Dendrimer FISH detection of single-copy intervals in acute promyelocytic leukemia.
Acute promyelocytic leukemia (AML-M3) is characterized by a translocation between chromosomes 15 and 17 [t(15;17)]. The detection of t(15;17) at the single cell level, is commonly done by fluorescence in situ hybridization (FISH) using recombinant locus specific genomic probes greater than 14 kilobases kb in length. To allow a more thorough study of t(15;17), we designed small (0.9-3.6 kb), target-specific, single-copy probes from the human genome sequence. A novel detection approach was evaluated using moieties possessing more fluorophores, DNA dendrimers (up to 375 fluorophores per dendrimer). Two detection approaches were evaluated using the dendrimers: (1) dendrimers modified with anti-biotin antibodies for detection of biotinylated bound probes, and (2) dendrimers modified with 45-base long oligonucleotides designed from the single-copy probes, for direct detection of the target region. The selectivity of the probes was confirmed via indirect labeling with biotin/digoxigenin by nick translation, with detection efficiencies between 50 and 90%. Furthermore, the scFISH probes were successfully detected on metaphase cells with anti-biotin dendrimer conjugates and on interphase cells with 45-base modified dendrimers. Our results bring up the possibility to detect target regions of less than 1 kb, which will be a great contribution to high-resolution analysis of genomic sequences.
Mora
Johanna R
JR
Department of Chemistry, University of Kansas, Lawrence, 66045, USA.
Knoll
Joan H M
JH
Rogan
Peter K
PK
Getts
Robert C
RC
Wilson
George S
GS
D045744:Cell Line, Tumor; D002884:Chromosomes, Human, Pair 15 / Q000235:genetics; D002886:Chromosomes, Human, Pair 17 / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry; D050091:Dendrimers / Q000737:chemistry; D015894:Genome, Human; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007399:Interphase; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics* / Q000473:pathology; D008677:Metaphase; D014178:Translocation, Genetic
D015342:DNA Probes; D050091:Dendrimers