Article

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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    • "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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    • "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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    • "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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