PMID- 20809313
OWN - NLM
STAT- MEDLINE
DCOM- 20101214
LR  - 20131121
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Linking)
VI  - 659
DP  - 2010
TI  - Simultaneous visualization of FISH signals and bromo-deoxyuridine incorporation 
      by formamide-free DNA denaturation.
PG  - 203-18
LID - 10.1007/978-1-60761-789-1_14 [doi]
AB  - The replication timing of different DNA sequences in the mammalian cell nucleus 
      is a tightly regulated system, which affects important cellular processes such as 
      genes expression, chromatin epigenetic marking, and maintenance of chromosome 
      structure. For this reason, it is important to study the replication properties 
      of specific sequences, to determine for example, if the replication timing varies 
      in different tissues, or in the presence of specific reagents, such as hormones, 
      or other biologically active molecules. In this chapter, we present a technique, 
      which allows identification of specific DNA sequences by fluorescence in situ 
      hybridization (FISH) and simultaneously analyses the incorporation of a thymidine 
      analogue, 5-bromo-2-deoxyuridine (BrdU), to mark DNA replication. First, tissue 
      culture cells are synchronized at the beginning of the S-phase. BrdU is then 
      added, either at specific time-points during S-phase or during the whole of the 
      cell cycle. After harvesting the cells, the chromosomal DNA is hybridized to FISH 
      probes that identify specific DNA sequences; this is performed without the 
      teratogen formamide normally used in FISH. Finally, the cell preparations are 
      analysed with an epifluorescence microscope to determine if the sequence of 
      interest incorporates BrdU and in which point of the S-phase.
FAU - Moralli, Daniela
AU  - Moralli D
AD  - Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
FAU - Monaco, Zoia L
AU  - Monaco ZL
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (DNA Probes)
RN  - 38966-21-1 (Aphidicolin)
RN  - 9007-49-2 (DNA)
RN  - G34N38R2N1 (Bromodeoxyuridine)
SB  - IM
MH  - Aphidicolin/metabolism
MH  - Bromodeoxyuridine/*metabolism
MH  - Cell Line
MH  - DNA/*genetics/metabolism
MH  - DNA Probes/metabolism
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Nucleic Acid Denaturation
MH  - Time Factors
EDAT- 2010/09/03 06:00
MHDA- 2010/12/16 06:00
CRDT- 2010/09/03 06:00
PHST- 2010/09/03 06:00 [entrez]
PHST- 2010/09/03 06:00 [pubmed]
PHST- 2010/12/16 06:00 [medline]
AID - 10.1007/978-1-60761-789-1_14 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2010;659:203-18. doi: 10.1007/978-1-60761-789-1_14.