scholarly journals Sample preparation and PCR amplification from paraffin-embedded tissues.

1994 ◽  
Vol 3 (6) ◽  
pp. S113-S122 ◽  
Author(s):  
C E Greer ◽  
C M Wheeler ◽  
M M Manos
Keyword(s):  
Sample Preparation ◽  
Pcr Amplification

scholarly journals Integrated, Automated, Fast PCR System for Point-Of-Care Molecular Diagnosis of Bacterial Infection

Sensors ◽  
2021 ◽  
Vol 21 (2) ◽  
pp. 377
Author(s):  
Dongkyu Lee ◽  
Deawook Kim ◽  
Jounghyuk Han ◽  
Jongsu Yun ◽  
Kang-Ho Lee ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Fluorescence Intensity ◽  
Pathogenic Bacteria ◽  
Point Of Care ◽  
Pcr Amplification ◽  
Sample Volume ◽  
Separation And Purification ◽  
Heating And Cooling ◽  
Automated Sample Preparation ◽  
Commercial Device

We developed an integrated PCR system that performs automated sample preparation and fast polymerase chain reaction (PCR) for application in point-of care (POC) testing. This system is assembled from inexpensive 3D-printing parts, off-the-shelf electronics and motors. Molecular detection requires a series of procedures including sample preparation, amplification, and fluorescence intensity analysis. The system can perform automated DNA sample preparation (extraction, separation and purification) in ≤5 min. The variance of the automated sample preparation was clearly lower than that achieved using manual DNA extraction. Fast thermal ramp cycles were generated by a customized thermocycler designed to automatically transport samples between heating and cooling blocks. Despite the large sample volume (50 μL), rapid two-step PCR amplification completed 40 cycles in ≤13.8 min. Variations in fluorescence intensity were measured by analyzing fluorescence images. As proof of concept of this system, we demonstrated the rapid DNA detection of pathogenic bacteria. We also compared the sensitivity of this system with that of a commercial device during the automated extraction and fast PCR of Salmonella bacteria.

scholarly journals Quantification of Toxic Cyanobacteria in Water by Use of Competitive PCR Followed by Sequence-Specific Labeling of Oligonucleotide Probes

1998 ◽  
Vol 64 (7) ◽  
pp. 2639-2643 ◽  
Author(s):  
Knut Rudi ◽  
Olav M. Skulberg ◽  
Frank Larsen ◽  
Kjetill S. Jakobsen
Keyword(s):  
Sample Preparation ◽  
Solid Phase ◽  
Bacterial Species ◽  
Simultaneous Detection ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Oligonucleotide Probes ◽  
Competitive Pcr ◽  
Wide Range ◽  
Preparation Strategy

ABSTRACT A complete nucleic-acid-based assay which consists of sample preparation, DNA amplification, and chromogenic detection was developed for quantifying potential toxin-producing cyanobacteria of interest to the public. The sample preparation strategy involves the same solid phase for cell concentration and DNA purification. For the detection step, we used a combination of competitive PCR amplification, sequence-specific labeling of oligonucleotide probes, hybridization of the labeled oligonucleotides to immobilized complements and, finally, chromogenic detection. The complete assay was tested with water containing toxin-producing cyanobacteria belonging to the genusMicrocystis. A detection limit of 100 cells/ml and a quantitative range of more than 3 orders of magnitude were obtained. This approach can easily be adapted to a wide range of bacterial species and has the potential for simultaneous detection and quantitation of several different target organisms by a single assay.

DNA amplification in mixed aqueous-organic media: chemical analysis of leading polymerase chain reaction compositions

2021 ◽  
Author(s):  
Jalel Neffati ◽  
Ioanna Petrounia ◽  
Rudy D. Moreira ◽  
Raj Chakrabarti
Keyword(s):  
Sample Preparation ◽  
Mass Spectra ◽  
Matrix Effects ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Polymerase Activity ◽  
Ion Source ◽  
Amplification Efficiency ◽  
Internal Standards ◽  
Polymerase Chain

