Amplification Of Target Sequences Research Articles

Genotyping Zebrafish Point Mutant by Allele-Specific Blocking PCR.

Genotyping zebrafish carrying wild-type, heterozygous, or homozygous copies of a mutant allele is often required for investigating gene specific functions, and is routinely performed to differentiate point mutants. In this study, we describe a modified allele-specific PCR method using an additional blocking primer to promote target sequence amplification while suppressing sequences with single mismatch. Using the tp53m214k point mutant as an example, we show that wild-type, heterozygous, and homozygous zebrafish can be easily distinguished using this simple PCR method, which could be widely adapted for genotyping zebrafish with point mutations or small nucleotide insertions/deletions.

Detection of Mtb and NTM: preclinical validation of a new asymmetric PCR-binary deoxyribozyme sensor assay.

Tuberculosis (TB) and infectious diseases caused by non-tuberculous mycobacteria (NTM) are global concerns. The development of a rapid and accurate diagnostic method, capable of detecting and identifying different mycobacteria species, is crucial. We propose a molecular approach, the BiDz-TB/NTM, based on the use of binary deoxyribozyme (BiDz) sensors for the detection of Mycobacterium tuberculosis (Mtb) and NTM of clinical interest. A panel of DNA samples was used to evaluate Mtb-BiDz, Mycobacterium abscessus/Mycobacterium chelonae-BiDz, Mycobacterium avium-BiDz, Mycobacterium intracellulare/Mycobacterium chimaera-BiDz, and Mycobacterium kansasii-BiDz sensors in terms of specificity, sensitivity, accuracy, and limit of detection. The BiDz sensors were designed to hybridize specifically with the genetic signatures of the target species. To obtain the BiDz sensor targets, amplification of a fragment containing the hypervariable region 2 of the 16S rRNA was performed, under asymmetric PCR conditions using the reverse primer designed based on linear-after-the-exponential principles. The BiDz-TB/NTM was able to correctly identify 99.6% of the samples, with 100% sensitivity and 0.99 accuracy. The individual values of specificity, sensitivity, and accuracy, obtained for each BiDz sensor, satisfied the recommendations for new diagnostic methods, with sensitivity of 100%, specificity and accuracy ranging from 98% to 100% and from 0.98 to 1.0, respectively. The limit of detection of BiDz sensors ranged from 12 genome copies (Mtb-BiDz) to 2,110 genome copies (Mkan-BiDz). The BiDz-TB/NTM platform would be able to generate results rapidly, allowing the implementation of the appropriate therapeutic regimen and, consequently, the reduction of morbidity and mortality of patients.IMPORTANCEThis article describes the development and evaluation of a new molecular platform for accurate, sensitive, and specific detection and identification of Mycobacterium tuberculosis and other mycobacteria of clinical importance. Based on BiDz sensor technology, this assay prototype is amenable to implementation at the point of care. Our data demonstrate the feasibility of combining the species specificity of BiDz sensors with the sensitivity afforded by asymmetric PCR amplification of target sequences. Preclinical validation of this assay on a large panel of clinical samples supports the further development of this diagnostic tool for the molecular detection of pathogenic mycobacteria.

Multiplex solid-phase RPA coupled CRISPR-based visual detection of SARS-CoV-2

The COVID-19 pandemic has presented a significant challenge to the world's public health and led to over 6.9 million deaths reported to date. A rapid, sensitive, and cost-effective point-of-care virus detection device is essential for the control and surveillance of the contagious severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. The study presented here aimed to demonstrate a solid-phase isothermal recombinase polymerase amplification coupled CRISPR-based (spRPA-CRISPR) assay for on-chip multiplexed, sensitive and visual COVID-19 DNA detection. The assay targets the SARS-CoV-2 structure protein encoded genomes and can simultaneously detect two specific genes without cross-interaction. The amplified target sequences were immobilized on the one-pot device surface and detected using the mixed Cas12a-crRNA collateral cleavage of reporter-released fluorescent signal when specific genes were recognized. The endpoint signal can be directly visualized for rapid detection of COVID-19. The system was tested with samples of a broad range of concentrations (20 to 2 × 104 copies) and showed analytical sensitivity down to 20 copies per microliter. Furthermore, a low-cost blue LED flashlight (∼$12) was used to provide a visible SARS-CoV-2 detection signal of the spRPA-CRISPR assay which could be purchased online easily. Thus, our platform provides a sensitive and easy-to-read multiplexed gene detection method that can specifically identify low concentration genes.

