Amplification Of DNA Templates Research Articles

Frequency of Pyrethroid Insecticide Resistance kdr Gene and Its Associated Enzyme Modulation in Housefly, Musca domestica L. Populations From Jhang, Pakistan

Housefly, Musca domestica, is considered responsible for transmitting a wide variety of human and veterinary diseases. Mostly, insecticides are being used for their control and more commonly, pyrethroid insecticides worldwide. However, resistance has been reported against various pyrethroid insecticides. Houseflies become resistant by two major mechanisms, i.e., target site insensitivity through knockdown resistance gene mutation (kdr) and enzyme detoxification. Thus, the current study was designed to monitor the frequency of pyrethroid resistance gene kdr in housefly populations of District Jhang. The flies were collected from seven sampling sites and then reared in the lab for molecular and biochemical assays. The amplification of template DNA was performed for knockdown resistance gene through the outer primers kdr1 and kdr4, and the inner primers kdr1 and kdr2 using PASA (PCR Amplification of Specific Alleles) method which specifically amplify the domain-II of kdr gene. Three populations were found homozygous susceptible (+/+; 42.85%), whereas two populations were found genetically homozygous resistant (−/−; 28.57%) which are insensitive to pyrethroid insecticides. Similarly, two populations were found heterozygous (+/−; 28.57%) for kdr suggesting thereby that at least 1/4th homozygous-resistant (−/−) housefly populations with insensitivity to pyrethroids would be produced in the future keeping in view the Mendelian ratio. Biochemical assay showed that homozygous-resistant populations had increased activity of Acetylcholinesterase (AChE), α-Carboxylesterases (α-Carboxyl), β-Carboxylesterase (β-Carboxyl), Alkaline Phosphatase (AkP), and Acidic Phosphatase (AcP) enzymes. In addition, heterozygous populations also showed increased activities of these enzymes. The current results would not only help avoid the indiscriminate load of insecticides onto the environment but also serve as a hallmark for the management of housefly populations in target areas in the future.

Miniaturized and IoT Enabled Continuous-Flow-Based Microfluidic PCR Device for DNA Amplification.

Herein, a continuous-flow driven microfluidic device has been designed and fabricated using the CO2 laser ablation method for polymerase chain reaction (PCR). The device consists of a polymethyl methacrylate (PMMA) microfluidic channel with 30 serpentine thermal cycles, an arduino board, two custom-made cartridge heaters, and thermocouple sensors. The portable thermal management system, with aluminium blocks placed on a wooden substrate, working on the PID controller principle, is low-cost, battery-powered, automated, integrated, and IoT-enabled. The device with dimensions 80×72×36 mm3 (L × W × H) has a temperature accuracy of ±0.2 °C. The IoT module enables accessing and storage of real-time temperature values directly onto the smartphone through ThingSpeak analytics. It was developed to achieve desirable accurate temperature at two thermal zones, denaturation and annealing (95 °C and 60 °C) on the microfluidic thermal management platform. A PCR mixture of [Formula: see text] was infused into the serpentine-based microchannel using a syringe pump. Amplification of DNA template with 594-base pair (bp) fragment of the rat GAPDH gene was successfully performed on the miniaturized thermal management system. The total time required for a complete PCR reaction was 32 min at an optimum flow rate of [Formula: see text]/min. The amplified sample of the target DNA obtained from the PCR microchannel was then separated by agarose gel electrophoresis and was further analyzed using a gel-doc system. Finally, the obtained results were compared to the conventional PCR instrument showing excellent performance.

Evaluation of different nested PCRs for detection of Anaplasma phagocytophilum in ruminants and ticks

