Quantification Of PCR Products Research Articles

Genomic Copy Number Analysis Using Droplet Digital PCR: A Simple Method with EvaGreen Single-Color Fluorescent Design.

Droplet digital PCR (ddPCR) is an emerging method for the absolute quantification of PCR products, and it can detect DNA copy numbers accurately. It analyzes the end-point absolute fluorescence signals of the PCR-positive droplets and calculates the target concentration. EvaGreen is a nonspecific double-stranded DNA-binding fluorescent dye, and the ddPCR system also supports assays using this cost-effective hydrolysis probe. Here, we describe a simple method of quantification for DNA copy numbers using the EvaGreen single-color fluorescent design.

Mechanism of Reproductive Effect of Acetyl acetate‐methanol Fraction of Adansonia digitata (Linn) Root Bark Extract in Female Wistar Rats

The aim of this work was to identify the exclusive components of Acetyl acetate‐methanol fraction (AAMF) of Adansonia digitata (Linn) root bark extract and to elucidate its mechanism of action in modulating hormone signaling. Gas chromatography and Mass spectrometry was done to identify the compounds present in fractions of A. digitata (Linn) root bark extract. Thirty female Wistar rats had their estrous cycles synchronized and were randomly distributed into three groups of ten rats each. Each group received 0, 150, and 300mg/kg body weight of AAMF per os from the day observed as proestrus by vaginal cytology to day‐seven. On day‐one, day‐four, day‐five, day‐six and day‐seven, blood was collected from rats in each group. Blood collected was analyzed for estrogen, progesterone and follicle stimulating hormone levels. Uterus and ovary were processed for histopathology and immunohistochemistry to study the regulation of estrogen and progesterone receptor proteins. Where a receptor protein was regulated, further investigation was done to determine its effect on the gene expression of that receptor in endometrial stromal cells through RNA extraction, OneStep reverse transcription‐pCR amplification, Agarose gel electrophoresis and quantification of pCR product. Results showed that AAMF uniquely contained 15.25% of oleic acid and caused mass atresia of antral follicles at proestrus. AAMF treated rats had significantly (p<0.05) lowered serum estradiol levels at proestrus when compared to control rats and had up‐regulated estrogen receptor beta proteins in endometrium and downregulated estrogen receptor alpha (ERα) gene expression in endometrial cells. It was concluded that the oleic acid component of AAMF may have lowered estrogen level by inhibiting aromatase and hydroxysteroid 17 beta dehydrogenase enzyme activities in estrogen steroidogenesis. Also, oleic acid may have induced overexpression of Tumour Suppressor Factor (P53) which promotes follicular apoptosis and indirectly downregulates ERα when p53 activities become exacerbated. It is recommended that AAMF be investigated as a possible therapy for some ERα sensitive clinical conditions like cancer.

Using real-time PCR as strategy to evaluate performance of PCR and Sanger sequencing reactions

Sanger sequencing is an indispensable technique for mitochondrial ancestry studies. However, it is a time consuming process that requires several resources to obtain high quality electropherograms. To optimize the efficiency of DNA analysis, we used real-time PCR (qPCR) to evaluate Sanger sequencing reaction performance before capillary electrophoresis. qPCR was performed to amplify Mitochondrial Control Region (CR). We determined that final PCR product quantification can be done using qPCR final fluorescence instead of visualization in agarose gel. Real-time sequencing reactions were done using BigDye Terminator v3.1 and SYBR Safe (Invitrogen); in addition, a dissociation curve step from 65 °C to 95 °C was added at the end of the thermal cycler protocol. The sequencing qPCR products were purified and run in a 3130 Genetic Analyzer (Applied Biosystems). The results demonstrated that fluorescence at 65 °C during the dissociation curve was directly related with DNA quantity in the sequencing qPCR reaction. Furthermore, samples that presented higher fluorescence at 65 °C generated better quality electropherograms. We suggest that electropherogram quality prediction before capillary electrophoresis is possible using sequencing qPCR. In this way, it is possible to increase workflow efficiency in laboratory procedures and identify errors during samples processing.

Development of a passive quenching SiPM-based detection system for quantification of real time PCR products based on fluorescence lifetime

This paper presents a technique for development of a high-performance and low cost real time PCR detection system based on fluorescence lifetime of fluorescent dyes by using silicon photomultipliers. A high gain passive quenching silicon photomultiplier is used as a detector in a relatively high photon rate condition along with a devised low cost discrete electronic circuitry to implement a detection system in which both time- and frequency-resolved measurements are performed. The requirements and restrictions to implement and test the system for quantification of the amplified dsDNA PCR products are discussed. The SYBR Green I as an intercalating double-stranded DNA fluoresenct-dye has been used to stain PCR products. It is demonstrated that the quantification of PCR products based on fluorescence lifetime by passive quenching silicon photomultiplier, without using any optical filter, is an efficient and cost effective method in real time PCR instrumentation.