VertU: universal multilocus primer sets for eDNA metabarcoding of vertebrate diversity, evaluated by both artificial and natural cases

Environmental DNA (eDNA) metabarcoding is a powerful tool for monitoring biodiversity in natural ecosystems. The accuracy of eDNA metabarcoding relies heavily on the PCR primers that amplify target sequences. For vertebrates, most available primers are designed for particular groups. There have been inadequate attempts to design “universal” primers applicable to all vertebrates. Here, we developed three new universal primer sets (V12S-U, V16S-U, and VCOI-U) targeting mitochondrial 12S, 16S, and COI genes, respectively. They work for all vertebrate groups, with amplification length of 200-250 bp, facilitating the use of short-read sequencing platforms. We evaluated and compared the species detection ability of our primer sets and seven previously published ones through bothin-silicoPCR and experiment tests with mock DNA and zoo eDNA. The species detection success of the new primer sets is over 90%, showing better performance than previously published primer sets. We also tested our new primers with water eDNA samples of natural environments, and detected a total of 895 vertebrate OTUs (BLAST identity ≥ 0.95), comprising 182 species, 195 genera, and 94 families. Our results indicated that utilizing multiple markers can achieve better species detection than using only a single marker, which is especially important for monitoring large biological communities like vertebrates. The new primer sets enable researchers to detect the presence of species of different vertebrate groups in one eDNA metabarcoding survey, which simplifies the workflow and reduces the cost. It has the potential to serve as an alternative/complementary tool for future eDNA metabarcoding studies targeting vertebrates.

Mutation Analysis and Restriction Site Mapping of GDF9 in Indonesian Bligon Goat

Bligon goat is one of the crossbred goats raised in Indonesia, which has a prolific nature, potentially increasing the economic benefits. The gene that particularly influences prolific traits is Growth Differentiation Factor 9 (GDF9). This study aims to identify the mutation analysis (SNPs and amino acid changes) and restriction enzymes map in the GDF9 of Bligon goats. Six pairs of primers were used to amplify target sequences of GDF9 by polymerase chain reaction method and continued by sequencing. The sequence products were analyzed to get information on the SNPs and restriction enzyme (RE) around the SNPs for genotyping by PCR-RFLP method. A total of 15 SNPs were found in position g.1956A>C, g.2248G>T, g.2470A>T, g.2172DelA (shift T, Heterozygote), g.2807C>T, g.2919C>T, g.2996C>T, g.3615T>C, g.3855A>C, g.3879A>G, g.3924A>G, g.3943G>T, g.3969G>A, g.3981G>A, and g.4314C>T. Eight out of fifteen SNPs are located at the exon. Thus, the amino acid shows one synonymous at Exon 1 (Leucine to Leucine) and seven non synonymous at exon 2 with varied amino acid alteration (Valine to Alanine, Glutamine to Proline, Lysine to Arginine, Lysine to Arginine, Glutamine to Histidine, Arginine to Lysine, and Serine to Asparagine, respectively). Two SNPs at position g.1956A>C in Exon 1 and g.3855A>C in exon 2 show the homozygote CC and heterozygote AC. The most sample at those two position 67% and 83% homozygote type, respectively. Recognitions of site restriction enzymes BsaI and BsmAI were found at g.1956A>C or g.667C>M (Exon I). SNP g.3855A>C or g.2566C>M was recognized by three restriction enzymes (MspI, HapII, and HpaII). Two SNPs were not recognized by the restriction enzyme, and two other SNPs have more than 12 fragmented sequences, and as a consequence, it is very difficult to analyze the genotype. In conclusion, fifteen site mutations were identified; however, only two potential genetic markers with transversion mutations in Exon 1 and Exon 2 were recognized by restriction enzymes (BsaI, HapII, and MspI). These enzymes were recommended as candidate markers for further genotype identification using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs.

