Determination of genomic copy number with quantitative microsphere hybridization.
We developed a novel quantitative microsphere suspension hybridization (QMH) assay for determination of genomic copy number by flow cytometry. Single copy (sc) products ranging in length from 62 to 2,304 nucleotides [Rogan et al., 2001; Knoll and Rogan, 2004] from ABL1 (chromosome 9q34), TEKT3 (17p12), PMP22 (17p12), and HOXB1 (17q21) were conjugated to spectrally distinct polystyrene microspheres. These conjugated probes were used in multiplex hybridization to detect homologous target sequences in biotinylated genomic DNA extracted from fixed cell pellets obtained for cytogenetic studies. Hybridized targets were bound to phycoerythrin-labeled streptavidin; then the spectral emissions of both target and conjugated microsphere were codetected by flow cytometry. Prior amplification of locus-specific target DNA was not required because sc probes provide adequate specificity and sensitivity for accurate copy number determination. Copy number differences were distinguishable by comparing the mean fluorescence intensities (MFI) of test probes with a biallelic reference probe in genomic DNA of patient samples and abnormal cell lines. Concerted 5' ABL1 deletions in patient samples with a chromosome 9;22 translocation and chronic myelogenous leukemia were confirmed by comparison of the mean fluorescence intensities of ABL1 test probes with a HOXB1 reference probe. The relative intensities of the ABL1 probes were reduced to 0.59+/-0.02 fold in three different deletion patients and increased 1.42+/-0.01 fold in three trisomic 9 cell lines. TEKT3 and PMP22 probes detected proportionate copy number increases in five patients with Charcot-Marie-Tooth Type 1a disease and chromosome 17p12 duplications. Thus, the assay is capable of distinguishing one allele and three alleles from a biallelic reference sequence, regardless of chromosomal context.
Laboratory of Genomic Disorders, Children's Mercy Hospitals and Clinics, University of Missouri-Kansas City, Kansas City, Missouri 64108, USA.
Heather L
HL
Newkirk
Peter K
PK
Rogan
Mauricio
M
Miralles
Joan H M
JH
Knoll
D002460:Cell Line; D002607:Charcot-Marie-Tooth Disease / Q000235:genetics; D002869:Chromosome Aberrations; D002872:Chromosome Deletion; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics; D002899:Chromosomes, Human, Pair 9 / Q000235:genetics; D015342:DNA Probes; D018628:Gene Dosage / Q000235:genetics*; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D006801:Humans; D008863:Microspheres; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012680:Sensitivity and Specificity; D014314:Trisomy / Q000235:genetics
D015342:DNA Probes