Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques
An. acad. bras. ciênc
; 90(1): 41-47, Mar. 2018. graf
Article
em En
| LILACS
| ID: biblio-886917
Biblioteca responsável:
BR1.1
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.
Palavras-chave
Texto completo:
1
Índice:
LILACS
Assunto principal:
Reação em Cadeia da Polimerase
/
Primers do DNA
/
Marcação in Situ com Primers
/
Análise Citogenética
Tipo de estudo:
Evaluation_studies
Limite:
Humans
Idioma:
En
Revista:
An. acad. bras. ciênc
Assunto da revista:
CIENCIA
Ano de publicação:
2018
Tipo de documento:
Article