Standard operating procedure for pulsenet pfge of listeria monocytogenes. Pulsedfield gel electrophoresis pfge is a macrorestriction profiling technique based on the digestion of dna with restriction enzymes that cut dna infrequently rarecutters of 6 or 8 bp recognition sites. Results all isolates were collected from hospitalized patients with an average age of. Pulsed field gel electrophoresis pfge technique and its use in molecular biology 406 introduction much of the rapid progress that is being made in molecular biology today depends upon the ability to separate, size and visualize dna molecules. Chromosomal fingerprinting by means of pulsed field gel electrophoresis is considered as a gold standard method for subtyping and the most reliable among molecular techniques. Examination of chromosome 4, the dot chromosome in drosophila, using pulsd field gel electrophoresis. Protocol for pulsed field gel electrophoresis pfge protocol griffith 2000 a 1520 liter natural seawater sample is passed through a glass fiber prefilter gelman ae and a 0. Pulsedfield gel electrophoresis pfge pulsenet methods.
Pulsed field gel electrophoresis pfge this technique was developed by shwartz and cantor in 1984. Scope to provide the pulsenet participants with the same procedure for performing pfge of botulinum toxin producing clostridia, thus ensuring interlaboratory comparability of the generated results. Pdf pulsedfield gel electrophoresis for disease monitoring and control. Use of pulsed field gel electrophoresis to determine the source of microbial contamination of central venous catheters m.
I am on sabatical leave january june99 and thus not teaching this term. Standardized rapid pulsed field gel electrophoresis pfge protocols for the subtyping of escherichia coli o157. Gel electrophoresis of digested dna documentation of pfge gel pulsed field gel electrophoresis pfge has enabled progress in cancer research, food safety, public health, quality control, and genome. Pulse field gel electrophoresis pfge is a powerful genotyping technique used for the separation of large dna molecules entire genomic dna after digesting. Use of pulsed field gel electrophoresis to determine the. C to wash plugs 2 times with 10 ml of heated distilled water. The digestion products are then analysed on an agarose gel by applying an electric field. Pulsefield gel electrophoresis pfge is the reference method employed for bacterial genotyping, but it is commonly used for typing mainly pathogenic bacteria. Pdf pulsed field gel electrophoresis pfge is a technique for the fractionation of highmolecularweight dna ranging from 10 kb to 10 mb by. Part of the methods in molecular biology book series mimb, volume 1. Place gel in chamber and equilibrate at the running temperature for 0. Pfge is a form of rflp typing in which the bacterial genome is digested with rare cutting enzymes. Pulsedfield gel electrophoresis typing of escherichia. In a uniform magnetic field, components larger than 50kb pass a route through the gel.
The scientist takes bacterial cells from an agar plate. Pulsed field gel electrophoresis pfge as dna larger than 1520kb migrating through a gel essentially moves together in a sizeindependent manner, the standard gel electrophoresis technique was unable to separate very large molecules of dna effectively which led to the practice of pulsed field gel electrophoresis. Gautom washington state department of health, public. The principle of pulsed field gel electrophoresis pfge. Professorlevel mthe fields ofsocialpersonality, cognitive psychologyincludingperception andinformation processing, andor behavioral neuroscience. Pulsed field gel electrophoresis pfge enables large dna molecules, including whole chromosomes, to be easily separated and visualized 1,2. The optimization of a rapid pulsed field gel electrophoresis protocol for the typing of acinetobacter baumannii, escherichia coli and klebsiella spp. Rapid pulsed field gel electrophoresis protocol for typing of escherichia coli o157.
The use of pulsed field gel electrophoresis in listeria. Electrophoresis and results the use of pulsed field electrophoresis. Pfge with endonuclease xbai has been widely recognized as a sensitive means for epidemiological analysis of salmonella serovars. Pulsedfield gel electrophoresis pfge technique and its.
Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid dna molecules by applying to a gel matrix an electric field. Standard operating procedure for pulsenet pfge of escherichia coli o157. Pulsedfield gel electrophoresis is a strategy for resolving large fragments of dna for analysis. Pulsed field gel electrophoresis is a procedure used for the separation of massive deoxyribonucleic acid dna molecules through applying to a gel matrix an electric subject that. Pulsed field gel electrophoresis pfge is considered as the gold standard for s. Pulsed field gel electrophoresis pfge has been widely applied to characterize numerous bacteria. Pulsed field gel electrophoresis pfge is a methodology developed by schwartz and cantor in 1984. This protocol describes pulsedfield gel electrophoresis pfge, a method developed for separation of large dna molecules. It permitted to resolve mixes of dna molecules larger than 20 kb and up to 5 mb in agarose gels, which narrowed the gap between the resolution of conventional electrophoresis. Pdf ingel dna radiolabelling and twodimensional pulsed. The primary infecting escherichia coli strains from 156 women with communityacquired uncomplicated urinary tract infection uti randomized to pivmecillinam or placebo and the e.
