PHOTOPROBE biotin: an alternative method for labeling archival DNA for comparative genomic hybridization.
Comparative genomic hybridization (CGH) represents a powerful method for screening the entire genome of solid tumors for chromosomal imbalances. Particularly it enabled the molecular cytogenetic analysis of archival, formalin-fixed, paraffin-embedded (FFPE) tissue. A well-known dilemma, however, is the poor DNA quality of this material with fragment sizes below 1000 bp. Nick translation, the conventionally used enzymatic DNA labeling method in CGH, leads to even shorter fragments often below a critical limit for successful analysis. In this study we report the alternative application of non-enzymatic, PHOTOPROBE biotin labeling for conjugation of the hapten to the DNA prior to in situ hybridization and fluorescence detection. We analyzed 51 FFPE tumor samples mainly from the upper respiratory tract by both labeling methods. In 19 cases, both approaches were successful. The comparison of hybridized metaphases showed a distinct higher fluorescence signal of the PHOTOPROBE samples sometimes with a discrete cytoplasm background which however did not interfere with specificity and sensitivity of the detected chromosomal imbalances. For further 32 cases characterized by an average DNA fragment size below 1000 bp, PHOTOPROBE biotin was the only successful labeling technique thus offering a new option for CGH analysis of highly degraded DNA from archival material.
Korinth
Dirk
D
Institute of Pathology, University Hospital Charité, Berlin, Germany.
Donhuijsen
Konrad
K
Bockmühl
Ulrike
U
Petersen
Iver
I
D001710:Biotin / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002869:Chromosome Aberrations; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism / Q000648:ultrastructure*; D015342:DNA Probes / Q000737:chemistry; D004273:DNA, Neoplasm / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization; D007621:Karyotyping; D008027:Light; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*
D015342:DNA Probes; D004273:DNA, Neoplasm; D001710:Biotin; D004247:DNA