of all target DNA fragments for each species were simultaneously assessed. Species-level identification was performed by sequence analysis of representative isolatesfrom each group. In the fruit samples, Hanseniaspora uvarum (Kloeckera apiculata) was found to be the most frequently isolated species followed by members of the Pichia genus, namely P. kluyveri, P. fermentans and P. kudriavzevii (Issatchenkia orientalis). Multifragment melting analysis provided a rapid and reliable approach for discrimination and grouping of a large number of yeast isolates recovered from fruit samples prior to sequence analysis-based identification. The MFMA has great potential for fast andaccurate investigation of yeast communities associated with food spoilage to determine their sources and routes and to prevent contamination.

Comparison of the myostatin (MSTN) gene in Russian Stavropol Merino sheep and New Zealand Merino sheep

The aim of this research was to study the myostatin gene (MSTN) in Russian Stavropol Merino sheep and compare them with Merino sheep from New Zealand (NZ). Thirty rams of the Stavropol Merino breed and ten NZ Merinos were investigated. In order to detect sequence variation in MSTN, enrichment and sequencing of targeted DNA fragments was undertaken using NimbleGen technology. Nucleotide sequence variations (n = 31) were found in MSTN, including 29 single nucleotide polymorphisms (SNPs), one deletion and one insertion. Of these, 21 of the SNPs, the insertion and the deletion, were found in the Stavropol Merino sheep and 17 were found in the NZ Merinos. Eight variations in MSTN are described for the first time: c.373+396T>C, c.374-362A>T, c.374-16delT, c.747+185C>A, c.748-194C>A, c.782_783insT, c.940G>T and c.*310G>T, and these were unique to the Stavropol Merinos. The presence of SNP c.940G>T leads to the formation of a stop-codon (GAA>TAA) and thus truncated protein at amino acid position 314, while the insertion c.782_783insT changes the reading frame and would cause an amino acid sequence change from amino acid position 263 onwards, with the formation of a stop-codon at position 265. The study confirms the presence of additional variability in both non-coding and coding regions of ovine myostatin, which might subsequently be analysed to ascertain whether it affects meat production.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection.

A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.

Development of a Tandem Repeat-Based Polymerase Chain Displacement Reaction Method for Highly Sensitive Detection of 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of 'Candidatus Liberibacter asiaticus', a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of 'Ca. L. asiaticus' with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of 'Ca. L. asiaticus'.

Detection of E2F-DNA Complexes Using Chromatin Immunoprecipitation Assays.

Chromatin immunoprecipitation (ChIP), originally developed by John T. Lis and David Gilmour in 1984, has been useful to detect DNA sequences where protein(s) of interest bind. ChIP is comprised of several steps: (1) cross-linking of proteins to target DNA sequences, (2) breaking genomic DNA into 300-1000bp pieces by sonication or nuclease digestion, (3) immunoprecipitation of protein bound to target DNA with an antibody, (4) reverse cross-linking between target DNA and the bound protein to liberate the DNA fragments, and (5) amplification of target DNA fragment by PCR. Since then, the technology has evolved significantly to allow not only amplifying target sequences by PCR, but also sequencing all DNA fragment bound to a target protein, using a variant of the approach called the ChIP-seq technique (1). Another variation, the ChIP-on-ChIP, allows the detection of protein complexes bound to specific DNA sequences (2).

DNA barcoding in concentrated Chinese medicine granules using adaptor ligation-mediated polymerase chain reaction

The use of DNA barcodes for species identification is a common laboratory practice. However, PCR amplification of full-length DNA barcode in processed material is difficult because of severe DNA fragmentation. In this study, an adaptor ligation-mediated PCR protocol was derived to amplify sets of target DNA fragments isolated from two CCMG products. The specially designed adaptor with asymmetric strands and terminal modification avoids amplification of non-target DNA sequences. DNA extracted from Angelica sinensis and Panax notoginseng CCMG were ligated with the adaptors and amplified by an adaptor primer and a single universal barcode primer to obtain partial ITS2 sequence. Results showed that various length of DNA fragments within the ITS2 region were amplified and could be used to identify the concerned species. The adaptor ligation-mediated PCR is therefore a promising universal method for species identification in highly processed herbal products.

A pilot study on PCR-based detection of four foodborne pathogenic microorganisms

To establish PCR-based detection methods for Staphylococcus aureus, Shigella, Pasteurella multocida and Pseudomonas aeruginosa, the nuc, ipah, ptfa and oprl genes were amplified by singleplex PCRs and multiplex PCR using specific primers that were designed according to the DNA sequences retrieved from GenBank. Then the annealing temperature was optimized, accompanied by a study of the specificity and sensitivity of the singleplex PCRs and multiplex PCR. The results showed that DNA fragments of 280, 474, 150 and 331 bp were specifically amplified from the four pathogenic bacteria mentioned above. No target DNA fragments were obtained from other pathogenic bacteria, including Salmonella typhimurium, Campylobacter jejuni, Clostridium perfringens and pathogenic Escherichia coli. The sensitivity of the singleplex PCRs were 100, 1, 1 and 10 pg/μL respectively. The detection limits of the four pathogenic bacteria in the multiplex PCR were 100, 1, 10 and 10 pg/µL respectively. These results showed that singleplex PCRs and multiplex PCR have good specificity and sensitivity. In conclusion, this experiment has laid a foundation for further research on rapid detection methods against these four pathogenic bacteria in food.