MilliporeSigma

Construction of repeat-free fluorescence in situ hybridization probes.

Nucleic acids research (2011-11-30)
Joost F Swennenhuis, Brad Foulk, Frank A W Coumans, Leon W M M Terstappen
ABSTRACT

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C(0)t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
GenomePlex® Whole Genome Amplification (WGA) Kit, Kit for whole genome amplification from a variety of DNA sources including FFPE tissue
Sigma-Aldrich
Thrombin from human plasma, lyophilized powder, ≥2,000 NIH units/mg protein (E1%/280, 18.3)
Sigma-Aldrich
Aminoallyl-dUTP sodium salt, ≥85%, lyophilized powder
Sigma-Aldrich
Dextran sulfate sodium salt from Leuconostoc spp., for molecular biology, average Mw >500,000 (dextran starting material), contains 0.5-2% phosphate buffer
Sigma-Aldrich
SSC Buffer 20× Concentrate, for Northern and Southern blotting, solution