DNA from Bacteria

Of all the various nucleic acid molecules within the cell the three that are most often isolated are chromosomal DNA, mRNA from tissue or cells and plasmid DNA from bacteria. In all cases the principles for isolation are similar and can be divided into three stages. 

Examine the procedure described in Experiment 21 for the isolation of plasmid DNA from bacteria. It is very different from the procedure used to isolate chromosomal DNA in this experiment. Could it be used to isolate chromosomal DNA from bacteria Why or why not Explain why such different procedures are used for isolating these two types of DNA. 

DNA from Vertebrates For the differentiation of DNA from bacteria and the DNA from vertebrates, analysis can utilize primers specific for genomic or mitochondrial DNA. Real-time PCR with primers specific for conserved sequences of the mitochondrial cytochrome b gene detects DNA from all mammals and most fishes. For the detection of chondrichtyes (ray, shark) and prawns, other primers have to be chosen. The analysis can be performed with unlabeled primers. Amplicons can be detected by SybrGreen. For most instruments, control samples with known concentrations can be used as markers, and using these the DNA concentration of samples is calculated automatically. 

Figure 16.1 Viruses vary in size and shape from the simplest satellite viruses (a) that need another virus for their replication to the T-even bacteriophages (d) that have developed sophisticated mechanisms for injecting DNA into bacteria. Four different virus particles are shown to scale.

In practice, the situation isn t quite that simple. DNA samples taken from a victim are almost certain to be contaminated with DNA from fungi or bacteria. Certain dyes can combine with restriction enzymes, causing them to cut in the wrong places. Finally, DNA may decay in a warm or moist environment. 

A third mechanism of plasmid transfer is by transformation, which is the ability of certain microorganisms to acquire naked DNA from the environment. This is limited to certain bacteria, notably Neisseria gonorrhoeae, which is naturally competent to acquire DNA in this manner. Neisseria gonorrhoeae strains have the ability to recognize DNA from their own species, and are thus selective in their acquisition of naked DNA from the environment. 

Ahmad D, R Masse, M Sylvestre (1990) Cloning, physical mapping and expression in Pseudomonas putida of 4-chlorobiphenyl transformation genes from Pseudomonas testosteroni strain B-356 and their homology to the genomic DNA from other PCB-degrading bacteria. Gene 86 53-61. 

DNA,83 which produces one million or more copies of amplified DNA in a short time. For identification of bacteria, PCR can be used to amplify DNA either after extraction from a sample or after lysis of the cells.83,84 Methods using washing, filtration, or magnetic beads with specific antibodies can be used to collect bacterial cells for PCR.85,86 PCR can be modified for the detection of bacteria from various sources32 and can even amplify DNA from dead cells.87... 

Most often proteins are the bacterial biopolymers studied using MALDI MS either from fractions or whole cells. They are not the only isolated cellular biopolymers studied by MALDI, nor the first. Very soon after the introduction of MALDI there were a few reports of the analysis of bacterial RNA or DNA from bacterial fractions. One of the first applications of MALDI to bacteria fractions involved analysis of RNA isolated from E. coli,4 Other studies included analysis of PCR-amplified DNA,5 6 DNA related to repair mechanisms7 and posttranscriptional modification of bacterial RNA.8 While most MALDI studies involve the use of UV lasers, IR MALDI has been reported for the analysis of double stranded DNA from restriction enzyme digested DNA plasmids, also isolated from E. coli.9... 

Plasmids and DNA Repair. Plasmids are extrachromosomal genetic elements that are composed of circular double-stranded DNA. In bacteria some can mediate their own transfer from cell to cell by conjugation they contain a set of Ira genes coding for tube-like structures, such as pili, through which a copy of plasmid DNA can pass during transfer. Plasmids range in size from 1.5 to 200 million daltons. The number... 

The newer applications involve the field of biotechnology. Proteins produced by genetically altered organisms such as bacteria must be examined to verify that they are identical to the same proteins produced by humans. Also, analysis of DNA from crime scenes is relatively recent. Indeed, DNA analysis and fingerprinting are powerful tools in modern forensics. 

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