PMID- 30656455
OWN - NLM
STAT- MEDLINE
DCOM- 20190813
LR  - 20200225
IS  - 1615-6102 (Electronic)
IS  - 0033-183X (Print)
IS  - 0033-183X (Linking)
VI  - 256
IP  - 3
DP  - 2019 May
TI  - A simple non-toxic ethylene carbonate fluorescence in situ hybridization 
      (EC-FISH) for simultaneous detection of repetitive DNA sequences and fluorescent 
      bands in plants.
PG  - 873-880
LID - 10.1007/s00709-019-01345-7 [doi]
AB  - The major drawbacks of standard plant fluorescence in situ hybridization (FISH) 
      designed for double-stranded DNA probes include requirement for experimentally 
      determined heat denaturation of chromosomes at high temperatures and at least 
      overnight hybridization. Consequently, processing with chromosomal preparations 
      may easily result in heat-induced deterioration of chromosomal structural 
      details, is time-consuming, and involves the use of toxic formamide and 
      formaldehyde. Here, I have described a simple and appealing non-toxic procedure 
      with ethylene carbonate (EC)-a formamide-substituting solvent and double-stranded 
      repetitive DNA probes. Applying EC as a component of the hybridization solution 
      at 46  degrees C not only allowed successful overnight hybridization but also gave a 
      possibility to reduce the hybridization time to 3 h, hence converting the 
      technique into a 1-day procedure. Importantly, the EC-FISH tended to preserve 
      well chromosome structural details, e.g., DAPI-positive bands, thus facilitating 
      simultaneous FISH mapping and chromosome banding on the same slide. The procedure 
      requires no formaldehyde and RNA-se treatment of chromosomes, and no heat 
      denaturation of chromosomal DNA. The key condition is to obtain high-quality 
      cytoplasm-free preparations. The method was reproducible in all the plants 
      studied (Allium, Nigella, Tradescantia, Vicia), giving a species-specific signal 
      pattern together with clear DAPI bands on chromosomes. The procedure described 
      here is expected to give a positive stimulus for improving gene-mapping 
      approaches in plants.
FAU - Golczyk, Hieronim
AU  - Golczyk H
AUID- ORCID: 0000-0001-6981-2144
AD  - Department of Molecular Biology, Institute of Biotechnology, John Paul II 
      Catholic University of Lublin, Konstantynow 1i, 20-708, Lublin, Poland. 
      h.golczyk@wp.pl.
LA  - eng
GR  - 2015/19/B/NZ2/01692/Polish National Science Center (NCN)/
PT  - Journal Article
DEP - 20190117
PL  - Austria
TA  - Protoplasma
JT  - Protoplasma
JID - 9806853
RN  - 0 (DNA Probes)
RN  - 0 (DNA, Ribosomal)
RN  - 0 (Dioxolanes)
RN  - 0 (Indoles)
RN  - 47165-04-8 (DAPI)
RN  - RGJ96TB7R7 (ethylene carbonate)
MH  - Base Sequence
MH  - DNA Probes/metabolism
MH  - DNA, Ribosomal/metabolism
MH  - Dioxolanes/*chemistry
MH  - Fluorescence
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Indoles/chemistry
MH  - Plants/*genetics
MH  - Repetitive Sequences, Nucleic Acid/*genetics
PMC - PMC6482133
OTO - NOTNLM
OT  - Chromosome DAPI banding
OT  - Ethylene carbonate
OT  - Fluorescence in situ hybridization
OT  - Heterochromatin
OT  - Plants
OT  - rDNA
COIS- The author declares that there is no conflict of interest.
EDAT- 2019/01/19 06:00
MHDA- 2019/08/14 06:00
PMCR- 2019/01/17
CRDT- 2019/01/19 06:00
PHST- 2018/11/13 00:00 [received]
PHST- 2019/01/01 00:00 [accepted]
PHST- 2019/01/19 06:00 [pubmed]
PHST- 2019/08/14 06:00 [medline]
PHST- 2019/01/19 06:00 [entrez]
PHST- 2019/01/17 00:00 [pmc-release]
AID - 10.1007/s00709-019-01345-7 [pii]
AID - 1345 [pii]
AID - 10.1007/s00709-019-01345-7 [doi]
PST - ppublish
SO  - Protoplasma. 2019 May;256(3):873-880. doi: 10.1007/s00709-019-01345-7. Epub 2019 
      Jan 17.