Amplification Of DNA Sequences Research Articles

Phylogenetic Biodiversity of Bacteria Community in the Gut of Diarrhoeic Patients in Rivers State, Nigeria

Background: The microbial ecosystem in the human intestine is complex and it plays a great role in health and nutrition. Cultural techniques have been used over the years to study the gut microbiota but studies suggest that a greater percentage of these bacteria found in the gut cannot be cultivated using the conventional methods of bacteria isolation.
 Aim: To increase understanding in this area, we characterized the bacterial diversity (both cultivated and non-cultivated bacteria) in the gut of diarrhoeic individuals using 16S rRNA gene (rDNA) sequences.
 Methodology: PCR amplification, sequencing and phylogenetic analysis of the 16S ribosomal DNA (rDNA) sequences were done on 10 diarrhoeic stool samples.
 Results: After quality filtering and chimeric sequence removal, 72313 sequences from all 10 diarrhoeic stool samples subjected to clustering generated 2767 Operational Taxonomic Units (OTUs) of which 2073 were new and unassigned.
 Representative sequences of the bacteria OTUs cluster were used to construct a bacteria phylogenetic tree which revealed a wide variety of bacteria Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Tenericutes and Cyanobacteria and others that could not be detected using the cultural techniques. The evolutionary relationship of the most abundant organisms and their contributions from each sample revealed the phylum Firmicutes to be most abundant and therefore have contributed most in the samples followed by Bacteroidetes. Fewer contributions were made by the other phyla Proteobacteria, Actinobacteria, Tenericutes and Cyanobacteria.
 Conclusion: This study was able to identify culturable and unculturable bacteria in the gut of diarrhoeic people in Rivers state and also show the biodiversity and interrelatedness of these microorganisms using molecular methods. Therefore, we can say that 16S rRNA techniques for detection and identification of predominant bacteria create new opportunities for non-cultivation studies of the human intestinal microflora, proper diagnosis of infectious diseases and new methods of treatments of diseases.

In-silico Specificity Comparison between GMF-GMR and JMF-JMR Primers for Detecting moaC Genes of Food Spoilage Bacteria Pseudomonas spp

Pseudomonas spp. have been known as notorious food spoilage bacteria with ability to produce thermo-tolerant enzymes. They pose serious risk to public health as its most pathogenic member, P. aeruginosa, could cause nosocomial infections affecting peoplewith immunodeficiency. The use of GMF-GMR primers had been reported capable for detecting bacterial moaC of Alcaligenes javaensis JG3. The gene is suspected to be related with dormancy of pathogenic bacteria. This study aimed to investigate specificity of the GMR-GMF as well as a newly designed JMF-JMR pairs of primers (JMF: 5’- GGCGTACATCATCCACACTG-3’ and JMR: 5’-GGCGTTGACCATCTATGACA-3’) for detecting moaC genes of 57 members of Pseudomonas spp. retrieved from http://insilico.ehu.eus/ database using in silico PCR (Polymerase Chain Reaction). The results showed that GMF-GMR primers could selectively amplify 271-bp in silico PCR products from 14 out of 57 members of Pseudomonas spp. tested. However, BLASTn analysis on these 14 amplified DNA sequences showed that they were not part of moaC, yet glpK gene fragment sequences. Meanwhile, the newly designed primers from moaC sequence of strain JG3, JMFJMR, could specifically amplify 214-bp in silico PCR products from 2 out of 57 members of Pseudomonas spp. matched to bacterial moaC gene fragment sequences. As conclusion, based on in silico study JMF-JMR primers are more specific than GMF-GMR ones for detecting moaC gene fragments of members of Pseudomonas spp. studied.

