RESUMO
We have developed a method to suppress the PCR amplification of repetitive sequences in whole chromosome painting probes by adding Cot-1 DNA to the amplification mixture. The repetitive sequences in the Cot-1 DNA bind to their homologous sequences in the probe library, prevent the binding of primers, and interfere with extension of the probe sequences, greatly decreasing PCR efficiency selectively across these blocked regions. A second labelling reaction is then done and this product is resuspended in FISH hybridization mixture without further addition of blocking DNA. The hybridization produces little if any non-specific binding on any other chromosomes. We have been able to successfully use this procedure with both human and rat chromosome probes. This technique should be applicable in producing probes for CGH, M-FISH and SKY, as well as reducing the presence of repetitive DNA in genomic libraries.
Assuntos
Coloração Cromossômica , Cromossomos de Mamíferos/genética , Sondas de DNA/genética , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Animais , Bandeamento Cromossômico , Biblioteca Gênica , Humanos , Hibridização in Situ Fluorescente , Metáfase , Hibridização de Ácido Nucleico , RatosRESUMO
As templates for chromosome microdissection, meiotic cells offer several advantages over mitotic cells. The pairing of homologous chromosomes at the metaphase plate of the first meiotic division allows the simultaneous isolation of two copies of the same chromosome, and the sex chromosomes are easy to identify in male meiotic cells. We report on a method for making fluorescence in-situ hybridization (FISH) probes from dissected meiotic chromosomes.
