PMID- 22911704
OWN - NLM
STAT- MEDLINE
DCOM- 20130419
LR  - 20240313
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 7
IP  - 7
DP  - 2012
TI  - Fast and non-toxic in situ hybridization without blocking of repetitive 
      sequences.
PG  - e40675
LID - 10.1371/journal.pone.0040675 [doi]
LID - e40675
AB  - Formamide is the preferred solvent to lower the melting point and annealing 
      temperature of nucleic acid strands in in situ hybridization (ISH). A key benefit 
      of formamide is better preservation of morphology due to a lower incubation 
      temperature. However, in fluorescence in situ hybridization (FISH), against 
      unique DNA targets in tissue sections, an overnight hybridization is required to 
      obtain sufficient signal intensity. Here, we identified alternative solvents and 
      developed a new hybridization buffer that reduces the required hybridization time 
      to one hour (IQFISH method). Remarkably, denaturation and blocking against 
      repetitive DNA sequences to prevent non-specific binding is not required. 
      Furthermore, the new hybridization buffer is less hazardous than formamide 
      containing buffers. The results demonstrate a significant increased hybridization 
      rate at a lowered denaturation and hybridization temperature for both DNA and PNA 
      (peptide nucleic acid) probes. We anticipate that these formamide substituting 
      solvents will become the foundation for changes in the understanding and 
      performance of denaturation and hybridization of nucleic acids. For example, the 
      process time for tissue-based ISH for gene aberration tests in cancer diagnostics 
      can be reduced from days to a few hours. Furthermore, the understanding of the 
      interactions and duplex formation of nucleic acid strands may benefit from the 
      properties of these solvents.
FAU - Matthiesen, Steen H
AU  - Matthiesen SH
AD  - Research and Development, Dako, Glostrup, Denmark. steen.matthiesen@dako.com
FAU - Hansen, Charles M
AU  - Hansen CM
LA  - eng
PT  - Journal Article
DEP - 20120724
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Buffers)
RN  - 0 (DNA Probes)
SB  - IM
MH  - Buffers
MH  - DNA Probes/chemistry
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Neoplasms/diagnosis/genetics
MH  - *Repetitive Sequences, Nucleic Acid
PMC - PMC3404051
COIS- Competing Interests: SHM is employed by Dako. CMH has worked as consultant for 
      Dako. SHM and CMH are inventors on the patent application WO 2009/144581 
      Hybridization Compositions and Methods. CMH holds financial interest in the 
      application. SHM is as well inventor on the following patent applications: WO 
      2009/147537 Compositions and Methods for Detection of Chromosomal Aberrations 
      with Novel Hybridization Buffers; WO 2010/097655 Compositions and Methods for RNA 
      Hybridization Applications; WO 2010/097656 Compositions and Methods for 
      Performing a Stringent Wash Step in Hybridization Applications; WO 2010/097707 
      Compositions and Methods for Performing Hybridizations with Separate Denaturation 
      of the Sample and Probe; WO 2011/067678 A2 Compositions and Methods for 
      Performing Hybridizations with No Denaturation. SHM holds no financial interest 
      in the patent applications. All applications are owned by Dako. This does not 
      alter the authors' adherence to all the PLoS ONE policies on sharing data and 
      materials.
EDAT- 2012/08/23 06:00
MHDA- 2013/04/23 06:00
PMCR- 2012/07/24
CRDT- 2012/08/23 06:00
PHST- 2012/03/06 00:00 [received]
PHST- 2012/06/12 00:00 [accepted]
PHST- 2012/08/23 06:00 [entrez]
PHST- 2012/08/23 06:00 [pubmed]
PHST- 2013/04/23 06:00 [medline]
PHST- 2012/07/24 00:00 [pmc-release]
AID - PONE-D-12-07283 [pii]
AID - 10.1371/journal.pone.0040675 [doi]
PST - ppublish
SO  - PLoS One. 2012;7(7):e40675. doi: 10.1371/journal.pone.0040675. Epub 2012 Jul 24.