2.7 million samples genotyped for HLA by next generation sequencing: lessons learned

BackgroundAt the DKMS Life Science Lab, Next Generation Sequencing (NGS) has been used for ultra-high-volume high-resolution genotyping of HLA loci for the last three and a half years. Here, we report on our experiences in genotyping the HLA, CCR5, ABO, RHD and KIR genes using a direct amplicon sequencing approach on Illumina MiSeq and HiSeq 2500 instruments.ResultsBetween January 2013 and June 2016, 2,714,110 samples largely from German, Polish and UK-based potential stem cell donors have been processed. 98.9% of all alleles for the targeted HLA loci (HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) were typed at high resolution or better. Initially a simple three-step workflow based on nanofluidic chips in conjunction with 4-primer amplicon tagging was used. Over time, we found that this setup results in PCR artefacts such as primer dimers and PCR-mediated recombination, which may necessitate repeat typing. Split workflows for low- and high-DNA-concentration samples helped alleviate these problems and reduced average per-locus repeat rates from 3.1 to 1.3%. Further optimisations of the workflow included the use of phosphorothioate oligos to reduce primer degradation and primer dimer formation, and employing statistical models to predict read yield from initial template DNA concentration to avoid intermediate quantification of PCR products. Finally, despite the populations typed at DKMS Life Science Lab being relatively homogenous genetically, an analysis of 1.4 million donors processed between January 2015 and May 2016 led to the discovery of 1,919 distinct novel HLA alleles.ConclusionsAmplicon-based NGS HLA genotyping workflows have become the workhorse in high-volume tissue typing of registry donors. The optimisation of workflow practices over multiple years has led to insights and solutions that improve the efficiency and robustness of short amplicon based genotyping workflows.

Solid-phase plate-reader quantification of specific PCR products by measurement of band-specific ethidium bromide fluorescence

Real-time PCR is widely employed to quantify PCR products across a range of applications. However, accurate real-time PCR is not always technically feasible, and alternative methods for PCR product quantification can be expensive and time consuming to validate. We have developed an inexpensive, rapid, and immediately accessible protocol to quantify PCR products, by measuring ethidium bromide fluorescence of PCR products excised from agarose gels. This protocol has relevance to a broad range of methods in molecular biology where quantification of PCR products is necessary.

Identification of New and UnusualrevandnefTranscripts Expressed by an HIV Type 1 Primary Isolate

We analyzed RNA splice site usage in three HIV-1 subtype B primary isolates through reverse transcriptase polymerase chain reaction (RT-PCR) amplification of spliced RNAs using a fluorescently labeled primer, with computerized size determination and quantification of PCR products, which were also identified by clone sequencing. In one isolate, P2149-3, unusual and unreported spliced transcripts were detected. This isolate preferentially used for rev RNA generation a 3' splice site (3'ss) located five nucleotides upstream of A4a, previously identified only in a T cell line-adapted virus and in a group O isolate, and designated A4d. P2149-3 also used an unreported 3'ss for rev RNA generation, designated A4h, located 20 nucleotides upstream of 3'ss A4c. Additionally, unusual nef RNAs using 3'ss A5a and A7a and with exon composition 1.3.7 were identified. The identification of several unusual and unreported spliced transcripts in an HIV-1 primary isolate suggests a greater diversity of splice site usage in HIV-1 than previously appreciated.

Testicular Nuclear Receptor 4 (TR4) Regulates UV Light-induced Responses via Cockayne Syndrome B Protein-mediated Transcription-coupled DNA Repair

UV irradiation is one of the major external insults to cells and can cause skin aging and cancer. In response to UV light-induced DNA damage, the nucleotide excision repair (NER) pathways are activated to remove DNA lesions. We report here that testicular nuclear receptor 4 (TR4), a member of the nuclear receptor family, modulates DNA repair specifically through the transcription-coupled (TC) NER pathway but not the global genomic NER pathway. The level of Cockayne syndrome B protein (CSB), a member of the TC-NER pathway, is 10-fold reduced in TR4-deficient mouse tissues, and TR4 directly regulates CSB at the transcriptional level. Moreover, restored CSB expression rescues UV hypersensitivity of TR4-deficient cells. Together, these results indicate that TR4 modulates UV sensitivity by promoting the TC-NER DNA repair pathway through transcriptional regulation of CSB. These results may lead to the development of new treatments for UV light-sensitive syndromes, skin cancer, and aging.