The use of microRNAs as clinical cancer biomarkers is hindered by the absence of accurate, fast and inexpensive assays for their detection in biofluids. Here we report a one-step and one-pot isothermal assay that leverages rolling-circle amplification and the endonuclease Cas12a for the accurate detection of specific miRNAs. The assay exploits the cis-cleavage activity of Cas12a to enable exponential rolling-circle amplification of target sequences and its trans-cleavage activity for their detection and for signal amplification. In plasma from patients with pancreatic ductal adenocarcinoma, the assay detected the miRNAs miR-21, miR-196a, miR-451a and miR-1246 in extracellular vesicles at single-digit femtomolar concentrations with single-nucleotide specificity. The assay is rapid (sample-to-answer times ranged from 20 min to 3 h), does not require specialized instrumentation and is compatible with a smartphone-based fluorescence detection and with the lateral-flow format for visual readouts. Simple assays for the detection of miRNAs in blood may aid the development of miRNAs as biomarkers for the diagnosis and prognosis of cancers.

Ichthyoplankton metabarcoding: An efficient tool for early detection of invasive species establishment.

Detection of invasive species is critical for management but is often limited by challenges associated with capture, processing and identification of early life stages. DNA metabarcoding facilitates large-scale monitoring projects to detect establishment early. Here, we test the use of DNA metabarcoding to monitor invasive species by sequencing over 5000 fishes in bulk ichthyoplankton samples (larvae and eggs) from four rivers of ecological and cultural importance in southern Canada. We were successful in detecting species known from each river and three invasive species in two of the four rivers. This includes the first detection of early life-stage rudd in the Credit River. We evaluated whether sampling gear affected the detection of invasive species and estimates of species richness, and found that light traps outperform bongo nets in both cases. We also found that the primers used for the amplification of target sequences and the number of sequencing reads generated per sample affect the consistency of species detections. However, these factors have less impact on detections and species richness estimates than the number of samples collected and analysed. Our analyses also show that incomplete reference databases can result in incorrectly attributing DNA sequences to invasive species. Overall, we conclude that DNA metabarcoding is an efficient tool for monitoring the early establishment of invasive species by detecting evidence of reproduction but requires careful consideration of sampling design and the primers used to amplify, sequence and classify the diversity of native and potentially invasive species.

Knowledge and DdeI Based Confirmation of Sickle Cell Anemia Among the Tharu Community.

Sickle cell anemia is an inherited blood disorder caused due to a point mutation at the sixth codon of the ?-globin gene of both alleles. Sickle cell traits occur when the mutation is in one of the two alleles of the ?-globin genes. This study was carried out in the Tharu community, which is an indigenous and minority group mostly residing in the Terai region of Nepal. They are also considered as the most vulnerable group for inheriting Sickle cell anemia. Purposive sampling, which included 130 Tharu individuals of Kanchanpur district of Nepal, was considered for the study. The survey was conducted using a descriptive questionnaire that contained relevant information including the family history of Sickle cell anemia. This was followed by the analysis of blood samples to determine the prevalence of Sickle cell anemia and Sickle cell traits. Primer-mediated enzymatic amplification of target sequences in genomic DNA followed by restriction endonuclease assay with an enzyme DdeI was carried out for the confirmation. Among 130 individuals, only 55.4% had basic knowledge about Sickle cell anemia. After screening for sickle cell anemia from 60 participants, 27 (45%) of them were found to be in the heterozygous state (carrier, Hb AS) and 28 (46.7%) were in the homozygous (normal, AA Hb) state with 5 (8.3%) having the diseased hemoglobin (Hb SS) variant of Sickle cell anemia. This study demonstrated a high prevalence of Sickle cell anemia and Sickle cell traits in the Tharu community. This study may be beneficial for concerned personnel policymakers to reduce sickle cell cases by improving genetic literacy among the Tharu community.

