Isopropanol preservation of biological samples for subsequent DNA extraction and reassociation studies
✍ Scribed by Adrian V. Rake
- Publisher
- Elsevier Science
- Year
- 1972
- Tongue
- English
- Weight
- 218 KB
- Volume
- 48
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
One of the problems connected with the storing of tissue in the field and the transportation of tissue by mail is the method of preservation of that tissue. Dry ice of course is impract'ical in the field and shipped tissue often arrives long after the ice has gone. On the other hand, various preservat,ives commonly used, such as formaldehyde, while preserving the gross integrity of the tissue reacts with the DNA4 and in so doing alters the Watson-Crick hydrogen bonding of the DNA strands (1). This reacted DNA has altered reassociation characterist'ics to such an extent, even after short periods of t,ime in formaldehyde, tbat DNA-DNA reassociation studies, homologous or heterologous, are meaningless.
Arrighi et al.
(2) published some information about the effects of other t,ypes of preservatives on DNA from mammalian tissues, cultured cells, and bacteria. The ultraviolet absorption specka, ratios, denaturation properties, or buoyant, density in cesium chloride gradients were evaluated for changes in the DNA due to these preservatives. They found that the most effective and convenient fixative studied was isopropanol. Unfortunately, although suggestive, their m&hods are not overly sensit,ive to Watson-Crick hydrogen bonding alterations and do not prove that the DNA fixed in isopropanol was in fact suitable for DNA-DNA reassociation techniques wit.11 lit,tle or no chemical alterations of the bases in the DNA strands. Here DNA is shown to be unaltered by fixation in isopropanol and useful for DNA-DNA reassociation studies.
MATERIALS
Escherichia coli bacteria (strain B,-,) were grown in nutrient broth to stationary phase, harvested by centrifugation, washed in dilute salt solution, and suspended in 95% isopropanol; 5 to 14 days later the cells were recentrifuged and washed twice with dilute salt and the DNA, after