A new serotyping method of S. pneumoniae using an automated microarray-based assay

Serotype replacement is a major concern following the introduction of polysaccharide-conjugated vaccine against S. pneumoniae and requires a close monitoring in the population. Antibody-based serotyping methods are expensive, semi quantitative, cross-reactions are common and a significant number of isolates cannot be typed. Multiplex PCR-based assays have been developed but quantification of PCR products remains problematic. To address these issues a novel PCR-based automated microarray assay was developed and tested on clinical samples.

Rapid label-free identification of mixed bacterial infections by surface plasmon resonance

BackgroundEarly detection of mixed aerobic-anaerobic infection has been a challenge in clinical practice due to the phenotypic changes in complex environments. Surface plasmon resonance (SPR) biosensor is widely used to detect DNA-DNA interaction and offers a sensitive and label-free approach in DNA research.MethodsIn this study, we developed a single-stranded DNA (ssDNA) amplification technique and modified the traditional SPR detection system for rapid and simultaneous detection of mixed infections of four pathogenic microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, Clostridium tetani and Clostridium perfringens).ResultsWe constructed the circulation detection well to increase the sensitivity and the tandem probe arrays to reduce the non-specific hybridization. The use of 16S rDNA universal primers ensured the amplification of four target nucleic acid sequences simultaneously, and further electrophoresis and sequencing confirmed the high efficiency of this amplification method. No significant signals were detected during the single-base mismatch or non-specific probe hybridization (P < 0.05). The calibration curves of amplification products of four bacteria had good linearity from 0.1 nM to 100 nM, with all R2 values of >0.99. The lowest detection limits were 0.03 nM for P. aeruginosa, 0.02 nM for S. aureus, 0.01 nM for C. tetani and 0.02 nM for C. perfringens. The SPR biosensor had the same detection rate as the traditional culture method (P < 0.05). In addition, the quantification of PCR products can be completed within 15 min, and excellent regeneration greatly reduces the cost for detection.ConclusionsOur method can rapidly and accurately identify the mixed aerobic-anaerobic infection, providing a reliable alternative to bacterial culture for rapid bacteria detection.

Quantitative evaluation of gene expression of IL-1 beta and IL-1 receptor antagonist (IL-1ra) from alveolar macrophages in idiopathic pulmonary fibrosis (IPF) and sarcoidosis

Our previous studies demonstrated the concomitant release of IL-1 and IL-1ra in the culture supernatants of alveolar macrophages in both healthy subjects and patients with interstitial lung diseases. IL-1ra was decreased in healthy smokers (HS), IPF, and sarcoidosis, compared to healthy nonsmokers (HNS), although an increase in IL-1 release could not be detected. In this study, we examined whether such findings could be found at the gene level, or reflect posttranscriptional regulation in patients with IPF (n = 8), sarcoidosis (n = 7), healthy smokers (n = 6) and healthy nonsmokers (n = 5). The expression of IL-1 beta, IL-1ra and a house keeping gene (glucose 6 phosphate dehydrogenase: G6PD) was investigated using reverse transcription polymerase chain reaction (RT-PCR). After RT-PCR, analysis and quantification of PCR products were performed by high-performance liquid chromatography. We used 26 cycle for IL-1 beta and IL-ra mRNA and 29 for G6PD, because amplification of mRNA was in the exponential phase on that cycles, respectively. The results were expressed as the ratio of each mRNA to G6PD mRNA. IL-1ra/G6PD ratio in patients with IPF and sarcoidosis, was lower than that in HNS. On the other hand, IL-1 beta/G6PD ratio was similar in all groups. IL-1ra/IL-1 beta ratio was decreased in patients with IPF and sarcoidosis, compared to HNS. These results suggest that decreased expression of IL-1ra gene may contribute to the development of chronic low grade inflammation of the lung.

Helminth Cysteine Proteases Inhibit TRIF-dependent Activation of Macrophages via Degradation of TLR3

Helminth pathogens prepare a Th2 type immunological environment in their hosts to ensure their longevity. They achieve this by secreting molecules that not only actively drive type 2 responses but also suppress type 1 responses. Here, we show that the major cysteine proteases secreted from the helminth pathogens Fasciola hepatica (FheCL1) and Schistosoma mansoni (SmCB1) protect mice from the lethal effects of lipopolysaccharide by preventing the release of inflammatory mediators, nitric oxide, interleukin-6, tumor necrosis factor alpha, and interleukin-12, from macrophages. The proteases specifically block the MyD88-independent TRIF-dependent signaling pathway of Toll-like receptor (TLR)4 and TLR3. Microscopical and flow cytometric studies, however, show that alteration of macrophage function by cysteine protease is not mediated by cleavage of components of the TLR4 complex on the cell surface but occurs by degradation of TLR3 within the endosome. This is the first study to describe a parasite molecule that degrades this receptor and pinpoints a novel mechanism by which helminth parasites modulate the innate immune responses of their hosts to suppress the development of Th1 responses.

