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
in English
| LILACS
| ID: biblio-886917
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.
Full text:
Available
Index:
LILACS (Americas)
Main subject:
Polymerase Chain Reaction
/
DNA Primers
/
Primed In Situ Labeling
/
Cytogenetic Analysis
Type of study:
Evaluation studies
Limits:
Humans
Language:
English
Journal:
An. acad. bras. ciênc
Journal subject:
Science
Year:
2018
Type:
Article
Affiliation country:
Brazil
Institution/Affiliation country:
Universidade Federal de Viçosa/BR
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