BackgroundAnaplasma phagocytophilum is a causative agent of granulocytic anaplasmosis in mammals, which has a broad geographical distribution and a high degree of clinical diversity. Currently, numerous PCR assays have been developed and used for the detection of A. phagocytophilum in various specimens. However, their performance varies. The aim of this study was to evaluate the performance of five nested PCR assays by detection of 363 ruminant and tick samples, and to select the most appropriate methods for the sensitive detection of A. phagocytophilum in environmental or clinical samples.ResultsPositive PCR results for A. phagocytophilum were obtained in 75 (20.7 %), 42 (11.6 %) and 19 (5.2 %) specimens with primer sets EC (EC9/EC12a and SSAP2f/SSAP2r), EE (EE1/EE2 and EE3/EE4) and ge (ge3a/ge10r, ge9f/ge2), respectively. The amplification of template DNA with the primer set MSP (MAP4AP5/MSP4AP3, msp4f/msp4r) could not be obtained in both ruminants and ticks, and a low specificity of the EL primers [EL(569)F/EL(1193)R, EL(569)F/EL(1142)R] in tick samples was observed. Our results revealed that the nested PCR with primer set EC complementary to the 16S rRNA gene was the most sensitive assay for detection of A. phagocytophilum in ruminant and tick specimens. A. phagocytophilum was detected in 47 (35.1 %) sheep, 12 (10.4 %) cattle, and 17 (14.9 %) ticks. Two A. phagocytophilum genotypes were identified, that varied between sheep and cattle in sample collection sites.ConclusionsThis report provides more valuable information for the diagnosis and management of granulocytic anaplasmosis in China.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0663-2) contains supplementary material, which is available to authorized users.

Real-time PCR-based genotyping from whole blood using Taq DNA polymerase and a buffer supplemented with 1,2-propanediol and trehalose

Amplification of DNA templates from whole blood with Taq DNA polymerase remains a difficult task worldwide. Using a real-time PCR setup and a buffer supplemented with 1M 1,2-propanediol, 0.2M trehalose, and SYBR green I we show a reliable technique for genotyping in mice and detection of single-nucleotide polymorphisms/mutations in humans. Elimination of DNA extraction and use of the common Taq DNA polymerase and DNA dye bring about substantial savings in labor and cost.

A Novel SNPs Detection Method Based on Gold Magnetic Nanoparticles Array and Single Base Extension

To fulfill the increasing need for large-scale genetic research, a high-throughput and automated SNPs genotyping method based on gold magnetic nanoparticles (GMNPs) array and dual-color single base extension has been designed. After amplification of DNA templates, biotinylated extension primers were captured by streptavidin coated gold magnetic nanoparticle (SA-GMNPs). Next a solid-phase, dual-color single base extension (SBE) reaction with the specific biotinylated primer was performed directly on the surface of the GMNPs. Finally, a “bead array” was fabricated by spotting GMNPs with fluorophore on a clean glass slide, and the genotype of each sample was discriminated by scanning the “bead array”. MTHFR gene C677T polymorphism of 320 individual samples were interrogated using this method, the signal/noise ratio for homozygous samples were over 12.33, while the signal/noise ratio for heterozygous samples was near 1. Compared with other dual-color hybridization based genotyping methods, the method described here gives a higher signal/noise ratio and SNP loci can be identified with a high level of confidence. This assay has the advantage of eliminating the need for background subtraction and direct analysis of the fluorescence values of the GMNPs to determine their genotypes without the necessary procedures for purification and complex reduction of PCR products. The application of this strategy to large-scale SNP studies simplifies the process, and reduces the labor required to produce highly sensitive results while improving the potential for automation.

DNA-templated combinatorial assembly of small molecule fragments amenable to selection/amplification cycles

The discovery of small molecule probes which selectively modulate biological pathways is a cornerstone in the development of new therapeutics. Progress in our ability to access libraries of biologically relevant small molecules in conjunction with streamlined screening technologies have also enabled a more systematic approach to chemical biology. Nevertheless, the current state of the art still requires a large infrastructure and only a small fraction of the proteome has been addressed thus far. The emergence of technologies based on nucleic acid encoding of small molecules presents a new screening paradigm. We describe a method based on DNA-templated combinatorial display of PNA-encoded drug fragments affording 62 500 combinations which can be amplified following a selection. This concept was demonstrated with a screen against a representative target, carbonic anhydrase, by iterative cycles of affinity selection, amplification of DNA template and “translation” back into selected library members. The results show a clear convergence towards combinations which, upon resynthesis as covalent adducts, proved to bind cooperatively to carbonic anhydrase.