Quantification of PCR products by phosphate measurement

Various techniques for quantification of PCR are available. Most frequently, the densitometric intensities of ethidium bromide-stained PCR products separated in gels are compared after normalizing to the levels of housekeeping gene products such as β-actin. More precise, but extremely time consuming, is the technique of competitive PCR. Newer methods, such as tracking amplification in real-time, have high start-up and maintenance costs (e.g., TaqMan, Applied Biosystems; LightCycler, Roche; I-Cycler, Bio-Rad). Here, I describe an alternative, simple technique to quantify PCR products by determining the entire phosphate released during PCR. The method can be performed using common laboratory equipment, and the reagents needed are extremely cheap. The method is validated by measuring the induction of inducible nitric oxide synthase gene expression in cell culture and comparing the results with data obtained by LightCycler experiments and RNase protection assays.

Large deletions account for an increasing number of mutations in SGCE

Myoclonus-dystonia (M-D) (MIM 159900) is a rare "dystonia plus" syndrome, characterized by rapid myoclonic jerks, predominantly in the neck and upper limbs, in combination with dystonia. Mutations in the gene epsilon-sarcoglycan (SGCE) are known to be responsible for approximately one-third of cases. We screened 21 probands diagnosed with M-D for large deletions who were mutation negative as determined by PCR-direct sequencing. Multiplex PCR and quantification of PCR products was performed using a modified application of denaturing high performance liquid chromatography (dHPLC). We have identified two novel large multiexonic deletions of SGCE, which were confirmed by amplifying and sequencing the deletion breakpoints. Five other families were found to harbor small mutations identified by direct sequencing. Analysis of the region surrounding the deletions demonstrates that both deletions are the result of nonhomologous recombination with homologous end joining. This is only the second report of intragenic deletions with SGCE and it highlights the need to include exonic copy number variation when performing mutational analysis of SGCE.

Use of Real-Time Quantitative Polymerase Chain Reaction to Monitor the Evolution of Brucella melitensis DNA Load During Therapy and Post-Therapy Follow-Up in Patients with Brucellosis

We performed quantitative real-time polymerase chain reaction (Q-PCR) to monitor the evolution of Brucella melitensis DNA load from initial diagnosis through post-therapy follow-up in patients with brucellosis. On the basis of real-time fluorometric quantification of PCR products, we used the ultra-rapid LightCycler system (Roche Diagnostics). We collected 180 peripheral blood samples from 18 patients with brucellosis. Analysis of bacterial DNA loads was performed for 2 groups: 11 patients who did not experience relapse and 7 patients who experienced relapse in the follow-up phase. Q-PCR was 100% specific for B. melitensis and showed an analytical sensitivity of 15 fg. Sensitivity of Q-PCR for both initial infections and relapses was 100%. There were no statistically significant differences between groups with respect to bacterial DNA load from initial diagnosis to the end of post-treatment follow-up (P > .05). Evolution of the bacterial DNA load throughout the treatment phase was similar among patients who relapsed and did not relapse. Despite positive response to treatment and a sharp decrease in bacterial DNA load after initiating therapy, the results of Q-PCR on finalizing treatment for 50% of the patients (7 from the relapse group and 2 from the nonrelapse group) were low-level positive. At the conclusion of follow-up, almost 40% of the patients (4 from the relapse group and 3 from the nonrelapse group), most of them asymptomatic, still maintained low bacterial DNA loads. Using Q-PCR techniques, we consistently detected B. melitensis DNA in the blood samples of patients with brucellosis throughout treatment and follow-up, despite apparent recovery from infection. These findings may have diagnostic, pathogenic, and therapeutic implications.

Label-Free Microelectronic PCR Quantification

We present a robust and simple method for direct, label-free PCR product quantification using an integrated microelectronic sensor. The field-effect sensor can sequentially detect the intrinsic charge of multiple unprocessed PCR products and does not require sample processing or additional reagents in the PCR mixture. The sensor measures nucleic acid concentration in the PCR relevant range and specifically detects the PCR products over reagents such as Taq polymerase and nucleotide monomers. The sensor can monitor the product concentration at various stages of PCR and can generate a readout that resembles that of a real-time fluorescent measurement using an intercalating dye but without its potential inhibition artifacts. The device is mass-produced using standard semiconductor processes, can be reused for months, and integrates all sensing components directly on-chip. As such, our approach establishes a foundation for the direct integration of PCR-based in vitro biotechnologies with microelectronics.

