Universal linkage system: an improved method for labeling archival DNA for comparative genomic hybridization.
Comparative genomic hybridization (CGH) has become a powerful technique for studying gains and losses of DNA sequences in solid tumors. Importantly, DNA derived from archival tumor tissue is also applicable in CGH analysis. However, DNA isolated from routinely processed, formalin-fixed, paraffin-embedded tissue is often degraded, with the bulk of DNA showing fragment sizes of only 400-750 bp. Enzymatic labeling of archival DNA by standard nick translation (NT) decreases DNA size even further, until it becomes too small for CGH (<300 bp). This study presents application in CGH of a commercially available, non-enzymatic labeling method, called Universal Linkage System (ULS), that leaves the DNA fragment size intact. To compare the effect of chemical labeling of archival DNA by ULS vs. enzymatic by NT on the quality of CGH, DNA derived from 16 tumors was labeled by both ULS and NT. In those cases (n = 8), in which the bulk of DNA had a fragment size of 400-1,000 bp, CGH was successful with ULS-labeled probes, but not with NT-labeled probes. In the DNA samples (n = 6) with a fragment size > 1 kb, the intensity of CGH signals was comparable for both ULS- and NT-labeled probes, but CGH with ULS-labeled samples showed a high, speckled, background, which seriously hampered image analysis. In the remaining two cases, which had evenly distributed DNA fragment sizes (range 250-5,000 bp), CGH was successful with both labeling methods. Using DNA fragment size < 1 kb as a selection criterion for ULS labeling, we were able to obtain good quality CGH of a large panel (n = 77) of a variety of archival solid tumors. We conclude that ULS is an excellent labeling method for performing CGH on small-fragment-sized DNA.
Alers
J C
JC
Department of Pathology, Erasmus University Rotterdam, The Netherlands. alers@path.fgg.eur.nl
Rochat
J
J
Krijtenburg
P J
PJ
van Dekken
H
H
Raap
A K
AK
Rosenberg
C
C
D001109:Archives; D004273:DNA, Neoplasm / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D005404:Fixatives; D005821:Genetic Techniques / Q000639:trends*; D006801:Humans; D008297:Male; D009369:Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D009693:Nucleic Acid Hybridization / Q000379:methods*
D004273:DNA, Neoplasm; D005404:Fixatives