Molecular Detection of Chlamydia Trachomatis in Patients with Pelvic Inflammatory Disease Visiting a Tertiary Care Hospital in Pakistan

Objective: To detect the frequency of Chlamydia trachomatis in urine samples of women with Pelvic Inflammatory Disease visiting a tertiary care hospital using a Polymerase Chain Reaction assay and find an association between different risk factors.
 Study Design: Cross-sectional study.
 Place and Duration of Study: Department of Microbiology, Army Medical College/National University of Medical Sciences Rawalpindi Pakistan, from Mar 2018 to Jan 2019.
 Methodology: Over eleven months, 60 diagnosed urine samples of married females with pelvic inflammatory disease between the ages of 14-49 years were included in the study. DNA of Chlamydia trachomatis from urine samples was extracted manually using the commercially available kit. It was detected by performing a real-time Polymerase Chain Reaction assay using a forward primer (5’-CATGAAAACTCGTTCCGAAATAGAA-3’) and a reverse primer (5’-TCAGAGCTTTACCTAACAACGCATA-3’) of sequence mentioned above for amplification of target sequences, of the Chlamydia trachomatis.
 Results: Out of sixty cases included in this study, Chlamydia trachomatis DNA was detected in 12 cases (20%), and 48 cases (80%) were negative. So, the frequency of Chlamydia trachomatis in our study population was estimated at 20%. In addition, age, socioeconomic status, education and no of sexual partners were all risk factors were evaluated for their role in acquiring infection.
 Conclusion: Females having Chlamydia trachomatis infection and other sexually transmitted infections are at an increased risk of developing Pelvic Inflammatory disease due to the presence of risk factors and the asymptomatic nature of the disease.

The Selective Function of Quantum Biological Electron Transfer between DNA Bases and Metal Ions in DNA Replication.

Metal ions play vital roles in the electron transfer between biological molecules in humans, animals, and plants. However, the electron transfer between metal ions and nucleic acids and its impact on DNA-ion binding during DNA replication has been ignored. Here, we present a long-range quantum biological electron transfer (QBET) between DNA bases and metal ions and its selective function of DNA-ion binding in DNA replication. We discover biophysical DNA-ion binding and create biological filters that allow selective DNA replication by dual modulators of the valence and concentration of metal ions. QBET-based DNA replication filters provide powerful tools for ultrasensitive polymerase chain reaction (PCR) to selectively amplify target sequences with a discrete concentration window of metal ions; for example, Au3+ exhibits a concentration window that is approximately 3 orders of magnitude lower than that of Na+. DNA-ion filters provide new perspectives into metal ion-mediated QBET in DNA replication and hold great potential in life sciences and medical applications.

DNA Metabarcoding Across Disciplines: Sequencing Our Way to Greater Understanding Across Scales of Biological Organization.

DNA metabarcoding describes the use of targeted DNA (i.e., amplicon) sequencing to identify community constituents from a complex sample containing genetic material from multiple organisms, such as water, soil, gut contents, microbiomes, or biofilms. This molecular approach for characterizing mixed DNA samples relies on the development of "universal primers" that allow for effective amplification of target sequences across a broad range of taxa. Armed with optimized lab protocols and rigorous bioinformatics tools, DNA metabarcoding can produce a wealth of information about the hidden biodiversity of various sample types by probing for organisms' molecular footprints. DNA metabarcoding has received considerable popular press over the last few years because of gut microbiome studies in humans and beyond. However, there are many other applications that are continually integrating molecular biology with other fields of study to address questions that have previously been unanswerable, for both prokaryotic and eukaryotic targets. For example, we can now sample mostly digested gut contents from virtually any organism to learn about ontogeny and foraging ecology. Water samples collected from different locations can be filtered to extract eDNA (i.e., environmental DNA), revealing the biodiversity of fish and other taxa targeted by carefully selected primer sets. This universal primer metabarcoding approach has even been extended to looking at diverse gene families within single species, which is particularly useful for complex immune system genetics. The purpose of this SICB symposium was to bring together researchers using DNA metabarcoding approaches to (a) showcase the diversity of applications of this technique for addressing questions spanning ecology, evolution, and physiology, and (b) to spark connections among investigators from different fields that are utilizing similar approaches to facilitate optimization and standardization of metabarcoding methods and analyses. The resulting manuscripts from this symposium represent a great diversity of metabarcoding applications and taxonomic groups of interest.

