GENOMIC DNA

This paper is a study of the genetic material derivable from the simplest (and among the oldest) life forms, principally bacteria. Many definitions from microbiology are developed and interrelated, and procedures for the study of DNA from simple organisms are described. The history of the elucidation of the microbiological significance of prokaryotic DNA, an early and persistent building block of life on earth, is revisited from many perspectives.

A genome consists of "all the genetic material in a cell" (8 :204). A prokaryote is an "organism that lacks a membrane-bound nucleus" (8:216); although some argue that there is much more to a prokaryote than that, and rather more subtle differences from eukaryotes than the latter having a membrane-bound nucleus (18:2-3). Heritage et al. give a six-page comparison (8:2-7).

DNA, centrally, is a nucleic acid (deoxyribonucleic acid, hence DNA), whose function along with other nucleic acids is to "store, maintain, and transmit genetic information" (16:103). Very fundamentally, should DNA and its sister nucleic acids not be able, for any reason, to pass along an organism's existing traits during reproduction or division, the existing organism must either change (mutate or evolve) or die, and possibly both. Otherwise, the offspring would be the same as the parents in all demonstrable characteristics. Among the possibilities of change, of course, is t

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ows: 1) Samples of bacteria are taken from a "wild-type strain" and a "mutant strain." 2) Segments of the genomic DNA of interest are amplified by PCR [methods unclear and unreported]. 3) "Base pair exchanges" are transferred in mismatches by mixing the wild-type and the mutant materials, and this is followed by a denaturation-renaturation cycle. 4) The sample is then analyzed by either parallel or perpendicular TGGE (temperature gradient gel electrophoresis). In detail, the isolation and analysis procedures described by Henco et al. (7:221-22) are the following: 1) Perform 35-40 cycles of PCR on 0.2-1.0 �g of boiled DNA: Initial cycle: 94(C for 180 s; 33-38 cycles: 94(C (denaturation) for 60 s; 1 cycle: 55(C for 60 s (primer annealing); 1 cycle: 72(C for 90 s (primer extension); Final cycle: 72(C for 240 s (more primer extension). 2) Transfer to a new Eppendorf tube. Add wild-type PCR product, if none is present in the genome; add TNE-buffer. 3) Mix with equal vol. of phenol/ chloroform and centrifuge (3-5 min.). Transfer aqueous top layer to new centrifuge tube with 2 vols. (300-400 �L) of chloroform: isoamyl alcohol. 4) Transfer supernatant into a screw-top Eppendorf tube; precipitate the DNA with 2.5-3

Category: Science - G