Mirror-image polymerase chain reaction.
Ontology highlight
ABSTRACT:
SUBMITTER: Jiang W
PROVIDER: S-EPMC5643884 | biostudies-literature | 2017
REPOSITORIES: biostudies-literature
Publications
Mirror-image polymerase chain reaction.
Jiang Wenjun W Zhang Baochang B Fan Chuyao C Wang Min M Wang Jiaxing J Deng Qiang Q Liu Xianyu X Chen Ji J Zheng Jishen J Liu Lei L Zhu Ting F TF
Cell discovery 20171017
The construction of mirror-image biological systems may open the next frontier for biomedical technology development and discovery. Here we have designed and chemically synthesized a mutant version of the thermostable <i>Sulfolobus solfataricus</i> P2 DNA polymerase IV (Dpo4) consisting of d-amino acids. With a total peptide length of 358 amino acid residues, it is the largest chemically synthesized d-amino acid protein reported to date. We show that the d-polymerase is able to amplify a 120-bp ...[more]
Similar Datasets
Project description:Biological evolution resulted in a homochiral world in which nucleic acids consist exclusively of d-nucleotides and proteins made by ribosomal translation of l-amino acids. From the perspective of synthetic biology, however, particularly anabolic enzymes that could build the mirror-image counterparts of biological macromolecules such as l-DNA or l-RNA are lacking. Based on a convergent synthesis strategy, we have chemically produced and characterized a thermostable mirror-image polymerase that efficiently replicates and amplifies mirror-image (l)-DNA. This artificial enzyme, dubbed d-Dpo4-3C, is a mutant of Sulfolobus solfataricus DNA polymerase IV consisting of 352 d-amino acids. d-Dpo4-3C was reliably deployed in classical polymerase chain reactions (PCR) and it was used to assemble a first mirror-image gene coding for the protein Sso7d. We believe that this d-polymerase provides a valuable tool to further investigate the mysteries of biological (homo)chirality and to pave the way for potential novel life forms running on a mirror-image genome.
| S-EPMC5605242 | biostudies-literature
Project description:Although faster and powerful, polymerase chain reaction (PCR) often failed to amplify targets efficiently. Numerous PCR enhancers have been used to increase the amplification efficiency of difficult DNA targets. However, there is no systematic comparison of their effects in normal and difficult PCR conditions. In this paper, we have selected nine different PCR enhancers that can promote the PCR amplification efficiency. We have compared their effect in Taq DNA polymerase thermostability, inhibitor resistance, and amplification of various DNA targets. Although the PCR enhancers more or less reduced the amplification efficiency of DNA fragments with moderate GC-content, they were able to improve the amplification efficiency and specificity of GC-rich fragments. Betaine outperformed the other enhancers in amplification of GC-rich DNA fragments, thermostabilizing Taq DNA polymerase, and inhibitor tolerance. Sucrose and trehalose showed similar effect in thermostabilizing Taq DNA polymerase and inhibitor tolerance, while they showed mildest inhibitory effect on normal PCR. For GC-rich region-containing long DNA fragment amplification, 1 M betaine, 0.5 M betaine + 0.2 M sucrose, or 1 M betaine + 0.1 M sucrose can be used to effectively promote the amplification, while keep their negative effect in amplification of normal fragment to a minimal level.
| S-EPMC11521273 | biostudies-literature
Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.
| S-EPMC7896886 | biostudies-literature
Project description:Polymerase chain reaction (PCR) kits have been used as common diagnosing tools during the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, with daily worldwide usage in the millions. It is well known that at the beginning of the pandemic, there was a shortage of PCR kits. So far, the ecosystem of a PCR kit is linear use; that is, kits are produced, used once, and disposed of as biolab waste. Here, we show that to mitigate the risk of future shortages, it is possible to envision recyclable PCR kits based on a more sustainable use of nucleic acid resources. A PCR kit is mainly composed of primers, nucleotides, and enzymes. In the case of a positive test, the free nucleotides are polymerized onto the primers to form longer DNA strands. Our approach depolymerizes such strands, keeping the primers and regenerating the nucleotides, i.e., returning the nucleic acid materials to the original state. The polymerized long DNA strands are hydrolyzed into nucleotide monophosphates that are then phosphorylated into triphosphates using a method that is developed from a recent publication. We used oligonucleotides with a 3'-terminal phosphorothioate (PS) backbone modification as nonhydrolyzable PCR primers, which are able to undergo the recycling process unchanged. The nuclease resistance of oligonucleotides with a ribose sugar modification was also evaluated, which showed worse recycling efficiency than PS-modified oligonucleotides. We successfully recycled both PCR primers and nucleotide monomers (∼75% yield). We demonstrate that the method allows for the direct reuse of PCR kits. We also show that the recycled primers can be isolated and then added to endpoint or quantitative PCR. This recycling approach provides a new path for circularly reusing nucleic acid materials in PCR kits.
| S-EPMC10081572 | biostudies-literature
Project description:Actinobaculum suis is an important agent related to urinary infection in swine females. Due to its fastidious growth characteristics, the isolation of this anaerobic bacterium is difficult, thus impairing the estimation of its prevalence. The purpose of this study was to develop and test a polymerase chain reaction (PCR) for the detection and identification of A. suis and then compare these results with traditional isolation methods. Bacterial isolation and PCR were performed on one hundred and ninety-two urine samples from sows and forty-five preputial swabs from boars. The results indicate that this PCR was specific for A. suis, presenting a detection limit between 1.0 × 10(1) CFU/mL and 1.0 × 10(2) CFU/mL. A. suis frequencies, as measured by PCR, were 8.9% (17/192) in sow urine samples and 82.2% (37/45) in preputial swabs. Assessed using conventional culturing techniques, none of the urine samples were positive for A. suis; however, A. suis was detected in 31.1% (14/45) of the swabs. This PCR technique was shown to be an efficient method for the detection of A. suis in urine and preputial swabs.
