PMID- 10889276
OWN - NLM
STAT- MEDLINE
DCOM- 20000914
LR  - 20190915
IS  - 0165-022X (Print)
IS  - 0165-022X (Linking)
VI  - 44
IP  - 1-2
DP  - 2000 Jul 10
TI  - Quantitative microscopy after fluorescence in situ hybridization - a comparison 
      between repeat-depleted and non-depleted DNA probes.
PG  - 59-72
AB  - Complex probes used in fluorescence in situ hybridization (FISH) usually contain 
      repetitive DNA sequences. For chromosome painting, in situ suppression of these 
      repetitive DNA sequences has been well established. Standard painting protocols 
      require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. 
      Recently, it has become possible to remove repetitive DNA sequences from library 
      probes by means of magnetic purification and affinity PCR. Such a 'repeat 
      depleted library probe' was hybridized to the q-arm of chromosome 15 of human 
      metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 
      DNA. Apart from this, 'standard' FISH conditions were used. After in situ 
      hybridization, microscope images were obtained comparable to those achieved with 
      the #15q library probe prior to depletion. The images were recorded by a true 
      color CCD camera. By digital image analysis using 'line scan' and 'area scan' 
      procedures, the painting efficiency expressed in terms of relative fluorescence 
      signal intensity was quantitatively evaluated. The painting efficiency using the 
      repeat depleted probe of chromosome 15q was compared to the painting efficiency 
      after standard FISH. The results indicate that both types of probes are 
      compatible to a high FISH efficiency. Using equivalent probe concentrations, no 
      significant differences were found for FISH with standard painting probes and 
      repeat depleted painting probes.
FAU - Rauch, J
AU  - Rauch J
AD  - Applied Optics and Information Processing, Kirchhoff Institute of Physics, 
      University of Heidelberg, Albert-Ueberle-Strasse 3-5, D-69120, Heidelberg, 
      Germany.
FAU - Wolf, D
AU  - Wolf D
FAU - Craig, J M
AU  - Craig JM
FAU - Hausmann, M
AU  - Hausmann M
FAU - Cremer, C
AU  - Cremer C
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - J Biochem Biophys Methods
JT  - Journal of biochemical and biophysical methods
JID - 7907378
RN  - 0 (DNA Probes)
RN  - 9013-20-1 (Streptavidin)
SB  - IM
MH  - Cell Nucleus/ultrastructure
MH  - Chromatography, Affinity/methods
MH  - Chromosomes, Human, Pair 15/ultrastructure
MH  - DNA Probes/*ultrastructure
MH  - Gene Library
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Microscopy/*methods
MH  - Microscopy, Video/methods
MH  - Polymerase Chain Reaction/methods
MH  - *Repetitive Sequences, Nucleic Acid
MH  - Streptavidin/metabolism
EDAT- 2000/07/13 11:00
MHDA- 2000/09/19 11:01
CRDT- 2000/07/13 11:00
PHST- 2000/07/13 11:00 [pubmed]
PHST- 2000/09/19 11:01 [medline]
PHST- 2000/07/13 11:00 [entrez]
AID - S0165022X00000476 [pii]
AID - 10.1016/s0165-022x(00)00047-6 [doi]
PST - ppublish
SO  - J Biochem Biophys Methods. 2000 Jul 10;44(1-2):59-72. doi: 
      10.1016/s0165-022x(00)00047-6.