PO 8441 EXPERIMENTAL COMPARISON OF SENSITIVITY OF LAMP AND REAL-TIME PCR

BackgroundHuman African trypanosomiasis, or sleeping sickness, remains a serious problem in tropical Africa. Timely diagnosis of this disease requires systematic population screening, particularly for Trypanosoma brucei gambiense, which has a long asymptomatic period.The lack of sensitivity and specificity of conventional diagnostic tests has led in recent years to the use of molecular tools. Amplification of parasite-specific DNA sequences significantly improved diagnosis of infection. However, these molecular tools still have some limitations especially in the case of low parasitaemia. Furthermore, research is still needed to make molecular detection a real control tool for the fight against sleeping sickness. The purpose of this study is to determine the threshold of sensitivity of real-time PCR using the 18S and TgsGp primers and of the LAMP technique, applied in the DiTECT-HAT project as molecular reference tests.MethodsWe used serial dilutions containing 0, 1, 10, 100, 103, 104, 105, 106 parasites per ml of blood. Samples were extracted, and DNA was amplified.ResultsThe analytical sensitivity of the 18S real-time PCR with the Taqman probe of the filter paper samples is 100 parasites/ml and that of the TgsGp real-time PCR with the Taqman probe of filter paper samples is 104 parasites/ml. For Lamp technique, the analytical sensitivity is 103 parasites/ml.ConclusionThis study shows that a ‘negative PCR’ would not mean ‘no parasite’. It suggests that DNA detection techniques should still be improved.

Identification of cassava bacterial blight-causing Xanthomonas axonopodis pv. Manihotis based on rpoD and gyrB genes

Cassava (Manihot esculenta Crantz) is one of the most important crops in Vietnam for providing food, starch sources, and raw materials for the production of bio-ethanol and other purposes. Cassava bacterial blight (CBB)disease, caused by Xanthonomas axonopodis pv. manihotis (Xam), is one of the most important factors affecting cassava production in Vietnam. A rapid and sensitive molecular tool is required to support the traditional method, primarily based on biochemical reactions, that was considered less sensitive and time-consuming. In the present study, in order to detect Xam, the Polymerase Chain Reaction (PCR) technique was applied with the two primerpairs, rpoD_17F/rpoD_1005R and XgyrconpcrF1/Xgyrconrpcr1, to amplify partial sequences of rpoD and gyrBgenes, respectively. The primers directed the amplification of about 900 bp DNA fragments from CBB-infected leaf and stem tissues. All the amplified DNA sequences were identical in each gene and to the Xam reference strains (accession numbers KP265376 and KP265378 for the rpoD gene, and KP265387 and KP265388 for the gyrB gene) from GenBank. The representative sequences of rpoD and gyrB genes were deposited in GenBank with accession numbers MF774491 and MF774490, respectively. DNA and phylogenetic analysis based on the sequences of rpoD and gyrB genes have confirmed that Xam is the causal agent of CBB in Dong Nai, Vietnam. This is the first confirmed identification of the causal pathogen of CBBdisease in Vietnam using molecular tools, and this method is a reliable tool for the detection and identification of other plant bacterial pathogens.

Tissue variations of mosaic genome-wide paternal uniparental disomy and phenotype of multi-syndromal congenital hyperinsulinism

Mosaic genome-wide paternal uniparental disomy (GW-pUPD) is a rarely recognised disorder. The phenotypic manifestations of multilocus imprinting defects (MLIDs) remain unclear. We report of an apparently non-syndromic infant with severe congenital hyperinsulinism (CHI) and diffuse pancreatic labelling by 18F*-DOPA-PET/CT leading to near-total pancreatectomy. The histology was atypical with pronounced proliferation of endocrine cells comprising >70% of the pancreatic tissue and a small pancreatoblastoma. Routine genetic analysis for CHI was normal in the blood and resected pancreatic tissue. At two years’ age, Beckwith-Wiedemann Syndrome (BWS) stigmata emerged, and at five years a liver tumour with focal nodular hyperplasia and an adrenal tumour were resected. pUPD was detected in 11p15 and next in the entire chromosome 11 with microsatellite markers. Quantitative fluorescent PCR with amplification of chromosome-specific DNA sequences for chromosomes 13, 18, 21 and X indicated GW-pUPD. A next generation sequencing panel with 303 SNPs on 21 chromosomes showed pUPD in both blood and pancreatic tissue. The mosaic distribution of GW-pUPD ranged from 31 to 35% in blood and buccal swap to 74% in the resected pancreas, 80% in a non-tumour liver biopsy, and 100% in the liver focal nodular hyperplasia and adrenal tumour. MLID features included transient conjugated hyperbilirubinaemia and lack of macrosomia from BWS (pUPD6); and behavioural and psychomotor manifestations of Angelman Syndrome (pUPD15) on follow-up. In conclusion, atypical pancreatic histology in apparently non-syndromic severe CHI patients may be the first clue to BWS and multi-syndromal CHI from GW-pUPD. Variations in the degree of mosaicism between tissues explained the phenotype.