| S-EPMC3544261 | biostudies-literature
Project description:BackgroundGnosis is a modality-specific ability to access semantic knowledge of an object or stimulus in the presence of normal perception. Failure of this is agnosia or disorder of recognition. It can be highly selective within a mode. self-images are different from others as none has seen one's own image except in reflection. Failure to recognize this image can be labeled as mirror image agnosia or Prosopagnosia for reflected self-image. Whereas mirror agnosia is a well-recognized situation where the person while looking at reflected images of other objects in the mirror he imagines that the objects are in fact inside the mirror and not outside.Material and methodsFive patients, four females, and one male presented with failure to recognize reflected self-image, resulting in patients conversing with the image as a friend, fighting because the person in mirror is wearing her nose stud, suspecting the reflected self-image to be an intruder; but did not have prosopagnosia for others faces, non living objects on self and also apraxias except dressing apraxia in one patient. This phenomena is new to our knowledge.ResultsMirror image agnosia is an unique phenomena which is seen in patients with parietal lobe atrophy without specificity to a category of dementing illness and seems to disappear as disease advances.DiscussionReflected self-images probably have a specific neural substrate that gets affected very early in posterior dementias specially the ones which predominantly affect the right side. At that phase most patients are mistaken as suffering from psychiatric disorder as cognition is moderately preserved. As disease becomes more widespread this symptom becomes masked. A high degree of suspicion and proper assessment might help physicians to recognize the organic cause of the symptom so that early therapeutic interventions can be initiated. Further assessment of the symptom with FMRI and PET scan is likely to solve the mystery of how brain handles reflected self-images.ConclusionA new observation involving failure to recognize reflected self-images is reported.
| S-EPMC4201793 | biostudies-literature
Project description:Droplet digital PCR (ddPCR) is accurate in nucleic acid quantification owing to its linearity and high sensitivity. Amplification of nucleic acid in droplets, however, is limited by the stability of droplets against thermal cycling. While the use of fluorinated oil or supplementation of surfactant could improve the stability of droplets, this process has also greatly increased the cost of ddPCR and limited post-PCR analysis. Here, we report a novel method known as gel capsule-based digital PCR (gc-dPCR) which includes a method to prepare hydrogel capsules encapsulating the PCR reaction mix, conducting PCR reaction, and readout by either quantitative PCR (qPCR) system or fluorescence microplate reader. We have compared the developed method to vortex ddPCR. Our approach results in higher fluorescence intensity compared to ddPCR suggesting higher sensitivity of the system. As hydrogel capsules are more stable than droplets in fluorinated oil throughout thermal cycling, all partitions can be quantified, thus preventing loss of information from low-concentration samples. The new approach should extend to all droplet-based PCR methods. It has greatly improved ddPCR by increasing droplets stability and sensitivity, and reducing the cost of ddPCR, which help to remove the barrier of ddPCR in settings with limited resources.
| S-EPMC10233522 | biostudies-literature
Project description:Nanoparticles (NPs) are attractive materials owing to their physical and electrochemical properties, which make them extremely useful in diagnostic applications. Photon upconversion is the phenomenon where high-energy photons are emitted upon excitation of low-energy photons. Nucleic acids detection based on upconversion nanoparticles (UCNPs), which display a high signal-to-noise ratio and no photobleaching, has been widely applied. We evaluated whether UCNPs can improve polymerase chain reaction (PCR) specificity and affect PCR amplification. The effects of UCNPs with a diameter size of 40, 70, and 250 nm were evaluated using 3 PCR kits (AccuPower PCR PreMix, AmpliTaq Gold 360 Master Mix, and HotStarTaq Plus Master Mix) and 3 real-time PCR kits (AccuPower GreenStar qPCR PreMix, SYBR Green PCR Master Mix, and QuantiTect SYBR Green PCR Kit). Quantum dots were used for comparison with the UCNPs. In the presence of an appropriate concentration of UCNPs, PCR specificity was optimized. UCNPs of 40-nm size improved PCR specificity more effectively than did UCNPs sized 70 or 250 nm. As the size and concentrations of the UCNPs were increased, PCR amplification was more severely inhibited. At lower annealing temperatures (25°C-45°C), addition of the 40 nm UCNP (1 µg/µL) to the PCR reagent produced specific PCR products without nonspecific sequence amplification. Therefore, UCNPs of different sizes, with different DNA polymerases used in the commercial kits, showed different inhibitory effects on PCR amplification. These results demonstrate that optimization of UCNPs, added to reaction mixtures at appropriate concentrations, can improve PCR specificity. However, the mechanism underlining UCNPs effect on PCR remains unclear and will require further investigation.
| S-EPMC3764166 | biostudies-literature
Project description: Not available
| S-EPMC3155763 | biostudies-literature
Project description:Reducing the time taken to run qPCR assays on today's qPCR cyclers is rather straightforward and requires no specialised reagents or instruments. As the first article in a new series of short technical reports, I demonstrate that it is possible to reduce significantly both denaturation temperatures and cycling times, whilst retaining sensitivity and specificity of the original qPCR conditions.
| S-EPMC5496742 | biostudies-literature