Identification of Circulating Tumor Cell Phenotype in Differentiated Thyroid Carcinoma

Objective: Circulating tumor cells (CTCs) have been considered as the origin of tumor metastasis and recurrence, which always indicate a poor prognosis. There are three phenotypes of CTCs according on different epithelial-to-mesenchymal transition (EMT) markers, including epithelial, mesenchymal, and epithelial/mesenchymal (mixed phenotypic) CTCs. We intended to explore the relationship among CTC phenotypes and the clinicopathological characteristics of patients with differentiated thyroid carcinoma (DTC). Methods: Peripheral blood samples from 58 patients with DTC were collected, and CTCs were isolated by cell sizes. To identify phenotypes of CTCs, branched DNA signal amplification technology was adopted to capture and amplify target sequences, and then multiplex RNA-in situ hybridization (RNA-ISH) assay was used to identify CTC phenotypes depended on epithelial-mesenchymal transition (EMT) markers. Results: The positive rate of CTCs was 77.59% in 58 DTC patients. Totally, 488 CTCs with detective phenotype were found. Among them, there were 121 (24.80%) epithelial CTCs, 67 (13.72%) mesenchymal CTCs, and 300 (61.48%) mixed phenotypic CTCs. An obvious increased epithelial CTCs was observed in male patients compared with female. Notably, CTCs were more prevailing in younger male patients with ETI and bilateral focus. Conclusions: The CTCs are common in DTC patients, and mixed phenotypic is the major phenotype, indicating that EMT is prevalent in DTC even though its prognosis was better than other epithelial tumors. Detection of CTC and its phenotypes might independently predict the prognosis of DTC.

Oligoribonucleotide interference-PCR: principles and applications.

Polymerase chain reaction (PCR) amplification of multiple templates using common primers is used widely for molecular biological research and clinical diagnosis. However, amplifying a specific DNA sequence harboring a mutation that is present in a small number of mutant cells within a large population of normal cells (e.g., as in cancer) in a tissue is difficult using the original PCR protocol. Thus, some measures are necessary to suppress amplification of background signals. To achieve this, we developed the oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) technology in which an ORN (short RNA) hybridizes with a complementary DNA sequence to inhibit PCR amplification across the specific target sequence. ORNs can be prepared inexpensively, and ORNi-PCR can be carried out easily by adding ORNs to the PCR reaction mixture. Suppressing amplification of target sequences by ORNi-PCR is useful for detecting target sequence mutations. We showed that ORNi-PCR can discriminate single-nucleotide mutations in cancer cells and indel mutations introduced by genome editing. We also showed that ORNi-PCR can identify the CpG methylation status of a target sequence within bisulfite-treated DNA, and can enrich DNA sequences of interest from a DNA mixture by suppressing amplification of unwanted sequences. Thus, ORNi-PCR has many potential applications in various fields, including medical diagnosis and molecular biology. In this review, we outline the principles of the ORNi-PCR method and its use to detect nucleotide mutations in a variety of specimens.

Simple and rapid detection of common fetal aneuploidies using peptide nucleic acid probe-based real-time polymerase chain reaction

To examine the detection performance of a peptide nucleic acid (PNA) probe-based real-time time polymerase chain reaction (PCR) assay to detect common aneuploidies. Using amniotic fluid samples, PNA probe based real-time PCR (Patio DEP Detection Kit; SeaSun Biomaterials, Korea) assay was performed. PNA probe was designed to hybridize to similar sequences located on different segments of target chromosomes (21, 18, and 13) and a reference chromosome. Amplification of target sequences and melting curve analysis was performed. When analyzing the melting curve, the ratio of the peak height of the target and reference chromosome was calculated and determined as aneuploidy if the ratio of peak height was abnormal. All the results from the PNA probe-based real-time PCR and melting curve analyses were compared to those from conventional karyotyping. Forty-two cases with common aneuploidies (24 of trisomy 21, 12 of trisomy 18, and 6 of trisomy 13) and 131 cases with normal karyotype were analyzed. When comparing the karyotyping results, the sensitivity and specificity of the PNA probe-based real-time PCR assay were both 100%. The level of agreement was almost perfect (k = 1.00). PNA real-time PCR assay is a rapid and easy method for detecting common aneuploidies.

Development of a Panel of Genotyping-in-Thousands by Sequencing in Capsicum.

