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PLASMID DNA.
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Definition, genetic roles, step-by-step isolation process, genetic engineering.... More...
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Paper Abstract:
Definition, genetic roles, step-by-step isolation process, genetic engineering.
Paper Introduction:
PLASMID DNA
Introduction
Plasmid DNA is one of three related genetic elements of all living organisms, chromosomal DNA, plasmids or plasmid-DNA, and the so-called "transposable elements" that co-exist with the other two and can enhance genetic instability among organisms (5:28). Plasmids have become crucial to medicine and genetics because they are capable of being transformed artificially and of transmitting on their own, from generation to generation, certain resistances or immunities to antibiotic chemicals or other environmental inputs.
The Role(s) of Plasmid DNA
Postgate introduces the study of microbiology by revealing that 90 percent of living material lies in the microorganisms; thus, they are responsible
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Applied and Environmental Microbiology. Totowa, NJ: Humane Press, Inc.; 1994: p. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. coli which contain one "segment ofchromosomal DNA" (and pass the "maleness" quality during conjugation); andsome genes usually found in plasmids are occasionally found within(bacterial) chromosomes (3:195). k) Mix gently by inversion or swirling, intermittently, 1 min. reported that "geographical differentiation accountsfor only about 2% of the total genetic diversity in E. Of the genomic elementsof chromosomes, plasmids, and transposable elements, plasmids appear tohold the greatest promise for humans, because they appear to be the mostrelated to medicinal and immunological traits that can be introduced andthen passed as hereditary characteristics. The method described occurred in parts or steps, as follows (2:549-55 ):1) Growth of cells (overnight) in an M-17 broth at 32 ?C. p) Centrifuge. And human insulin was cleared formarketing in both the United States and the United Kingdom in 1982 (7:187). Of interest, this method also exposed to view the (one) chromosomalDNA band, which the authors suggest "may mask the presence of plasmid DNAduring screening experiments" [because the chromosomal (genomic) DNA isabout the same size--about 3 Mdal--as the large plasmids being sought,which in this case ranged from 6 to 8 Mdal] (2:552).The Atomic Force Microscope Method. l) Add 2 Molar tris-hydrochloride (pH = 7. report that transfering the entire chromosome of E. During World War II, while penicillin was being developed andexploited first in Great Britain and then in the United States, scientistsalso learned that use of single drugs to treat an illness (specificallysulphanamides to treat gonorrhea) could have disastrous effects, becausethe gonorrhea organism was able to develop immunity fairly quickly (7:9 ).The whole lesson had to be relearned as late as the 198 s, when atetracycline-resistant gene spread to a variety of bacteria, and it settledwithin Neisseria gonorrhoeae; as a direct result, cases of drug-resistantgonorrhea fail to respond to treatment even today on every continent of theworld (5:53-54). Some of thebasic methodologies for isolating and studying plasmid DNA, which areeasily recognizable as elaborate and costly, are summarized below. In 1996, workers at the Oak RidgeNational Laboratory reported procedures by which they isolated, viewed, andphotographed plasmid DNA, connected to a "dimeric globular protein" ofknown sequence and a molecular weight of 62, --known as EcoRI (1:8826).The method obviously requires the use of an atomic force microscope,available to very few, which additionally had to be used within anenvironmental chamber to maintain (by dry nitrogen purging) the relativehumidity at 15 percent (1:8827). E. s) Incubate at ?C. g) Mix immediately. Philosophical Transactions of the Royal Soc. Further payoffs for medicine, plastic/fibers/and othermaterials and for the study of natural history continue to beckon toresearchers, promising both riches and acclaim. Some plasmids contain genes normally located in the chromosome,such as the F' plasmids of E. 4) Examine 5 to 1 µL by agarose gel electrophoresis [essentiallySouthern blotting and hybridization from the 197 's]. Hartl et al. Cambridge, UK: Cambridge University Press; 1992. The basic DNA elements--chromosomes, plasmids, and the transposableelements--are not completely and neatly isolated with respect to theirfunctions. t) Centrifuge. The first, chemical method describedhere is said by its developers to be both rapid and simple (2:549). of London, series B. Protocols for Gene Analysis. Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules. New York: John Wiley & Sons; 199 . Allison, D. J. Among the better known plasmid-borne genes are ones that specify (passdirections for) resistance to various antibiotics (7:18 ); and someplasmids can pass between genera and even families of bacteria, so a drug-resistant Escherichia coli, for example, can pass the property of drugresistance to a Salmonella and vice versa without the second organism everbeing exposed to the drug chemical directly and then having to produce aresistance, if it could (7:181). Postgate, however, has in one context referred to aplasmid as a "mini-chromosome"--anywhere from a quarter the size of achromosome to less than a hundredth (7:18 ). Kingsbury, D. Entire industries ofmedicine, biochemistry, and genetic engineering have ensued. m) Gently mix for 3 min. History of Methodological Developments Gene transfer in bacteria has been understood since (was discoveredin) the 194 s, when experiments with E. G. Thereader may judge; but it appears to be neither, requiring elaborateequipment and considerable time (days). coli showed that certain mutantstrains were unable to live on a rather minimal growth medium thatsupported the host population very adequately (5:54). of isopropanol. Mol. The methods of isolating and viewing plasmid DNA reported aboveherein by Anderson and McKay in 1983 (2) and by Allison et al. M. 5. They are now being seen andphotographed by remarkable microscopy. Introductory Microbiology. in 1996 (1)both indicate that the methods are elaborate, expensive, machineryintensive, time-consuming, and tedious; and their development continues tothe present time. P.; Kerper, P. 312:191-2 4; 1986. ). J. of the Natl. Literature Cited1. Therefore, several biotechnology or geneticengineering firms set about finding ways to clone the gene for humaninsulin. coli gene to keep the bacteriumfrom killing it as an unwanted alien. u) Remove excess isopropanol and resuspend in EDTA (pH = 7.5). T.; and Wagner, G. Interferon, "the material involved in immunity," is now another miracledrug developed in a similar fashion which awaits further testing,licensing, and clearance for the treatment of various viral diseases andeven cancer (7:187). 4. J.; Spain, J. "High frequency strains" of bacteria(called Hfr strains), during conjugation, pass their maleness factors(rapidly, at high frequency) within the DNA of the chromosome (7:18 ).Heritage et al. In: Harwood, A. of Sci. Hartl, D. L.; Medhora, M.; Green, L.; and Dykhuizen, D. The gels were then stainedwith .5 µg of ethidium bromide per mL., and photographs were taken througha red filter on Polaroid 1 7 film (2:55 ). Cambridge, UK: Cambridge University Press; 1996. They did indeed persuade a strain of E. M NaCl. 93: 8826-29; 1996. Heritage, J.; Evans, E. A. 2nd ed. Temperature gradient gel electrophoresis (TGGE) for the detection of polymorphic DNA and RNA. The result is a new plasmid, and the alien DNA in the newplasmid is said to have been "cloned" (7:185). 2. V.; and Killington, R. Inthe majority of conjugating bacteria, however, which are rare in toto,maleness is passed by the extrachromosomal plasmid DNA (7:18 ). e) Add EDTA. In the past 2 years or so, genetic engineers have found methods ofusing "restriction enzymes" to cut DNA strands at predictable places, addon "alien DNA" parts--even from another species, and smooth over or repairthe damaged DNA parts, if any, with other enzymes called "DNA ligases"(7:184-85). W.; Thundat, T.; and Warmack, R. The steps are: a) Resuspend the pelleted cells in a sucrose-EDTA (pH =8. S.; Doktycz, M. 8. A.; Modrich, P.; Larimer, F. USA. Proc. Microbiology. h Incubate for 5 - 1 min. d) Incubate for 5 min. Postgate, J. In natural E. 98:5 3-17; 1975. Postgate rather quickly dismisses the genetic engineering "alarms"generated by microbiologists fooling about with various cloning experimentsin the 197 s, but he gives a thoughtful and prosaic delivery on what isprobably still the greatest success story: the production in the laboratoryin 198 , and now on a commercial scale, of artificial insulin (7:187).Insulin, a hormone essential for diabetics, of whom there are more than 6 million in the world, was once extracted straightaway from pigs, but therewere side-effects (7:187). M. PLASMID DNA Introduction Plasmid DNA is one of three related genetic elements of all livingorganisms, chromosomal DNA, plasmids or plasmid-DNA, and the so-called"transposable elements" that co-exist with the other two and can enhancegenetic instability among organisms (5:28). Detection of specific sequences among DNA fragments separated by gel electrophoresis. ) b) Warm to 37?C. G.; and McKay, L. Southern, E. 3. L. Isolation of Plasmid DNAThe Method of Anderson and McKay. q) Remove upper phase and extract with chloroform-isoamyl alcohol. 