Multiplex ligation-dependent probe amplification using a completely synthetic probe set

Department of Medical and Molecular Genetics, Guy's, King's and St. Thomas' School of Medicine, London, UK.
BioTechniques (Impact Factor: 2.75). 10/2004; 37(3):399-405.
Source: PubMed

ABSTRACT The recent development of multiplex ligation-dependent probe amplification (MLPA) has provided an efficient and reliable assay for dosage screening of multiple loci in a single reaction. However, a drawback to this method is the time-consuming process of generating a probe set by cloning in single-stranded bacteriophage vectors. We have developed a synthetic probe set to screen for deletions in a region spanning 18.5 Mb within chromosome 3q. In a pilot study, we tested 15 synthetic probes on 4 control samples and on 2 patients previously found to possess a heterozygous deletion in the region 3q26-q28. These synthetic probes detected deletions at all previously known deleted loci. Furthermore, using synthetic probes, the variability of results within samples was similar to that reported for commercially available M13-derived probes. Our results demonstrate that this novel approach to MLPA provides a generic solution to the difficulties of probe development by cloning; such synthetically generated probes may be used to screen a large number of loci in a single reaction. We conclude that the use of synthetic probes for MLPA is a rapid, robust, and efficient alternative for research (and potentially diagnostic) deletion and duplication screening of multiple genomic loci.

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Available from: Roland G Roberts, Jul 30, 2015
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    • "However, in comparison to standard MLPA, in our strategy short oligonucleotide probes instead of long MLPA probes are used. The use of short MLPA probes for detection of large mutations in various human genes was shown before (Kozlowski et al., 2007b; Stern et al., 2004; White et al., 2004). It allows easy custom design and generation of assays for almost any genomic region of interest. "
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    ABSTRACT: Copy number variation has recently been recognized as an important type of genetic variation that modifies human phenotypes. Copy number variants (CNVs) are being increasingly associated with various human phenotypes and diseases. However, the lack of an appropriate method that allows fast, inexpensive and, most importantly, accurate CNVs genotyping significantly hampers CNV analysis. This limitation especially affects the analysis of multi-allelic CNVs that frequently modify various phenotypes. Recently, we developed a multiplex ligation-dependent probe amplification (MLPA)-based strategy for multiplex copy number genotyping and the validation of candidate CNV-miRNAs. Here we present the adaptation and optimization of this recently developed method for high-resolution genotyping of individual disease-related multi-allelic CNVs. We developed appropriate assays for three well-known and extensively studied CNVs: CNV-CCL3L1, CNV-DEFB, and CNV-UGT2B17, which have been associated with various human phenotypes including inflammation-related and infectious diseases. With the use of these assays we identified several general factors that allow to increase the resolution of the copy number genotyping. Performed experiments confirmed the high reproducibility and accuracy of the obtained genotyping results. The reliability of the results and relatively low per-genotype cost makes this strategy an attractive method for large-scale experiments such as genotype-phenotype association studies.
    Gene 06/2014; 546(2). DOI:10.1016/j.gene.2014.05.072 · 2.08 Impact Factor
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    • "All copy number variations that remained after exclusion criteria were confirmed by MLPA [38]. Probes were designed according to the recommendations by Stern et al. [39] using two probes per aberration. Probes were combined in several probe sets, all including four control probes (RELN2, PCLN16, RB1 and CREBBP) and a sex chromosome specific probe (Supplementary Table 2). "
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    ABSTRACT: Half of all patients with a disorder of sex development (DSD) do not receive a specific molecular diagnosis. Comparative genomic hybridization (CGH) can detect copy number changes causing gene haploinsufficiency or over-expression that can lead to impaired gonadal development and gonadal DSD. The purpose of this study was to identify novel candidate genes for 46,XY gonadal dysgenesis (GD) using a customized 1M array-CGH platform with whole-genome coverage and probe enrichment targeting 78 genes involved in sex development. Fourteen patients with 46,XY gonadal DSD were enrolled in the study. Nine individuals were analyzed by array CGH. All patients were included in a follow up sequencing study of candidate genes. Three novel candidate regions for 46,XY GD were identified in two patients. An interstitial duplication of the SUPT3H gene and a deletion of C2ORF80 were detected in a pair of affected siblings. Sequence analysis of these genes in all patients revealed no additional mutations. A large duplication highlighting PIP5K1B, PRKACG and FAM189A2 as candidates for 46,XY GD, were also detected. All five genes are expressed in testicular tissues, and one is shown to cause gonadal DSD in mice. However detailed functional information is lacking for these genes.
    European journal of medical genetics 09/2013; 56(12). DOI:10.1016/j.ejmg.2013.09.003 · 1.49 Impact Factor
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    • "Probes for each locus were prepared as described by Schouten et al. (2002), with the exception that all probes were made synthetically (Eurofins MWG Operon, Huntsville , AL) as described previously (Stern et al. 2004). All probes were tested individually, and amplification was checked on a 2% agarose gel. "
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    ABSTRACT: Chromosomal microarray analysis has identified many novel microdeletions or microduplications that produce neurodevelopmental disorders with a recognizable clinical phenotype and that are not observed in normal individuals. However, imbalance of other genomic regions is associated with a variable phenotype with intellectual disability (ID) or autism in some individuals but are also observed in completely normal individuals. Several large studies have reported the prevalence of copy number (CN) variants in people with particular features (e.g., ID, autism, schizophrenia, or epilepsy); few studies have investigated the prevalence of genomic CN changes in the general population. We used a high-throughput method to screen 6813 consecutive cord blood samples from a predominantly French-Canadian population to assess genomic CN in five genomic regions: 1p36, 15q11-q13, 16p11.2, 16p11.2-p12.2, and 22q11.2. We identified one deletion and one duplication within 1p36, two deletions of 15q11-q13, eight deletions of 16p11.2-p12.2, two deletions and five duplications of 16p11.2, and six duplications of 22q11.2. This study provides estimates of the frequency of CN variants in an unselected population. Our findings have important implications for genetic counseling.
    07/2013; 1(2):87-97. DOI:10.1002/mgg3.12
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