Determination of genomic copy number with quantitative microsphere hybridization

Abstract

Communicated by Jing ChengWe 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 0 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 0.5970.02 fold in three different deletion patients and increased 1.4270.01 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. Hum Mutat 27(4), [376][377][378][379][380][381][382][383][384][385][386] 2006.

Analysis of the Prader‐Willi syndrome chromosome region using quantitative microsphere hybridization (QMH) array

“…Each probe was evaluated for potential stable secondary sequence conformations as predicted by MFold software (http://www.bioinfo.rpi/edu/applications/mfold/old/dna) and any probe not meeting defined criteria (ΔG < 50, ΔH < −1,000, ΔS < −3,500) was discarded from design of the PWS QMH multiplex assay (Table I). These precise parameters were determined by in vitro testing of probes with varying secondary structure characteristics and previous studies found that probes not meeting these criteria produced MFI ratios deviating from the predicted ratio of 1 for normal loci (two copies per diploid genome), 0.5 for deleted loci (one copy), and 1.5 for duplicated loci (three copies) [Newkirk et al, 2006]. Each probe was synthesized using a 5′-C6-amino modification (Integrated DNA Technologies, Coralville, IA) for coupling to carboxylated microspheres.…”

“…Each probe was synthesized using a 5′-C6-amino modification (Integrated DNA Technologies, Coralville, IA) for coupling to carboxylated microspheres. Probes were conjugated to spectrally distinct microsphere levels of Luminex XMAP microspheres (Miraibio, San Francisco, CA) via a carbodiimide coupling reaction as described previously [Dunbar et al, 2003; Newkirk et al, 2006]. To minimize the initial expenditure on microspheres for assay development, only ten different levels were purchased, however previous studies have demonstrated successful multiplex analysis with up to 20 or more microsphere levels in a single reaction [Dunbar et al, 2003].…”

“…Previously, we described an assay for the detection of genomic copy number changes called quantitative microsphere hybridization [QMH; Newkirk, patent pending, 2008; Newkirk et al, 2005, 2006]. This assay utilizes unique sequence probes attached to spectrally distinct microspheres in multiplex hybridization to detect homologous target sequences in genomic DNA.…”

“…Hybridized test and reference probes are detected using flow cytometry and geometric mean fluorescence intensity (MFI) ratios are calculated to determine copy number differences. QMH is an attractive option for a clinical diagnostic test since it offers a high-throughput platform which is highly reproducible and accurate in part due to the lack of competitor DNA in assays [Newkirk et al, 2005, 2006]. It utilizes a flow cytometer for data acquisition which is found in most clinical settings.…”

“…The input sample requirement is minimal (10 ng), and capable of detecting deletions as small as 3 basepairs (BP) in the case of cystic fibrosis (manuscript in preparation). Examples of other previously developed QMH assays include those for chronic myelogenous leukemia [Newkirk et al, 2006], Charcot–Marie–Tooth syndrome, Alzheimer disease, velocardiofacial syndrome, and Duchenne muscular dystrophy (unpublished data). For the present study, we developed a series of single copy (sc) probes spanning the PWS chromosome region in an effort to delineate type I and type II deletion subjects and to identify deletions in the IC region.…”