ABSTRACT
ABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.
Subject(s)
Humans , Polymerase Chain Reaction/methods , DNA Primers/genetics , Primed In Situ Labeling/methods , Cytogenetic Analysis/methods , DNA Probes/genetics , Reproducibility of Results , In Situ Hybridization, Fluorescence/methods , Chromosomes, Human, X/genetics , Microdissection/methodsABSTRACT
Fragile sites (FS) are chromosomal regions where the normal compactation of chromatine is not observed. FRAXA (Fra Xq27.3, X sexual chromosome) is one of the most studied FS in humans. FRAXA is an expansion of the trinucleotide CGG located in the gene FMR-1. In cattle, sites of chromosomal fragility were reported in BTAX, associated with different pathologies and fertility impairment. Chromosomal microdissection has became a valuable tool for isolating chromatine fragments. In this work, it was combined the chromosomal microdissection technique with DOP-PCR in order to carry out a molecular analysis of the fragile chromosomal region BTAXq31-34. In that region, polymorphic DNA-RAPD sequences (GC rich) are present and sequences of the gene FMR-1 are missing. The results showed the usefulness of the microdissection-DOP-PCR technique for molecular characterization of fragile chromosomal sites in cattle.
Os sítios frágeis (FS) são regiões de cromossomo onde a compactação normal da cromatina não é realizada. O FRAXA (Fra Xq27.3, cromossomo sexual X) é um dos FS mais estudados em seres humanos. O FRAXA apresenta expansão do trinucleotídeo CGG localizado no gene FMR-1. Em bovinos, existem estudos informando sobre fragilidade cromossômica em BTAX associada com diversas patologias e alterações na fertilidade. A microdissecação cromossômica é uma valiosa técnica para isolar fragmentos de cromatina. Neste trabalho, combinou-se a técnica de microdissecação de cromossomo com DOP-PCR para executar a análise molecular da região do sitio frágil cromossômico BTAXq31-34. Naquela região estão presentes seqüências do polimorfo DNA-RAPD (rico em GC), em que as seqüências do gene FMR-1 estão ausentes. Os resultados mostram a utilidade da técnica de microdissecação-DOP-PCR para a caracterização molecular de sítios frágeis cromossômicos em bovinos.
Subject(s)
Animals , Cattle , Chromosome Fragile Sites , Chromatin/isolation & purification , Microdissection/methods , Microdissection/veterinary , X ChromosomeABSTRACT
Degenerate oligonucleotide primed PCR is an useful technique to amplify whole genome and its the applications for fluorescent in situ hybridization and comparative genomic hybridization (CGH) were reported. For the CGH, topoisomerase and sequenase were recommended to use for the better hybridization. But adding the enzymes to PCR reaction per every cycle is labor-intensive and can easily contaminate PCR reaction. This study was carried out to prove the possibility of application of DOP-PCR to CGH without use of sequenase. Several combinations of CGH e.g., DOP-PCR amplified normal DNA vs. DOP-PCR amplified normal DNA, DOP-PCR amplified normal DNA vs. non-DOP normal DNA, DOP-PCR amplified normal DNA vs. DOP-PCR amplified MCF-600 cell line DNA, and non-DOP normal DNA vs. non-DOP MCF-600 DNA were performed. In addition, randomly selected microsatellite loci were tested to know whether DOP-PCR covers whole genome amplification. Apparently the DOP-PCR provides enough amount and size of DNA for CGH application and covers whole genome amplification. These results suggest that DOP-PCR can be used for CGH and genotyping.