PMID- 14513562
OWN - NLM
STAT- MEDLINE
DCOM- 20040511
LR  - 20191210
IS  - 0736-6205 (Print)
IS  - 0736-6205 (Linking)
VI  - 35
IP  - 3
DP  - 2003 Sep
TI  - COMBO-FISH: specific labeling of nondenatured chromatin targets by 
      computer-selected DNA oligonucleotide probe combinations.
PG  - 564-70, 572-7
AB  - Here we present the principle of fluorescence in situ hybridization (FISH) with 
      combinatorial oligonucleotide (COMBO) probes as a new approach for the specific 
      labeling of genomic sites. COMBO-FISH takes advantage of 
      homopurine/homopyrimidine oligonucleotides that form triple helices with intact 
      duplex genomic DNA, without the need for prior denaturation of the target 
      sequence that is usually applied for probe binding in standard FISH protocols. An 
      analysis of human genome databases has shown that homopurine/homopyrimidine 
      sequences longer than 14 bp are nearly homogeneously distributed over the genome, 
      and they represent from 1% to 2% of the entire genome. Because the observation 
      volume in a confocal laser-scanning microscope equipped with a high numerical 
      aperture lens typically corresponds to an approximate 250-kb chromatin domain in 
      a normal mammalian cell nucleus, this volume should contain 150-200 
      homopurine/homopyrimidine stretches. Using DNA database information, one can 
      configure a set of distinct, uniformly labeled oligonucleotide probes from these 
      stretches that is expected to exclusively co-localize within a 250-kb chromatin 
      domain. Due to the diffraction-limited resolution of a microscope, the 
      fluorescence signals of the configured oligonucleotide probe set merge into a 
      typical, nearly homogenous FISH spot. Using a set of 32 homopyrimidine probes, we 
      performed experiments in the Abelson murine leukemia region of human chromosome 9 
      as some of the very first proofs-of-principle of COMBO-FISH. Although the 
      experimental protocol currently contains several steps that are incompatible with 
      living cell conditions, the theoretical approach may be the first methodological 
      advance toward the long-term but still elusive goal of carrying out specific FISH 
      in high-resolution fluorescence microscopy of vital cells.
FAU - Hausmann, Michael
AU  - Hausmann M
AD  - Kirchhoff-Institute of Physics, University of Heidelberg, Heidelberg. 
      mihaus@ukl.uni-freiburg.de
FAU - Winkler, Ralph
AU  - Winkler R
FAU - Hildenbrand, Georg
AU  - Hildenbrand G
FAU - Finsterle, Jutta
AU  - Finsterle J
FAU - Weisel, Andrea
AU  - Weisel A
FAU - Rapp, Alexander
AU  - Rapp A
FAU - Schmitt, Eberhard
AU  - Schmitt E
FAU - Janz, Siegfried
AU  - Janz S
FAU - Cremer, Christoph
AU  - Cremer C
LA  - eng
PT  - Comparative Study
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PT  - Validation Study
PL  - England
TA  - Biotechniques
JT  - BioTechniques
JID - 8306785
RN  - 0 (Chromatin)
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (Oncogene Proteins)
SB  - IM
MH  - Chromatin/*genetics
MH  - Chromosomes, Human, Pair 9/genetics
MH  - Combinatorial Chemistry Techniques/*methods
MH  - Feasibility Studies
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Nucleic Acid Denaturation
MH  - Oligonucleotide Array Sequence Analysis/*methods
MH  - *Oligonucleotide Probes/*chemical synthesis
MH  - Oncogene Proteins/genetics
MH  - Reproducibility of Results
MH  - Sensitivity and Specificity
MH  - Sequence Analysis, DNA/*methods
MH  - *Software
EDAT- 2003/09/30 05:00
MHDA- 2004/05/12 05:00
CRDT- 2003/09/30 05:00
PHST- 2003/09/30 05:00 [pubmed]
PHST- 2004/05/12 05:00 [medline]
PHST- 2003/09/30 05:00 [entrez]
AID - 10.2144/03353rr03 [doi]
PST - ppublish
SO  - Biotechniques. 2003 Sep;35(3):564-70, 572-7. doi: 10.2144/03353rr03.