Inverse Polymerase Chain Reaction (PCR).

The standard polymerase chain reaction (PCR) is used to amplify a segment of DNA that lies between two inward-pointing primers. In contrast, inverse PCR (also known as inverted or inside-out PCR) is used to amplify DNA sequences that flank one end of a known DNA sequence and for which no primers are available. Inverse PCR DNA involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region. The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in PCR. The unknown sequence is amplified by two primers that bind specifically to the known sequence and point in opposite directions. The product of the amplification reaction is a linear DNA fragment containing a single site for the restriction enzyme originally used to digest the DNA. This site marks the junction between the previously cloned sequence and the flanking sequences. The size of the amplified fragment depends on the distribution of restriction sites within known and flanking DNA sequences.

Evolution of the S-Genomes in Triticum-Aegilops Alliance: Evidences From Chromosome Analysis.

Five diploid Aegilops species of the Sitopsis section: Ae. speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, and Ae. bicornis, two tetraploid species Ae. peregrina (= Ae. variabilis) and Ae. kotschyi (Aegilops section) and hexaploid Ae. vavilovii (Vertebrata section) carry the S-genomes. The B- and G-genomes of polyploid wheat are also the derivatives of the S-genome. Evolution of the S-genome species was studied using Giemsa C-banding and fluorescence in situ hybridization (FISH) with DNA probes representing 5S (pTa794) and 18S-5.8S-26S (pTa71) rDNAs as well as nine tandem repeats: pSc119.2, pAesp_SAT86, Spelt-1, Spelt-52, pAs1, pTa-535, and pTa-s53. To correlate the C-banding and FISH patterns we used the microsatellites (CTT)10 and (GTT)9, which are major components of the C-banding positive heterochromatin in wheat. According to the results obtained, diploid species split into two groups corresponding to Emarginata and Truncata sub-sections, which differ in the C-banding patterns, distribution of rDNA and other repeats. The B- and G-genomes of polyploid wheat are most closely related to the S-genome of Ae. speltoides. The genomes of allopolyploid wheat have been evolved as a result of different species-specific chromosome translocations, sequence amplification, elimination and re-patterning of repetitive DNA sequences. These events occurred independently in different wheat species and in Ae. speltoides. The 5S rDNA locus of chromosome 1S was probably lost in ancient Ae. speltoides prior to formation of Timopheevii wheat, but after the emergence of ancient emmer. Evolution of Emarginata species was associated with an increase of C-banding and (CTT)10-positive heterochromatin, amplification of Spelt-52, re-pattering of the pAesp_SAT86, and a gradual decrease in the amount of the D-genome-specific repeats pAs1, pTa-535, and pTa-s53. The emergence of Ae. peregrina and Ae. kotschyi did not lead to significant changes of the S*-genomes. However, partial elimination of 45S rDNA repeats from 5S* and 6S* chromosomes and alterations of C-banding and FISH-patterns have been detected. Similarity of the Sv-genome of Ae. vavilovii with the Ss genome of diploid Ae. searsii confirmed the origin of this hexaploid. A model of the S-genome evolution is suggested.

A copy number variation generated by complicated organization of PCDHA gene cluster is associated with egg performance traits in Xinhua E-strain.

