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

469sequences. DGGE and TGGE detect DNA sequence diversity based on theirstability toward denaturing conditions. Sequences differing in G+C content orneighbouring nucleotide interaction may produce different banding-patterns.For denaturing conditions, DGGE uses chemical agents (eg. urea) while TGGEutilises temperature. Given the ease of high-resolution outcomes, both methodshave been widely used for comparison of DNA structure (especially based on16S rDNA – PCR products) within complex microbial communities (Heuer etal. 1999).Diversity of thermal denaturation and reassociation of soil microbial DNASoil DNA may provide genetic information about the non-culturable bacteriain soil. DNA heterogeneity can be determined by thermal denaturation andreassociation. Denaturation of homologous, single-stranded DNA followssecond-order reaction kinetics. The fraction of denatured DNA is usuallyexpressed as a function of the product (Co t) of DNA nucleotide concentration(Co) in moles per liter and the reaction time ( t) in seconds. Under definedconditions Co tfor a half-completed reaction (Co t1/2) is proportional to the genomesize or the complexity of DNA. The complexity is defined as the number ofnucleotides in the DNA of a haloid cell, without repetitive DNA. The diversitycan be expressed by using Co t1/2as a diversity index (Torsvik et al. 1990).Molecular microbial diversity usingrRNA)small-subunit rRNA genes (SSUThe limitations for culture- or physiological- based measurements of soil microbialdiversity are results much less than real or estimated population. Developmentsin molecular ecology provide nucleic acid-based methods that do not requirecell cultivation and/or physiological measurement. The methods thenrevolutionise the present concept of diversity that also covers non-culturableorganisms (Amann et al. 1991, Amann et al. 1995, Ward et al. 1995).In eubacteria, rRNA genetic loci include 16S, 23S, and 5S genes, whichare separated by two internal transcribed spacer (ITS) regions. DNA sequencesin the 16S-23S spacer are known to exhibit a great deal of sequence and lengthvariation (Jensen et al. 1993). Previous researchers have shown that thepresence of uncultured microorganisms can be recognised using analyses ofthe small-subunit rRNA genes. SSU rDNA clonal libraries are constructed byusing DNA extracted from soil. SSU rRNA genes are amplified using theuniversal SSU rDNA primers. The PCR products are then sequenced. Thesequences are compared to sequence databases and statistically analysed. Themicrobial diversity is illustrated by a phylogenetic tree analysis of 100 SSUrDNA sequences (Borneman & Tripplet 1997, Borneman et al. 1996)