Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 3 de 3
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
PLoS One ; 8(10): e77172, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24194867

RESUMO

Mutations tagged by transposon insertions can be readily mapped and identified in organisms with sequenced genomes. Collections of such mutants allow a systematic analysis of gene function, and can be sequence-indexed to build invaluable resources. Here we present Mu-seq (Mutant-seq), a high-throughput NextGen sequencing method for harnessing high-copy transposons. We illustrate the efficacy of Mu-seq by applying it to the Robertson's Mutator system in a large population of maize plants. A single Mu-seq library, for example, constructed from 576 different families (2304 plants), enabled 4, 723 novel, germinal, transposon insertions to be detected, identified, and mapped with single base-pair resolution. In addition to the specificity, efficiency, and reproducibility of Mu-seq, a key feature of this method is its adjustable scale that can accommodate simultaneous profiling of transposons in thousands of individuals. We also describe a Mu-seq bioinformatics framework tailored to high-throughput, genome-wide, and population-wide analysis of transposon insertions.


Assuntos
Biologia Computacional/métodos , Elementos de DNA Transponíveis/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação/genética , Sequência de Bases , Biblioteca Gênica , Dados de Sequência Molecular , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase/métodos
2.
Methods Mol Biol ; 1057: 157-66, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23918427

RESUMO

The UniformMu transposon population is a large public resource for reverse genetics and functional genomics of maize. Users access the collection of UniformMu genetic stocks that are freely distributed by the Maize Cooperation Stock Center using online tools maintained at MaizeGDB.org. Genetic and molecular analyses of UniformMu stocks (UFMu insertion lines) typically require development of genotyping assays that use a gene-specific polymerase chain reaction (PCR) to follow segregation of transposon insertions in genes of interest. Here we describe methods for accessing the resource and recommended protocols for genotyping of transposon insertion alleles.


Assuntos
Elementos de DNA Transponíveis/genética , Técnicas de Genotipagem/métodos , Mutagênese Insercional/genética , Polinização , Reação em Cadeia da Polimerase , Zea mays/genética , Zea mays/crescimento & desenvolvimento , Zea mays/fisiologia
3.
BMC Genomics ; 8: 116, 2007 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-17490480

RESUMO

BACKGROUND: Gene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations. RESULTS: Transposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill. CONCLUSION: We show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.


Assuntos
Bases de Dados Genéticas , Inativação Gênica , Mutagênese Insercional/métodos , Sitios de Sequências Rotuladas , Zea mays/genética , Sequência de Bases , Análise por Conglomerados , Biologia Computacional , Elementos de DNA Transponíveis/genética , Dados de Sequência Molecular , Fosfogluconato Desidrogenase/genética , Sementes/genética , Análise de Sequência de DNA
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...