Detecting Copy Number Changes in Genomic DNA: MAPH and MLPA

White, Stefan J.; Breuning, Martijn H.; Dunnen, Johan T. den

doi:10.1016/s0091-679x(04)75032-3

Cited by 26 publications

(24 citation statements)

References 72 publications

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“…Oligonucleotide probes may, in some cases, also lack adequate sensitivity and specificity for reliable determination of genomic target levels (see Results). By contrast, longer, single-copy DNA probes hybridized to unamplified, directlylabeled genomic targets can be used to unequivocally determine genomic copy number [Southern, 1975;White et al, 2004].…”

Section: Introductionmentioning

confidence: 99%

Determination of genomic copy number with quantitative microsphere hybridization

et al. 2006

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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.

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Section: Introductionmentioning

confidence: 99%

Determination of genomic copy number with quantitative microsphere hybridization

et al. 2006

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“…Similarly, samples with reduced probe binding for a single exon should be verified with a non-MLPA method because point variations, deletions, and insertions that span where probes bind or are close to the ligation site of a pair of probes may affect probe ligation and cause reduced signals (27,28 ).…”

Section: Discussionmentioning

confidence: 99%

“…Several methods have been used to analyze MLPA dosage data (27 ). In global normalization, individual peak areas are divided by the combined peak area of all probes and the resulting relative peak area is divided by relative peak areas from control DNA.…”

Section: Discussionmentioning

confidence: 99%

Multiplex Ligation-Dependent Probe Amplification for Rapid Detection of Proteolipid Protein 1 Gene Duplications and Deletions in Affected Males and Carrier Females with Pelizaeus–Merzbacher Disease

Warshawsky

Chernova²,

Hübner

et al. 2006

Clinical Chemistry

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Background: Pelizaeus-Merzbacher disease is a rare X-linked neurodegenerative disorder caused by sequence variations in the proteolipid protein 1 gene (PLP1). PLP1 gene duplications account for ϳ50%-75% of cases and point variations for ϳ15%-20% of cases; deletions and insertions occur infrequently. We used multiplex ligation-dependent probe amplification (MLPA) to detect PLP1 gene alterations, especially gene duplications and deletions. Methods: We performed MLPA on 102 samples from individuals with diverse PLP1 gene abnormalities and from controls, including 50 samples previously characterized for the PLP1 gene by quantitative PCR but which were anonymized for prior results and sex. Results: All males with PLP1 gene duplications (n ‫؍‬ 13), 1 male with a triplication, 2 males with whole gene deletions, and all controls (n ‫؍‬ 72) were unambiguously assigned to their correct genotype. Of 4 female carriers tested by MLPA and quantitative PCR, 3 were duplication carriers by both methods, and 1 was a triplication

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“…When the number of targets is higher, for instance up to 100, other technologies are of interest. Examples are bead-based platforms or multiplex ligation-dependent probe amplification (MLPA) (10). As this test format requires a 5th to 10th of labelled DNA to hybridize, consequently the use of a mini-array system could reduce the labelling related costs to the same extent.…”

Section: Discussionmentioning

confidence: 99%

Rapid detection of genomic imbalances using micro-arrays consisting of pooled BACs covering all human chromosome arms

Knijnenburg

Burg²,

Nilsson³

et al. 2005

Nucleic Acids Research

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A strategy is presented to select, pool and spot human BAC clones on an array in such a way that each spot contains five well performing BAC clones, covering one chromosome arm. A mini-array of 240 spots was prepared representing all human chromosome arms in a 5-fold as well as some controls, and used for comparative genomic hybridization (CGH) of 10 cell lines with aneusomies frequently found in clinical cytogenetics and oncology. Spot-to-spot variation within five replicates was below 6% and all expected abnormalities were detected 100% correctly. Sensitivity was such that replacing one BAC clone in a given spot of five by a BAC clone from another chromosome, thus resulting in a change in ratio of 20%, was reproducibly detected. Incubation time of the mini-array was varied and the fluorescently labelled target DNA was diluted. Typically, aneusomies could be detected using 30 ng of non-amplified random primed labelled DNA amounts in a 4 h hybridization reaction. Potential application of these mini-arrays for genomic profiling of disseminated tumour cells or of blastomeres for preimplantation genetic diagnosis, using specially designed DNA amplification methods, are discussed.

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Detecting Copy Number Changes in Genomic DNA: MAPH and MLPA

Cited by 26 publications

References 72 publications

Determination of genomic copy number with quantitative microsphere hybridization

Determination of genomic copy number with quantitative microsphere hybridization

Multiplex Ligation-Dependent Probe Amplification for Rapid Detection of Proteolipid Protein 1 Gene Duplications and Deletions in Affected Males and Carrier Females with Pelizaeus–Merzbacher Disease

Rapid detection of genomic imbalances using micro-arrays consisting of pooled BACs covering all human chromosome arms

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