RESUMO
A method is described that uses arbitrarily primed PCR followed by many cycles of amplification under stringent conditions and selection by computational means to obtain a set of sequence tags that can be used for the comparison of metagenomes. Relative to unselective shot-gun sequencing, the results are small data sets that can be csompared electronically or plotted as scattergrams that are simple to interpret. The method can be used to compare groups of samples of any size to build in-house databases from which, for example, the provenance of trace soil samples may be inferred. The method also allows for selection of primers with locus-specificity and an example is given in which a South Australian sequence-related to a Portuguese thermophile (Rubrobacter radiotolerans) is extracted and tested on a set of soils.
Assuntos
Actinobacteria/genética , Metagenoma/genética , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Actinobacteria/isolamento & purificação , Austrália , Primers do DNA/genética , Microbiologia do SoloRESUMO
The Neurospora homologue msh-2 of the Escherichia coli mismatch repair gene mutS was mutated by repeat-induced point mutation (RIP) of a 1.9-kb duplication covering 1661bp of the coding sequence and 302 bp 5' of the gene. msh-2(RIP-LK1) exhibited a mutator phenotype conferring a 17-fold increase in the frequency of spontaneous mitotic reversion of his-3 allele K458. In msh-2(RIP-LK1) homozygotes, recombination frequency at the his-3 locus increased up to 2.9-fold over that in msh-2(+) diploids. Progeny of crosses homozygous msh-2(RIP-LK1), like those from crosses homozygous msh-2(+) frequently had multiple patches of donor chromosome sequence, suggesting that patchiness in msh-2(+) crosses is not explained by incomplete repair of heteroduplex DNA by MSH-2. These findings are consistent with data from the analysis of events in a Neurospora translocation heterozygote that suggested multiple patches of donor chromosome sequence arising during recombination reflect multiple template switches during DNA repair synthesis.