Fabrication and characterization of a polymethyl methacrylate continuous-flow PCR microfluidic chip using CO2 laser ablation

An improved microfabrication method was used to fabricate a continuous-flow PCR (polymerase chain reaction) microfluidic chip on the PMMA substrate using the low-power CO2 laser ablation technique. The use of the low-power CO2 laser and the PMMA material could reduce the cost and the time of the fabrication process, especially at the step of laboratory research because of the high flexibility of the laser fabrication technique and the low cost of PMMA. A CO2 laser output power of 4.5 W and a laser scanning velocity of 76.2 mm/s were chosen to fabricate the chip in this work. The micromachining quality could satisfy the microfluidic requirement of the PCR mixture within the microchannel. Good temperature distribution and gradient were obtained on the PMMA chip with a home-built integrated heating system. An amplification of DNA template with a 990 base pair fragment of Pseudomonas was successfully performed with this chip to characterize its availability and performance with various flow rates.

Efficient PCR amplification by an unnatural base pair system

Expansion of the genetic alphabet by an unnatural base pair system enables the site-specific incorporation of extra functional components into nucleic acids and proteins. In this system, PCR amplification of DNA templates containing unnatural base pairs is essential for modern biotechnology. We present a new unnatural base pair system, in which DNA duplexes containing the unnatural base pairs can be efficiently amplified by PCR. The system also provides a method for the site-specific incorporation of functional components into amplified DNA fragments by PCR, using unnatural base substrates linked with functional groups of interest.

Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

Rolling circle amplification (RCA) of plasmid or genomic DNA using random hexamers and bacteriophage phi29 DNA polymerase has become increasingly popular in the amplification of template DNA in DNA sequencing. We have found that the mutant protein of single-stranded DNA binding protein (SSB) from Thermus thermophilus (Tth) HB8 enhances the efficiency of amplification of DNA templates. In addition, the TthSSB mutant protein increased the specificity of phi29 DNA polymerase. We have overexpressed the native and mutant forms of TthSSB protein in Escherichia coli and purified them to homogeneity. In vitro, these proteins were found to bind specifically to single-stranded DNA. Addition of TthSSB mutant protein to RCA halved the elongation time required for phi29 DNA polymerase to synthesize DNA fragments in RCA. Furthermore, the presence of the TthSSB mutant protein essentially eliminates nonspecific DNA products in RCA reactions.

Molecular markers for identification of the hyperparasitoids Dendrocerus carpenteri and Alloxysta xanthopsis in Lysiphlebus testaceipes parasitizing cereal aphids.

Polymerase chain reaction (PCR)-based molecular markers have been developed to detect the presence of primary parasitoids in cereal aphids and used to estimate primary parasitism rates. However, the presence of secondary parasitoids (hyperparasitoids) may lead to underestimates of primary parasitism rates based on PCR markers. This is because even though they kill the primary parasitoid, it's DNA can still be amplified, leading to an erroneous interpretation of a positive result. Another issue with secondary parasitoids is that adults are extremely difficult to identify using morphological characters. Therefore, we developed species-specific molecular markers to detect hyperparasitoids. A 16S ribosomal RNA mitochondrial gene fragment was amplified by PCR and sequenced from two secondary parasitoid species, Dendrocerus carpenteri (Curtis) (Hymenoptera: Megaspilidae) and Alloxysta xanthopsis (Ashmead) (Hymenoptera: Charipidae), four geographic isolates of the primary parasitoid, Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae), and six aphid species common to cereal crops. Species-specific PCR primers were designed for each insect on the basis of these 16S rRNA gene sequences. Amplification of template DNA, followed by agarose gel electrophoresis, successfully distinguished D. carpenteri and A. xanthopsis from all four isolates of L. testaceipes and all six cereal aphid species in this laboratory test.

Improved simple generation of GTG-band specific painting probes

We developed an improved, simple method of generating chromosome-region-specific probes from only a few microdissected chromosomes. One to five dissected fragments from a defined chromosomal region were processed with a PEG/proteinase K cycling deproteinization step and directly amplified with a two-step amplification system using a degenerate oligonucleotide primed shuttle polymerase chain reaction (DOP-Shuttle-PCR). This modified method offered three advantages over previously reported methods: relaxation of the highly condensed chromosomal DNA, reduction of the risk of endogenous and exogenous contamination, and high efficiency amplification of template DNA. High intensity in the fluorescence in situ hybridization (FISH) signals from normal metaphase chromosomes, as well as regional specificity of these probes, corresponding to regions on R-banded chromosomes, were observed.