In recent years, a mass of duplicated and deleted DNA sequences have been found in human and animal genomes following the prevalence of employing high-throughput sequencing and SNP array. However, few copy number variation (CNV) studies have been performed on egg performance traits of chicken. In this study, 17 loci reported in previous studies were selected for CNV detection in the Xinhua E-strain by using the CNVplex kit, and the detection results showed that locus14 exhibited CNV. Further association analysis indicated the copies of locus14 could be significantly associated with age at first egg (AFE; P<0.0086) and egg number at 250 d (250EN; P<0.036). DNA sequence amplification showed the loss of a 260-bp-long fragment in the upstream of locus14, which mainly occurred in normal or copy-gain individuals. The qPCR results showed that subjects with gain of copies could promote the total expression level of the PCDHA gene cluster in the pituitary gland of adult individuals. Additionally, PCR amplification with randomly combined primers revealed a larger number of chicken variable exons than that previously reported, indicating the complexity of the organization of the PCDHA gene cluster. Those variable exons are divergent in their distribution among the populations of Xinhua E-strain, Chahua, Tibetan, and Tulufan Game Chicken, and most individuals only possess part of variable exons. Overall, the copies of locus14 reflect the variable exon dosage effects on the total expression level of the PCDHA gene cluster, which may regulate the layer egg production by affecting the development of the neural system.

Study of the xenobiotics detoxication system in patients with stone kidney disease

The aim of the study was to evaluate the distribution of the variant alleles and genotypes of the polymorphic loci of the xenobiotics biotransformation system gene NAT2 in patients with urolithiasis (SKD) and to determine the nature of their polymorphism in the healthy population. 60 patients aged 20–65, with SKD who were treated in the urological department of the Ivano-Frankivsk central city hospital in 2016–2018 were examined. The control group comprised healthy individuals of the same age, selected by random sampling, living in different regions of Ivano-Frankivsk oblast (157 people). The material for the study was a DNA isolated from leukocytes in the peripheral blood of patients. In subsequent phases of the study, amplification of DNA sequences in vitro was performed using the polymerase chain reaction method. The analysis of the frequency of NAT2 genotypes combinations in patients with SKD showed that homozygous mutant deletion of 481CC was more common in patients than in healthy subjects. Comparison of allelic frequencies and 481TC genotypes in patients revealed that the heteronucleases were 77.8% versus 41.0% in healthy subjects, with a significant difference (PN <0.05). Under such conditions, it can be assumed that the presence of a homozygous mutant deletion of the CC locus of the T481C and the GG of the A803G locus of the NAT2 gene, with high probability, suggests an adverse, recurrent CKD. The heterozygous version of the TC of the locus T481C determines partial control of the ailment. The heterozygous variant of the AG locus A803G determines the increased susceptibility to the disease, without clearly affecting its phenotypic variant. In this case, the homozygous variant of the AA allele of the A803G locus, the homozygous TT, and the heterozygous TC deletion of the T481C allele are protectors for the development of SKD.

Detection and prevalence of adenoviruses from free-ranging black howler monkeys (Alouatta pigra).

Adenoviruses are important pathogens known to infect vertebrate hosts, including a wide range of primates. Despite its importance, data on the diversity of these viruses in non-human primates living in their natural habitat remain scarce. In this study, we conducted a surveillance of adenoviral infection in wild black howler monkeys from two protected natural areas in Mexico. This was achieved by analyzing 67 fecal samples using a nested PCR that targets the adenovirus DNA polymerase gene. Adenoviral DNA was detected in 12 samples from both study sites, with an overall prevalence of 17.9%. The amplified DNA sequences shared 100% nucleotide identity and phylogenetic analyses revealed that the haplotype detected was novel, and clustered with Platyrrhini mastadenovirus A, which was previously described in captive New World monkeys. Our data, along with the previous evidence, confirm that monkeys native to the Americas are the original hosts of these adenoviruses.

Lateral flow test for meat authentication with visual detection

A new lateral flow assay has been developed for meat authentication. The assay is based on gold nanoparticles and enables visual detection, by naked eye, of meat species-specific DNA sequences. The procedure includes DNA isolation from fresh meat samples, amplification of specific DNA sequences and detection by the lateral flow assay. The proposed assay is rapid and has been applied for the identification of four animal species: horse, pork, beef and sheep. The detection is completed within 25–30 min after amplification. The assay offers high detectability and good selectivity and reproducibility. As low as 0.01% of horse and 0.02% of pork DNA were detectable in binary mixtures by the reported lateral flow device.