Genotyping by sequencing (GBS) enables genotyping of multiple loci at low cost. However, the single nucleotide polymorphisms (SNPs) revealed by GBS tend to be randomly distributed between individuals, limiting their direct comparisons without applying the various filter options to obtain a comparable dataset of SNPs. Here, we developed a panel of a multiplex targeted sequencing method, genotyping-in-thousands by sequencing (GT-seq), to genotype SNPs in Capsicum spp. Previously developed Fluidigm® SNP markers were converted to GT-seq markers and combined with new GT-seq markers developed using SNP information obtained through GBS. We then optimized multiplex PCR conditions: we obtained the highest genotyping rate when the first PCR consisted of 25 cycles. In addition, we determined that 101 primer pairs performed best when amplifying target sequences of 79 bp. We minimized interference of multiplex PCR by primer dimer formation using the PrimerPooler program. Using our GT-seq pipeline on Illumina Miseq and Nextseq platforms, we genotyped up to 1,500 (Miseq) and 1,300 (Nextseq) samples for the optimum panel size of 100 loci. To allow the genotyping of Capsicum species, we designed 332 informative GT-seq markers from Fluidigm SNP markers and GBS-derived SNPs. This study illustrates the first application of GT-seq in crop plants. The GT-seq marker set developed here will be a useful tool for molecular breeding of peppers in the future.

Single nucleotide polymorphisms and copy-number variations in the Trypanosoma brucei repeat (TBR) sequence can be used to enhance amplification and genotyping of Trypanozoon strains.

The Trypanosoma brucei repeat (TBR) is a tandem repeat sequence present on the Trypanozoon minichromosomes. Here, we report that the TBR sequence is not as homogenous as previously believed. BLAST analysis of the available T. brucei genomes reveals various TBR sequences of 177 bp and 176 bp in length, which can be sorted into two TBR groups based on a few key single nucleotide polymorphisms. Conventional and quantitative PCR with primers matched to consensus sequences that target either TBR group show substantial copy-number variations in the TBR repertoire within a collection of 77 Trypanozoon strains. We developed the qTBR, a novel PCR consisting of three primers and two probes, to simultaneously amplify target sequences from each of the two TBR groups into one single qPCR reaction. This dual probe setup offers increased analytical sensitivity for the molecular detection of all Trypanozoon taxa, in particular for T.b. gambiense and T. evansi, when compared to existing TBR PCRs. By combining the qTBR with 18S rDNA amplification as an internal standard, the relative copy-number of each TBR target sequence can be calculated and plotted, allowing for further classification of strains into TBR genotypes associated with East, West or Central Africa. Thus, the qTBR takes advantage of the single-nucleotide polymorphisms and copy number variations in the TBR sequences to enhance amplification and genotyping of all Trypanozoon strains, making it a promising tool for prevalence studies of African trypanosomiasis in both humans and animals.

Pathogen surveillance in Southern Ocean pinnipeds

Knowledge of the health status and potential effect of disease outbreaks among Southern Ocean fauna may be decisive for its conservation. We assessed the exposure and infection of Antarctic fur seals (Arctocephalus gazella, AFS) and Southern elephant seals (Mirounga leonine, SES) to parapoxvirus, Phocid alphaherpesvirus-1 (PhHV-1), smooth Brucella spp. and Toxoplasma gondii. AFS (n = 65) serum and swab samples, and SES (n = 13) serum samples from the sub--Antarctic island of Bouvetøya (54°25’S, 03°22’E) were collected during two austral summers (2014/15, 2017/18). Three polymerase chain reaction (PCR) tests amplifying the DNA polymerase, B2L and GIF parapoxvirus genomic regions were performed, investigating DNA from mucosal swab samples. The glycoprotein B gene was targeted to detect PhHV-1 viral DNA. Sera were assayed for T. gondii and smooth Brucella spp. antibodies with indirect enzyme-linked immunosorbent assays. Parapoxvirus PCR amplicons of the expected size were generated in two of the 29 AFS pups (nasal swabs, 2014/15), targeting the B2L (n = 2) and DNA polymerase (n = 1) genes, whereas the GIF PCR did not amplify target sequences. The PCR amplicons were sequenced and blasted in GenBank, revealing highest similarity with a seal parapoxvirus, confirming the presence of the virus in AFS for the first time. No PhHV-1 amplicons were generated, and antibodies against T. gondii or smooth Brucella spp. were not detected. Our data indicate that these seals are host for parapoxvirus but are neither exposed to smooth Brucella spp. nor T. gondii. Evidence of PhHV-1 shedding was not detected.

