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221.
Addition of gold nanoparticles to real-time PCR: effect on PCR profile and SYBR Green I fluorescence
Haber AL Griffiths KR Jamting AK Emslie KR 《Analytical and bioanalytical chemistry》2008,392(5):887-896
Real-time quantitative polymerase chain reaction (qPCR) is the industry standard technique for the quantitative analysis of
nucleic acids due to its unmatched sensitivity and specificity. Optimisation and improvements of this fundamental technique
over the past decade have largely consisted of attempts to allow faster and more accurate ramping between critical temperatures
by improving assay reagents and the thermal geometry of the PCR chamber. Small gold nanoparticles (Au-NPs) have been reported
to improve PCR yield under fast cycling conditions. In this study, we investigated the effect of Au-NPs on optimised real-time
qPCR assays by amplifying DNA sequences from genetically modified canola in the presence and absence of 0.9 nM Au-NPs of diameter
12 ± 2nm. Contrary to expectations, we found that Au-NPs altered the PCR amplification profile when using a SYBR Green I detection
system due to fluorescence quenching; furthermore, high-resolution melt (HRM) analysis demonstrated that Au-NPs destabilised
the double-stranded PCR product. The results indicate that effects on the assay detection system must be carefully evaluated
before Au-NPs are included in any qPCR assay.
Figure Raw amplification profiles in the presence and absence of gold nanoparticles 相似文献
222.
Hung CC Lee CN Lin CY Cheng WF Chen CA Hsieh ST Yang CC Jong YJ Su YN Lin WL 《Electrophoresis》2008,29(3):618-625
We evaluated the efficacy of PCR-RFLP, competitive multiplex PCR, and a commercially available system of multiplex ligation-dependent probe amplification (MLPA) for the determination of deletion and duplication genotypes of the PMP22 gene. We compared the methods for efficiency, sensitivity, and specificity. We determined the gene dosage of the PMP22 gene via PCR-RFLP, competitive multiplex PCR, and MLPA. To demonstrate the sensitivity and accuracy of these three methods, a total of 185 samples from 42 patients with hereditary neuropathy with liability to pressure palsies (HNPP), 57 patients with Charcot-Marie-Tooth disease type 1A (CMT1A), and 86 unaffected individuals, were analyzed. Molecular diagnosis by PCR-RFLP was performed on all 185 samples; 24 HNPP deletions and 33 CMT1A duplications were identified. In contrast, 25 HNPP deletions and 38 CMT1A duplications were identified correctly using competitive multiplex PCR and MLPA. Six samples were incorrectly identified by PCR-RFLP (one HNPP deletion and five CMT1A duplications). Competitive multiplex PCR and MLPA demonstrated reliability and relative speed compared to PCR-RFLP; they were superior to PCR-RFLP for gene dosage quantification. Multiplex PCR and MLPA should be the methods of choice for detection of deletion and duplication genotypes in molecular genetic diagnoses. 相似文献
223.
Mir Mohammad Hassan Kahila Amin Mahdi Najy Mohammad Mir-Derikvand 《Natural product research》2018,32(15):1858-1862
Thymoquinone is the most important secondary metabolite in black Cumin, which has several pharmaceutical applications. In this study, effect of TiO2 and SiO2 nanoparticles as new elicitors, on expression of Geranyl diphosphate synthase gene (GPPS gene), as a key gene involved in thymoquione biosynthesis pathway was investigated in two Iranian accessions. Plants were treatment in the early flowering stage and after 24 h of 50 and 100 mg/L of each nanoparticle, separately. After RNA extraction, GPPS gene expression was analysed by qRT-PCR method. The results showed that the TiO2 and SiO2 nanoparticles, generally stimulates the GPPS expression. The TiO2 nanoparticles were more effective than SiO2 for the induction of GPPS expression. Also, 100 mg/L treatment of nanoparticles raised gene expression more than 50 mg/L concentration. It can be concluded these nanoparticles can be used as robust elicitors to enhance the production of Thymoquinone in black cumin through up-regulation of related metabolic pathway genes. 相似文献
224.
《Electrophoresis》2018,39(17):2270-2276
Sudden cardiac death (SCD) occurs frequently in forensic practice and results in no visible pathological changes that can be detected in an autopsy. In recent years, the genetic background has been emphasized when examining SCD cases. The aim of this study is to establish a feasible system to detect SCD‐related genes for forensic DNA laboratories. Forty‐five reported SCD‐associated SNPs from sodium voltage‐gated channel alpha subunit 5 (SCN5A) were considered in our experiment. We established a SNaPshot assay for the typing of 45 SNPs using multiplex PCR and the minisequencing technique. Two multiplex PCRs were performed and optimized to cover 14 and 16 DNA fragments. The SCD victims came from the Chinese Han population residing in Shanxi and Chongqing provinces and were examined and compared with a non‐SCD group and with normal healthy individuals. A missense mutation at rs1805124 (H558R) was detected in the Chinese Han population in this study. A SNaPshot assay can be performed in any forensic DNA laboratory and would be capable of meeting the increasing demand for SCD detection. This method would also be beneficial for screening at‐risk in family members of SCD victims. 相似文献
225.