AbstractPCR amplification of GC-rich regions often leads to low yield and specificity. Addition of PCR-enhancing compounds is employed in order to overcome these obstacles. PCR-enhancing additives are low molecular polar organic compounds that are included as undisclosed co-solvents in commercial PCR buffers. In the interest of transparency and to permit further optimization by researchers of PCR compositions for challenging amplification problems, we studied eight PCR buffers by GC/MS to identify and quantify their co-solvents. Buffer specificity, both rich in water and salified substances, required a suitable sample preparation before injection into the GC/MS system. The aqueous phase of each buffer was replaced by an organic solvent to remove, by precipitation and filtration, salified substances which are detrimental to the GC/MS analysis. This approach has demonstrated the advantage of eliminating both water and salified substances without any loss of co-solvents. The sensitivity of the developed method was demonstrated as the main co-solvents were easily detected, identified and quantified. The methodology for identifying the co-solvents is mainly based on comparison of both library matching of acquired MS spectra with NIST library and experimental mass spectra obtained from authentic chemical standards. For the quantification of each co-solvent, deuterated Internal standards of similar structure to the cosolvents were used to correct the variable recovery caused by sample preparation, matrix effects, and ion source variability. The recovery ratio of the developed method was verified and found to be in the range 90-120 %. We then characterized the effects of specific organic co-solvents identified during PCR amplification -- using DNA melting, polymerase thermostability, polymerase activity and real-time PCR methods -- in order to elucidate their mechanism of action and to permit further optimization of their effects on amplification efficiency and specificity.

Pollutant Identification by Selected Area Electron Diffraction

1974 ◽  
Vol 32 ◽  
pp. 532-533
Author(s):  
R. E. Ferrell ◽  
G. G. Paulson ◽  
C. W. Walker
Keyword(s):  
Thermal Treatment ◽  
Electron Diffraction ◽  
Sample Preparation ◽  
Crystal Structures ◽  
Water Samples ◽  
Environmental Samples ◽  
Short Review ◽  
Selected Area Electron Diffraction ◽  
Multiple Component

Selected area electron diffraction (SAD) has been used successfully to determine crystal structures, identify traces of minerals in rocks, and characterize the phases formed during thermal treatment of micron-sized particles. There is an increased interest in the method because it has the potential capability of identifying micron-sized pollutants in air and water samples. This paper is a short review of the theory behind SAD and a discussion of the sample preparation employed for the analysis of multiple component environmental samples.

Preparation of Thin Cadmium Telluride Samples for Electron Microscope Studies

1974 ◽  
Vol 32 ◽  
pp. 548-549
Author(s):  
T. J. Magee ◽  
J. Peng ◽  
J. Bean
Keyword(s):  
Electron Microscope ◽  
Sulfuric Acid ◽  
Solid State ◽  
Sample Preparation ◽  
Cadmium Telluride ◽  
Potassium Dichromate ◽  
Optical Components ◽  
The Past ◽  
Solid State Devices

Cadmium telluride has become increasingly important in a number of technological applications, particularly in the area of laser-optical components and solid state devices, Microstructural characterizations of the material have in the past been somewhat limited because of the lack of suitable sample preparation and thinning techniques. Utilizing a modified jet thinning apparatus and a potassium dichromate-sulfuric acid thinning solution, a procedure has now been developed for obtaining thin contamination-free samples for TEM examination.

A Freeze Shadow Technique for the Preparation of Soft Paint Latexes for Electron Microscopy

1977 ◽  
Vol 35 ◽  
pp. 314-315
Author(s):  
Earl R. Walter ◽  
Glen H. Bryant
Keyword(s):  
Sample Preparation ◽  
High Vacuum ◽  
Vacuum System ◽  
Latex Particles ◽  
New Methods ◽  
Diffusion Pump ◽  
Film Forming ◽  
Routine Basis ◽  
Distribution Studies ◽  
Preparation Techniques

With the development of soft, film forming latexes for use in paints and other coatings applications, it became desirable to develop new methods of sample preparation for latex particle size distribution studies with the electron microscope. Conventional latex sample preparation techniques were inadequate due to the pronounced tendency of these new soft latex particles to distort, flatten and fuse on the substrate when they dried. In order to avoid these complications and obtain electron micrographs of undistorted latex particles of soft resins, a freeze-dry, cold shadowing technique was developed. The method has now been used in our laboratory on a routine basis for several years.The cold shadowing is done in a specially constructed vacuum system, having a conventional mechanical fore pump and oil diffusion pump supplying vacuum. The system incorporates bellows type high vacuum valves to permit a prepump cycle and opening of the shadowing chamber without shutting down the oil diffusion pump. A baffeled sorption trap isolates the shadowing chamber from the pumps.

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