46:549-52; 1983. Changes occur principally through metabolism,growth, and reproduction, of course; and deoxyribonucleic acid (DNA) isessential to the coding and passage of genetic facts and fashions.Plasmids are extra-chromosomal elements of DNA and occur in all livingspecies (6:22). coli" (3:193). Anderson, D. Biol. For example, the majority of conjugating bacteria pass their"maleness" gene to a "female" or recipient, cell not by chromosomal orgenomic DNA transfer, but through the smaller, simpler plasmid DNA (7:18 ). c) Add lysozyme. Plasmids have become crucialto medicine and genetics because they are capable of being transformedartificially and of transmitting on their own, from generation togeneration, certain resistances or immunities to antibiotic chemicals orother environmental inputs. o) Add phenol, saturated with 3% NaCl, mix thoroughly. The electrophoresis was performed in a "multipart EDTA buffer (pH =8.1);" the gels contained .6% agarose; and the electrophoresis wasperformed at "1 V (3.6V/cm) for 5 h" (2:55 ). 6. The evolution of DNA sequences in Escherichia coli. 7. coli to make it, havingto first sandwich the gene into another E. The function of many otherplasmids remains unknown to microbiologists, who nonetheless have botheredto name them: "cryptic plasmids" (3:195). N NaOH. E. Resultingculture provided a "2% inoculum" [presumably by weight].2) The inoculum was placed in a "modified Elliker broth medium designatedlysis broth," and the strains of Streptococcus lacti were then "propagated"for 4 hr at 32 ?C, harvested by centrifugation (pelleted).3) A plasmid purification protocol ensued, in numerous steps, which theauthors elaborate in greater detail than reproduced here. r) Remove upper phase, precipitate with 1 vol. Microbes and Man. The electrophoretic techniques of E. at 37 ?C "to complete lysis." i) Vortex at highest setting for 3 s in "appropriate tube." j) Add fresh 3. [See below herein.] Plasmids have a variety of roles: some are rudimentary and arcane,truly prehistorical; others are modern, powerful, disturbing, andinvaluable. Plasmids contain genes for the replication of their host, for theirown functions (such as the formation of transfer tunnels duringconjugation, called "pili"), and for regulating host metabolism (e.g., theantibiotic resistance business) (3:195). n) Add 5. The Role(s) of Plasmid DNA Postgate introduces the study of microbiology by revealing that 9 percent of living material lies in the microorganisms; thus, they areresponsible for "most of the chemical changes that living things bringabout on this planet" (7:3). Henco, K.; Harders, J.; Wiese, U.; and Riesner, D. Southern for the isolation,blotting, hybridization, and viewing of DNA elements--still used in variousways to this day--were first reported in 1975 (8). 3rd ed. Previousto this (pre-1983), it seems, methods that had revealed very few plasmidshad included the following: lysis of the cells (to free the intracellularelements, genomic or otherwise); purification by "cesium-chloride/ ethidiumbromide density gradient centrifugation"; and electron microscopy. f) Add sodium docecyl sulfate-EDTA. coli, plasmids have been found to range in size from afew hundred base pairs to a few hundred kilobase pairs ("kilobases" orkbs); generally they fall into a "large" group (>4 kbs) or a "small" group(<7.5 kbs) (3:194). It is a method specificallyderived to isolate plasmid DNA larger than "3 megadaltons"; and previouslyundetected large plasmids were observed via this method (2:549). coli inthis way takes about 95 minutes, and that time is remarkably constant(5:54). Conclusions Plasmid DNA is an amazing organic material, dating from prehistoryand found today in all organisms in forms at least representative andfamiliar to that of its parents and progenitors. J., ed. Acad. 211-228.
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