Human interferon ω1 (huIFN-ω1) gene was isolated and cloned from chromosome DNA derived from a Chinese fetal liver via polymerase chain reaction (PCR). By determining its nucleotide sequence we proved that the 88th codon should be GGA, coding for Gly. After engineering the original IFN-ω1 gene clone to a form that may be expressed as a nonfused protein, we also took the IFN-ω1 gene under the control of the PRPL promoter with an expression vector pBV220 in E. coli. The antivirus activity of the recombinant IFN-ω1 is about 6.5×10~7 units/L CULTURE (OD_(600)=0.75). Since IFN-ω1 not only has antivirus activity but also shows considerably high homology with animal trophoblast proteins which have been proved antiluteolysins as a maternal recognition signal for pregnancy, we believe that study on it will be practically and theoretically significant. 相似文献
226.
Paper-based analytical devices have become lately “must have” components in equipment and instrumental designed for point-of-care applications, especially when they are used in tandem with microfluidic platforms. Nowadays, paper-based electrochemical devices (PEDs) represent the first choice in the development of lab-on-a-chip biosensors because of their benefits in biomedical diagnosis in terms of simplicity, affordability, portability, and disposability. Moreover, cellulose is a biodegradable and biocompatible substrate, ideal for building disposable devices for use in remote locations or low-resource settings. Despite their low costs and simplicity, PEDs must face a tough challenge—meeting the affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end users criteria. The latest achievements in microfluidic PEDs for clinical diagnosis will be critically discussed, putting emphasis on innovative assay formats and methods for surface modification. 相似文献
227.
Determination of total antioxidant capacity in green tea by near-infrared spectroscopy and multivariate calibration 总被引:3,自引:0,他引:3
A principal component regression (PCR) model is built for prediction of total antioxidant capacity in green tea using near-infrared (NIR) spectroscopy. The modelling procedures are systematically studied with the focus on outlier detection. Different outlier detection methods are used and compared. The root mean square error of prediction (RMSEP) of the final model is comparable to the precision of the reference method. 相似文献
228.
Jiří Drábek Michaela Smolíková Ruslan Kalendar Fernando A. Lopes Pinto Pavel Pavloušek Karel Klepárník Ivo Frébort 《Electrophoresis》2016,37(23-24):3059-3067
Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser‐induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high‐throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling. 相似文献
229.
Jordan O. Cox Teresa Sikes DeCarmen Yiwen Ouyang Briony Strachan Hillary Sloane Cathey Connon Kemper Gibson Kimberly Jackson James P. Landers Tracey Dawson Cruz 《Electrophoresis》2016,37(23-24):3046-3058
This work describes the development of a novel microdevice for forensic DNA processing of reference swabs. This microdevice incorporates an enzyme‐based assay for DNA preparation, which allows for faster processing times and reduced sample handling. Infrared‐mediated PCR (IR‐PCR) is used for STR amplification using a custom reaction mixture, allowing for amplification of STR loci in 45 min while circumventing the limitations of traditional block thermocyclers. Uniquely positioned valves coupled with a simple rotational platform are used to exert fluidic control, eliminating the need for bulky external equipment. All microdevices were fabricated using laser ablation and thermal bonding of PMMA layers. Using this microdevice, the enzyme‐mediated DNA liberation module produced DNA yields similar to or higher than those produced using the traditional (tube‐based) protocol. Initial microdevice IR‐PCR experiments to test the amplification module and reaction (using Phusion Flash/SpeedSTAR) generated near‐full profiles that suffered from interlocus peak imbalance and poor adenylation (significant ?A). However, subsequent attempts using KAPA 2G and Pfu Ultra polymerases generated full STR profiles with improved interlocus balance and the expected adenylated product. A fully integrated run designed to test microfluidic control successfully generated CE‐ready STR amplicons in less than 2 h (<1 h of hands‐on time). Using this approach, high‐quality STR profiles were developed that were consistent with those produced using conventional DNA purification and STR amplification methods. This method is a smaller, more elegant solution than current microdevice methods and offers a cheaper, hands‐free, closed‐system alternative to traditional forensic methods. 相似文献
230.