Analysis of selected medicinal plants used in the treatment of malaria and typhoid fever in Ebonyi State, Nigeria

The study was aimed at evaluating selected medicinal plants used in the treatment of malaria and typhoid fever. Materials used include medicinal plants used locally in treating malaria and typhoid fever sourced from different villages in Ebonyi State. The analysis of the medicinal plants was conducted using random amplified polymorphic DNA (RAPD) markers and NTSYSpc software version 2.02. Different RAPD markers including OPB-1, OPB-2, OPB-3, OPB-5, OPB-12 and OPH-12 were used to amplify the DNA of these plants. These markers were found to be polymorphic except OPB-3 which did not produce any band. It was observed that RAPD markers can effectively amplify DNA sequences of different medicinal plants. The data matrix of RAPD profiles obtained from fragments of each amplicon were scored as 1 (presence of alleles) or 0 (absence of alleles). A dendrogram of the plants using unweighted pair group mean (UPGMA) clustered the genotypes into groups. The dissimilarity values were 0.26 and 1 as minimum and maximum with an edge length of 1.32. Principal component analysis of the generated amplicons resulted to clusters with unique genetic identity. The polymorphism detected among the plants genotypes will be useful in selecting genetically diverse species in future breeding programme. Keywords: Medicinal plants, Malaria/Typhoid, RAPD, Ebonyi State, Nigeria

Assessment of sampling and DNA extraction methods for identification of grapevine trunk microorganisms using metabarcoding

For a deeper understanding of grapevine trunk disease (GTD) in New Zealand, a cheap, rapid, sensitive method for identifying within-vine microbial communities is required. Wood tissue from grapevine trunks was collected and three different DNA extraction methods were compared: a cetyltrimethylammonium bromide (CTAB) method, the Geneaid Plant Genomic DNA Mini Kit and the Qiagen DNeasy Plant Mini Kit. DNA samples from the CTAB and Geneaid methods were used for MiSeq DNA metabarcoding targeting the ribosomal internal transcribed spacer 1 (ITS1) region. DNA produced by the CTAB method was of a greater quantity and quality than for the other two methods, although the majority of the DNA samples provided polymerase chain reaction (PCR) amplification of fungal DNA sequences. Fungal metabarcoding profiles from the CTAB and Geneaid samples indicated the presence of fungi normally associated with GTD in New Zealand. The CTAB method was chosen for subsequent work due to its low-cost, simplicity and effective detection of typical GTD fungi. The complete process of sampling through to metabarcoding is now used annually as part of a wider ecological study, screening more than 600 vines at 12 Marlborough vineyards.

Prognostic stratification of adult primary glioblastoma multiforme patients based on their tumor gene amplification profiles.

Several classification systems have been proposed to address genomic heterogeneity of glioblastoma multiforme, but they either showed limited prognostic value and/or are difficult to implement in routine diagnostics. Here we propose a prognostic stratification model for these primary tumors based on tumor gene amplification profiles, that might be easily implemented in routine diagnostics, and potentially improve the patients management. Gene amplification profiles were prospectively evaluated in 80 primary glioblastoma multiforme tumors using single-nucleotide polymorphism arrays and the results obtained validated in publicly available data from 267/347 cases. Gene amplification was detected in 45% of patients, and chromosome 7p11.2 including the EGFR gene, was the most frequently amplified chromosomal region – either alone (18%) or in combination with amplification of DNA sequences in other chromosomal regions (10% of cases). Other frequently amplified DNA sequences included regions in chromosomes 12q(10%), 4q12(7%) and 1q32.1(4%). Based on their gene amplification profiles, glioblastomas were subdivided into: i) tumors with no gene amplification (55%); ii) tumors with chromosome 7p/EGFR gene amplification (with or without amplification of other chromosomal regions) (38%); and iii) glioblastoma multiforme with a single (11%) or multiple (6%) amplified DNA sequences in chromosomal regions other than chromosome 7p. From the prognostic point of view, these amplification profiles showed a significant impact on overall survival of glioblastoma multiforme patients (p>0.001). Based on these gene amplification profiles, a risk-stratification scoring system was built for prognostic stratification of glioblastoma which might be easily implemented in routine diagnostics, and potentially contribute to improved patient management.

