In situ and Ex situ Conservation of Commercial Tropical Trees - ITTO

468Nucleic acid isolation and extraction from environmental samplesWithout culturing, diversity of un-culturable soil microbes could be assessed bynucleic acid information. Some methods have been developed to provide quickand easy steps for nucleic acid isolation and extraction from environmentalsamples (Torsvik et al. 1990, 1994, 1996, Zhou et al. 1996). The extractedDNA, which is a complex mixture of microbial community origin, may be usedfor direct amplification or for nucleic acid hybridisation. Purification from humicacids or clay interactions were the most difficult tasks in the past; however, insome cases extraction (e.g., using full CTAB or phenol-chloroform) is not alwaysnecessary, especially for PCR template (Thies et al. 1999). Simple methods ofsoil DNA extraction were developed by Zhou et al. (1996) thanks to commercialdevelopment of soil DNA extraction kits by which extraction could be completedwithin half an hour, even if sample with high content of humic acids (e.g.,MoBio TM products).Polymerase Chain Reactions (PCR)Molecular methods have revolutionised the study of microorganisms in situ,none more so than PCR, since its specificity and its ability to detect the presenceof very low target organisms within a large background of other microbialpopulations (Ward et al. 1995). The development of this method as a powerfultool is supported by the development of target-specific primers. However,random primers (such as arbitrary – primer with G: C> 50%) could be used forgenerating random PCR (RAPDs).PCR produces a fingerprint or profile for any particular target genome.Nevertheless, there are limitations to this technique and the fingerprint patternof the PCR product sequence may vary due to subtle variation during theamplification conditions.Further analysis of PCR productsFurther electrophoresis of PCR products in polyacrylamide gels also providesa finer resolution of discrimination on DNA sample diversity. Unlike agarosegel, polyacrylamide gel can distinguish the electrophoretic mobility ofmacromolecules on the basis of their shape, which is beyond the capacity ofroutine hybridisation methods.Several methods including single-strand conformational polymorphism(SSCP) (Dewit & Klatser, 1994), denaturing gradient gel electrophoresis(DGGE) (Sheffield et al. 1989), temperature gradient gel electrophoresis(TGGE) (Wartell et al. 1998), and hetero-duplex mobility assays (HMA) (Espejo& Romero 1998) can be used for single-base discrimination of amplified DNA