Developing a Low-Cost, Simple-to-Use Electrochemical Sensor for the Detection of Circulating Tumour DNA in Human Fluids.

It is well-known that two major issues, preventing improved outcomes from cancer are late diagnosis and the evolution of drug resistance during chemotherapy, therefore technologies that address these issues can have a transformative effect on healthcare workflows. In this work we present a simple, low-cost DNA biosensor that was developed specifically to detect mutations in a key oncogene (KRAS). The sensor employed was a screen-printed array of carbon electrodes, used to perform parallel measurements of DNA hybridisation. A DNA amplification reaction was developed with primers for mutant and wild type KRAS sequences which amplified target sequences from representative clinical samples to detectable levels in as few as twenty cycles. High levels of sensitivity were demonstrated alongside a clear exemplar of assay specificity by showing the mutant KRAS sequence was detectable against a significant background of wild type DNA following amplification and hybridisation on the sensor surface. The time to result was found to be 3.5 h with considerable potential for optimisation through assay integration. This quick and versatile biosensor has the potential to be deployed in a low-cost, point-of-care test where patients can be screened either for early diagnosis purposes or monitoring of response to therapy.

Detection of Copy Number of Mitochondrial DNA in Iraq Population

Mitochondrial DNA copy number (mtDNA-CN), a measure of the number of mitochondrial genomes percell. A cell contains two copies of genomic DNA compared to a single copy of DNA per chromosome. Thequantitative real-time PCR (qPCR) is the gold standard method for measuring mtDNA-CN, the quantificationafter DNA extraction it is an important step, the quantitation of DNA plays a central role inmedical andforensic DNA analysis, which provides the information about the amount of DNA present in unknownsamples. In this study the quantitative results showed that the higher value 74941 and the lower value 190.A multiplex quantitative PCR assay developed to amplify target sequences of different length, which allowsfor the assessment of DNA degradation in samples of forensic interest. The quantification of DNA by qPCRrelies on the detection of amplified product (amplicon) at each cycle of the PCR.

Degenerate PCR primers for assays to track steps of nitrogen metabolism by taxonomically diverse microorganisms in a variety of environments

Steps in the global nitrogen cycle are mainly catalyzed by microorganisms. Accordingly, the activities of these microorganisms affect the health and productivity of ecosystems. Their activities are also used in wastewater treatment systems to remove reactive nitrogen compounds and prevent eutrophication events triggered by nutrient discharges. Therefore, tracking the activities of these microorganisms can provide insights into the functioning of these systems. The presence and abundance of genes encoding nitrogen-metabolizing enzymes can be traced via polymerase chain reaction (PCR); however, this requires primers that are sensitive to a heterogenous gene pool yet specific enough to the target biomarker. The ever-expanding diversity of sequences available from databases includes many sequences relevant to nitrogen metabolism that match poorly with primers previously designed to track their presence and/or abundance. This includes genes encoding ammonia monooxygenase (AMO) of ammonia oxidizing microorganisms, nitrite oxidoreductase (NXR) of nitrite oxidizing bacteria, and nitrous oxide reductase (NOS) of denitrifying bacteria. Some primers are also not designed to generate the short (~200 nucleotides) amplicons required for real-time quantitative PCR (qPCR) and reverse-transcriptase qPCR (qRT-PCR). In this study, genes collected from the Integrated Microbial Genomes database (IMG) were aligned to design PCR primers that could capture more sequence diversity than is possible using existing primers. Primers were designed to target three clades of AMO (Betaproteobacteria, Chrenarchaeota, and complete ammonia oxidizing Nitrospira), periplasmic NXR and two clades of NOS (Proteobacteria and Bacteroidetes/Firmicutes). These primers successfully amplified target sequences from two wastewater treatment plants with biological nitrogen removal (one with simultaneous nitrification/denitrification and one with distinct anoxic/oxic zones) and estuary sediment. Nucleotide sequences of the amplicons retrieved homologs when used to query GenBank by BLAST. While convincingly identified as target sequences for these primer pairs, these amplicons were divergent from each other, and quite divergent (as low as 73%) from those present in GenBank, suggesting these primers are capable of capturing a diverse range of sequences. A direct comparison showed that primers designed here are better suited to environmental samples, such as wastewater treatment facilities, by producing a greater number of amplicons from the same sample than primers currently established in literature.