Polymorphisms in protamine 1 and 2 genes in asthenozoospermic men: A case-control study

Asthenozoospermia is one of the etiologies for male factor infertility. It was shown that any abnormality in protamines genes, reduction of protamines transcript and protamines deficiency may play a key role in asthenozoospermia. The aim of the current study was the evaluation of protamine-1 and 2 genes (PRM1 and PRM2) polymorphisms in asthenozoospermic men. In this case-control study, the samples were corresponded to asthenozoospermic specimens of infertile men. The normozoospermic samples were considered as the control group. DNA sequence amplification was performed using four PRM1 and PRM2 primers, designed from 5' to 3' flank regions. The human PRM1 and PRM2 gene sequences were screened in search of potential mutations in highly prevalent polymorphism regions in asthenozoospermia versus normozoospermia. Totally, nine highly prevalent polymorphism regions between the forward and reverse primers were screened. Three of them corresponded to PRM1 and six to PRM2. The most prevalent polymorphism regions in PRM1 were related to 102G>T (rs35576928), 49C>T (rs140477029) and 139C>A (rs737008). In the PRM2, 6 highly prevalent polymorphisms regions were screened, including 248C>T (rs779337774), 401G>A (rs545828790), 288C>T (rs115686767), 288G>C (rs201933708), 373C>A (rs2070923), and 298G>C (rs1646022). The allele frequencies of three upper mentioned single nucleotide polymorphisms in asthenozoospermic men including 373C>A, 298G>C and 139C>A was higher than the control group. Our findings indicated that the frequency of some altered genotypes in asthenozospermia was slightly higher than control group. We proposed more extensive studies to be sure that; these genotypes can precisely be related to diagnosis of asthenozoospermia, as the molecular markers.

Bovine foetal sex determination-Different DNA extraction and amplification approaches for efficient livestock production.

Foetal sex determination using polymerase chain reaction (PCR) in mammals is based on the amplification of gender-specific foetal DNA sequences circulating in maternal blood. The bovine synepitheliochorial placenta does not allow a direct contact between the trophoblast and the maternal blood, resulting in difficult passage of foetal DNA and, consequently, its very small amounts in maternal bloodstream. Circulating cell-free foetal DNA (ccffDNA) encompasses short nucleotide fragments (300-600bp) in maternal circulation. The aim of this study was to assess this non-invasive method in accurate prenatal sexing in early and late gestational periods in comparison with ultrasound diagnostics. As various DNA isolation and amplification methods were tested, their success in obtaining reliable results was evaluated. Two groups were tested, each consisting of 20 pregnant cows. Blood of a bull and a non-pregnant heifer was the controls. Extraction of foetal DNA was accomplished by three different methods: using tubes with silicone membranes, a single-tube extraction without silicone membranes and phenol-chloroform extraction. Following each extraction method, foetal DNA was amplified using PCR and real-time PCR with both bAML and TSPY primers in a separate reaction. Positive results were obtained only after amplification of foetal DNA extracted with a single-tube extraction kit. In comparison with ultrasound examination results and foetal gender recorded at birth, the sensitivity of the PCR test was 90% in Group I, but the technique failed to detect male foetuses in Group II. The real-time PCR test sensitivity in Group I was 90% and in Group II 91.6%.

PCR amplification of DNA sequence related to the hrpD gene of Xanthomonas cucurbitae in leaf spot disease of pumpkin and their antagonism by soil bacteria

A sensitive and specific assay was developed to detect and biological control of bacterial leaf spot of pumpkin, Xanthomonas cucurbitae was identified on the basis of the morphological, biochemical and molecular assay. The antibiotic sensitivity of the isolate showed that, Carbenicillin revealed highest antibacterial activity with 29 ± 0.00 mm zone of inhibition against isolated bacterial strain. Isolated bacterial strains from soil were also identified by biochemical and molecular characterisation. By analysing morphological and biochemical characteristics and 16S rDNA of three bacterial strains isolated from soil was matched 96% with Bacillus subtilis, 98% with Bacillus brevis and 97% with Pseudomonas fluorescens strain. They were subjected to the antagonistic activity against Xanthomonas cucurbitae by disc diffusion method. Among them, B. subtilis showed significant positive antagonistic activity with 17.0 ± 0.28 mm zone of inhibition against Xanthomonas cucurbitae. The presence of DNA sequence related to the hrpD gene successfully amplified in some isolates of Xanthomonas cucurbitae.

