RESUMO
We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes.
Assuntos
5-Metilcitosina/análise , Enzimas de Restrição do DNA , DNA Bacteriano/isolamento & purificação , DNA Fúngico/isolamento & purificação , DNA de Plantas/isolamento & purificação , DNA de Protozoário/isolamento & purificação , DNA Viral/isolamento & purificação , Desoxirribonuclease HpaII , Proteínas de Escherichia coli , Genética Microbiana/métodos , Genômica/métodos , Metagenoma , Ilhas de CpG/genética , Metilação de DNA , Enzimas de Restrição do DNA/isolamento & purificação , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/genética , DNA Fúngico/genética , DNA de Plantas/genética , DNA de Protozoário/genética , DNA Viral/genética , Desoxirribonuclease HpaII/isolamento & purificação , Desoxirribonuclease HpaII/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Biblioteca Gênica , Humanos , Microbiota/genética , Análise de Sequência de DNA , Escarro/microbiologia , Especificidade por SubstratoRESUMO
The MspI restriction endonuclease is a type II restriction enzyme. Unlike all other restriction enzymes with known structures, MspI recognizes the palindromic tetranucleotide sequence 5'-C/CGG and cleaves it as indicated by the '/' to produce DNA products with 5' two-base overhangs. Owing to the nature of its cleavage pattern, it is likely that MspI would represent a new structural class of restriction endonucleases. Crystals of the dimeric MspI restriction enzyme bound to a duplex DNA molecule containing the specific recognition sequence have been obtained by vapor-diffusion techniques in the presence of polyethylene glycol as precipitant. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 50.2, b = 131.6, c = 59.3 A, beta = 109.7 degrees. The crystals contain one dimeric complex in the asymmetric unit. A complete native data set has been collected to a resolution of 2.05 A by cryo-crystallographic methods, with an R(merge) of 4.0%.