Multiplexed RT- PCR for High Throughput Screening Applications

To increase efficiency of high throughput gene expression profiling, we established a new TaqMan RT-PCR (real-time reverse transcriptase-polymerase chain reaction with internal probes for the quantification of PCR products) method for quantitative mRNA expression analysis. In this procedure, we utilized poly-A mRNA capture plates and validated a multiplexed single tube RT-PCR assay for cell culture applications, including compound testing via gene induction measurement. In the described procedure, all steps including RNA extraction, RT and PCR are performed in the same tube, thus significantly enhancing throughput of this method. Optimization of conditions, including the number of cells necessary for detection of mRNA signal was performed. With a relatively abundant message such as GAPDH, we saw a linear response for all of the concentrations tested, from 10,000 cells to 10 cells. We have also demonstrated multiplexing of different targets within the PCR reactions. In these experiments, we combined VIC-labeled probes for GAPDH with several FAM-labeled probes obtained from Assays On Demand (Applied Biosystems). In the reported experiments, multiplexing did not affect the efficiency of RT-PCR. We also demonstrated the utility of this technology for compound screening applications. The described technology also has the potential to accelerate studies on target and biomarker identification and toxicity assessment in ADMET (absorption, distribution, metabolism, elimination, and toxicity) testing.

Quantitative Measurement of Fungal DNA Extracted by Three Different Methods Using Real-Time Polymerase Chain Reaction

Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount of A. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships ( R 2 =0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e =0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 × 10 9 copies/μl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 × 10 8 and 2.7 × 10 8 copies/μl for woodland and grassland, respectively), high DNA yield (6.4 μg/g and 1.8 μg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.

Quantitative measurement of fungal DNA extracted by three different methods using real-time polymerase chain reaction

Quantification of fungal populations in the environment is important for gaining a better understanding of various microbial processes. Recently, the development of real-time quantitative PCR (RTQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thereby allowing the routine and reliable quantification of PCR products. Thus, in this present study, RTQ-PCR was used to quantify the fungal target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in the presence of liquid nitrogen, and hot detergent SDS based enzymatic lysis combined with bead beating) to determine the suitability of the quantification of target DNA. For the purpose of this study, pure culture of Aspergillus fumigatus (model organism), sterilized soil seeded with a known amount of A. fumigatus (model soil system), and woodland and grassland soil samples (environmental samples) were chosen to extract DNA by the above three different protocols. The extracted DNA was then quantified by spectroscopy and a RTQ-PCR system. 18S rDNA specific universal fungal primers were used to quantify the target part and then amplification products were verified by agarose gel electrophoresis. Standard curves used for the quantification by RTQ-PCR revealed strong linear relationships ( R 2=0.9994 for the primer pair NS1 and NS2 and 0.9938 for the primer pair nu-SSU-0817 and nu-SSU-1196) with a higher amplification efficiency, e=0.983 for the primer pair NS1 and NS2 and 0.956 for the primer pair nu-SSU-0817 and nu-SSU-1196. Although for pure culture the hot detergent SDS based enzymatic lysis combined with bead beating method showed the highest target DNA copy number (1.5 × 10 9 copies/μl), for the model soil system and both environmental samples the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (6.16 × 10 8 and 2.7 × 10 8 copies/μl for woodland and grassland, respectively), high DNA yield (6.4 μg/g and 1.8 μg/g of soil for woodland and grassland, respectively), and high recovery on the basis of the target DNA copy number (39.2%), suggesting an overall high extraction efficiency.

Determination of tyrosinase mRNA in melanoma by reverse transcription-PCR and optical mirror resonance biosensor

Tyrosinase transcript in the blood is known as the marker of malignant melanoma and it has been often determined by using reverse transcription-polymerase chain reaction (RT-PCR). However, after the PCR process, the quantification of amplified cDNA by the gel electrophoresis is not reliable and time-consuming. For this reason, we tried to quantify the PCR product using a cuvette-type biosensor, where the oligonucleotide probe was immobilized on the cuvette surface and the single strand cDNA, the denatured PCR product, was then hybridized onto the immobilized probe to give a response signal. The response was immediate and takes 15 min to obtain a stable signal. The biosensor was much more sensitive comparing to the gel electrophoresis method. The quantification of PCR product using a cuvette-type biosensor was feasible and rapid.