Chromosomal fingerprinting by means of pulsed field gel electrophoresis is considered as a gold standard method for subtyping and the most reliable among molecular techniques 15. Pulsedfield gel electrophoresis pfge is a technique by which genomic dna is isolated from the organism of interest followed by restriction enzyme analysis. In gel dna radiolabelling and twodimensional pulsed field gel electrophoresis procedures suitable for fingerprinting and mapping small eukaryotic genomes. We will be revising the content, order of the lectures, and text for this course during the summer 99.
Pulsed field gel electrophoresis pfge is a powerful molecular typing technique by which genomic dna is isolated from the organism of. Request pdf pulsedfield gel electrophoresis conventional agarose gel electrophoresis is capable of separating dna fragments with sizes of up to 2030 kbp. Modified pulsenet procedure for pulsedfield gel electrophoresis of select gram negative bacilli. This procedure can be used to evaluate genomic dna gdna to ensure that it is of high quality. Pulsed field gel electrophoresis pfge is a technique for the fractionation of highmolecularweight dna ranging from 10 kb to 10 mb by electrophoresis in agarose gel with an electric field that alternates pulsates in two directions. Since dna is a large molecule, it would end up migrating to a single band. Preparation and separation of mammalian dna by pulsed. Hence, dna is cut using specific restriction endonucleases. This procedure can be used to evaluate genomic dna gdna to ensure that it is of high quality and high. Conventional electrophoresis can effectively separate dna fragments up to 20 kb. Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new twodimensional pulsed field gel electrophoresis 2dpfge protocols of.
H7, salmonella serotypes, and shigella species are described. Plasmid profile and pulsedfield gel electrophoresis. Pulsed field gel electrophoresis pfge is a technique for the fractionation of highmolecularweight dna ranging from 10 kb to 10 mb by electrophoresis in agarose gel with an electric field. Rapid pulsedfield gel electrophoresis protocol for typing. Also the issues commonly influencing the quality of pfge data and its analysis are discussed.
Pulsenet investigates bacterial isolates from sick people, contaminated food, and the places where food is produced. Ingel dna radiolabelling and twodimensional pulsed field. C shaker incubator and adjust shakerincubator to 50c. Silvia arancia, paola chiani, clarissa ferreri, antonella maugliani, fabio. Rapid pulsedfield gel electrophoresis protocol for. The dna molecules migrate in straight lanes, due to the homogeneous fields, and dna molecules from 50 kb to 6,000 kb can be separated by adjusting the frequency of the gel rotation. Standardization of pulsedfield gel electrophoresis. Standardized rapid pulsedfield gel electrophoresis pfge protocols for the subtyping of escherichia coli o157. Molecular typing of methicillinresistant staphylococcus. Pdf this protocol describes pulsedfield gel electrophoresis pfge, a method developed for separation of large dna molecules. H7, escherichia coli nono157 stec, salmonella serotypes, shigella sonnei and shigella flexneri. H7 and other gramnegative organisms in 1 day romesh k. A bacterial isolate is a group of the same type of bacteria. Electrophoresis system instruction manual and application guide.
Pulsedfield gel electrophoresis pfge is a technique used for the separation of high molecular weight restriction fragments from digested bacterial genomic dna on a gel matrix by applying an. In 1984, schwartz and cantor invented pulsed field gel electrophoresis. Application of pulsedfield gel electrophoresis pfge in bacillus. To provide the pulsenet participants with a standardized procedure. Pulsed field gel electrophoresis pfge is a technique for the fractionation of highmolecularweight dna ranging from 10 kb to 10 mb by electrophoresis in agarose gel with an electric field that alternates pulsates. This protocol describes pulsedfield gel electrophoresis pfge. A 15 20 liter natural seawater sample is passed through a glass fiber prefilter. These protocols are used by laboratories in pulsenet, a network of state and local health departments, and other public health laboratories.
Key words central venous catheter 9 pulsed field gel electrophoresis introduction. However, larger fragments will comigrate and appear as a large band at the top of the gel when imaged. To describe the oneday 2426 hour standardized laboratory protocol for molecular subtyping of. This video provides the best explanation about pulsed field electrophoresis, it helps you to fully understand the principle and the technique. Pulsed field gel electrophoresis pfge is considered the gold standard for molecular typing of methicillinresistant staphylococcus aureus mrsa. The most common technique for this purpose is that of standard agarose gel electrophoresis. The application of pulsed field gel electrophoresis in. Whereas standard dna gel electrophoresis commonly resolves fragments up. Drug susceptibility test, biofilm formation, and pulsedfield gel electrophoresis pfge were also performed. Dna, being negatively charged moves towards anode in an electric field during electrophoresis.
1445 26 744 148 735 1251 833 979 645 590 274 352 47 1091 135 61 979 589 225 289 374 1016 549 915 1120 875 923 1428 793 718 939 352 50 725 711 1254 727 973 150 641 895 982 724 44