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
AIMS AND OBJECTIVE: Primed in situ labeling/synthesis (PRINS) technique is an alternative to fluorescent in situ hybridization for chromosome analysis. This study was designed to evaluate the application of PRINS for rapid diagnosis of common chromosomal aneuploidy. MATERIALS AND METHODS: We have carried out PRINS using centromere specific oligonucleotide primers for chromosome X, Y, 13, 18 and 21 on lymphocyte metaphase and interphase cells spread. Specific primer was annealed in situ, followed by elongation of primer by Taq DNA polymerase in presence of labeled nucleotides. Finally, reaction was stopped and visualized directly under fluorescent microscope. RESULTS: Discrete centromere specific signals were observed with each primer. CONCLUSION: PRINS seems to be a rapid and reliable method to detect common chromosome aneuploidy in peripheral blood lymphocyte metaphase and interphase cells.
Subject(s)
Aneuploidy/genetics , Chromosomes, Human/genetics , Chromosomes, Human, Pair 13/genetics , Chromosomes, Human, Pair 18/genetics , Chromosomes, Human, Pair 21/genetics , Humans , Primed In Situ Labeling/methods , X Chromosome/genetics , Y Chromosome/geneticsABSTRACT
O diagnostico genetico pre-implantacional (PGD) e um novo procedimento que pode ser utilizado como diagnostico precoce pre-natal em casais com risco de transmissao de doencas geneticas. Utilizando "polymerase chain reaction" (PCR), "fluorescence in-situ hybridization" (FISH) e "primed in-situ" (PRINS) pode-se determinar o genotipo ou sexo genetico do embriao biopsiado obtido por...