Evaluation of Plasma Cell‐Free Circulating DNA Integrity in Patients with Prostate Cancer in Jamaica

Cell free circulating DNA (cfcDNA) is a promising diagnostic tool for prostate cancer (PCa). The study aimed to measure the cfcDNA concentration and cfcDNA integrity in prostate cancer patients using quantitative polymerase chain reaction (qPCR) analysis on plasma samples. The study also assessed correlation between cfcDNA concentration and integrity with baseline PSA, Gleason score, total prostate volume, age and the number of presenting comorbidities. Eleven patients with PCa and 9 patients with Benign Prostatic Hyperplasia (BPH) were enrolled. Plasma samples were collected before prostate biopsy and quantified by qPCR amplification of ALU115 DNA sequence, and the quantitative ratio of ALU 247 to ALU 115 reflected the integrity of cfcDNA. There were no significant differences in median (IQR) plasma cfcDNA concentration or its integrity between the patients with PCa [47.9 (214.93) ng/mL, 0.61 (0.49)] and patients with BPH [41.5 (55.13) ng/mL, 0.67 (0.45); p = 0.382, p = 0.342]. There was no correlation between cfcDNA and cfcDNA integrity with baseline PSA, Gleason score, total prostate volume, age and the number of presenting comorbidities.ConclusionThe small sample size of the current study did not allow for the differentiation of PCa from BPH using cfcDNA concentration and integrity. There was also no correlation with cfcDNA concentration and integrity with the selected clinical and pathological variables.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sensitive NPM1 Mutation Quantitation in Acute Myeloid Leukemia Using Ultradeep Next-Generation Sequencing in the Diagnostic Laboratory.

Context Detection of measurable residual disease after therapy is an important predictor of outcome in acute myeloid leukemia. Objective To investigate the feasibility of using next-generation sequencing (NGS) in the diagnostic laboratory to perform quantitative NPM1 mutation assessment using ultradeep (approximately 300 000×-500 000×) sequencing (NGS-q NPM1) as a method of assessing residual disease burden in patients with acute myeloid leukemia. Design A flexible NGS-based assay for the detection and quantitation of NPM1 mutations was developed by polymerase chain reaction amplification of target DNA sequences, sequencing on an Illumina (San Diego, California) MiSeq, and analyzing data with an in-house-designed bioinformatic pipeline. NGS-q NPM1 was compared with current NPM1 quantitation methods (real-time quantitative-polymerase chain reaction and multiparameter flow cytometry). Results The NGS-q NPM1 assay had a sensitivity of between 10-4 and 10-5 and showed high concordance and correlation with reference methodologies. Moreover, the NGS-q NPM1 assay was able to be integrated into the laboratory's existing, targeted amplicon-based sequencing workflow. Conclusions An NGS-based, quantitative NPM1-mutation assessment can be used to monitor patients with acute myeloid leukemia, and it has some practical advantages over existing modalities.

Helper-Dependent Chain Reaction (HDCR) for Selective Amplification of Methylated DNA Sequences.

Over the last few years a number of restriction enzymes that cut DNA only if cytosines within their recognition sequences are methylated have been characterized and become commercially available. Cleavage with these enzymes to release DNA fragments in a methylation-dependent manner can be combined with a novel method of amplification, Helper Dependent Chain Reaction (HDCR), to selectively amplify these fragments. HDCR uses "Helper" oligonucleotides as templates for extension of the free 3' end of target fragments to incorporate tag sequences at the ends of fragments. These tag sequences are then used for priming of amplification of target fragments. Modifications to the amplification primers (Drivers) and the Helpers ensure that there is selection for the sequences within target fragments with each cycle of amplification. The combination of methylation-dependent enzymes and HDCR allows the sensitive and selective amplification of methylated DNA sequences without the need for bisulfite treatment.