Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

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.

Plant Chromosome-Specific Probes by Microdissection of a Single Chromosome: Is That a Reality?

“…Our research group has been concentrating efforts to improve the chromosome painting technique, aiming especially to reduce the amount of target DNA necessary for the first round of DOP-PCR. To this end, we combined an in situ PCR (PRINS) prior to microdissection with the DOP-PCR and successfully constructed a complex probe from ten copies of a small fragment of human chromosome X (Passamani et al, 2018). Advancing in our main goal, the most critical steps were resolved: the chromosome microdissection, the transferring of the chromosome to the microtube and the initial low stringency amplification.…”

“…In human cytogenetics, it is common to perform G-banding to correctly identify chromosomes during microdissection. Nonetheless, the staining process is a source of contamination itself and should be avoided in the process of constructing chromosome-specific probes (Christian et al, 1999;Passamani et al, 2018). The high morphological quality of the obtained human chromosomes dismissed the need of staining because chromosome 2 was easily identified.…”

“…Human mitotic chromosomes used as cytogenetic model for probe construction were obtained from lymphocytes culture stored in absolute methanol fixative (Merck R ) at −20 • C. The cell bank is maintained in Laboratório de Citogenética e Citometria at Departamento de Biologia Geral (according to Passamani et al, 2018), safety standards and criteria of Ethics in Human Research, Resolution 196/96 of the National Health Council). Seeds of Zea mays and Capsicum annuum were obtained commercially.…”

“…The mitotic chromosomes were obtained from samples of the three taxa by treatment with antimitotic agents. Human lymphocytes, which were previously treated with the antitubulin agent colchicine by Passamani et al (2018), provided a high proportion of prometaphases/metaphases. For Z. mays and C. annuum, hydroxyurea (inhibitor of ribonucleotide redutase) was previously applied to arrest root meristematic cells in S phase.…”

“…Usually, a relatively high number of copies (∼20-50) of the target chromosome must be collected, which is considered a limiting technical factor (Christian et al, 1999;Henning et al, 2008). Nonetheless, efforts have been made to adapt protocols in which a reduced amount of target DNA is enough to obtain the chromosomespecific probes (Christian et al, 1999;Henning et al, 2008;Passamani et al, 2018). In plants, the need to dissect more than one copy of the same chromosome is often hampered by the highly homomorphic karyotypes.…”