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.
There have been several recent advances in PCR-mediated approaches to the detection of genomic copy number changes of multiple loci. Methods such as quantitative fluorescence PCR (QF-PCR) (1,2) and multiplex amplifiable probe hybridization (MAPH) (3) offer a considerable advantage over more established quantitative techniques such as Southern blot analysis and fluorescence in situ hybridization (FISH) because they require only small quantities of DNA and allow for multiple loci to be tested in a single reaction. A more recently devised method is multiplex ligation-dependent probe amplification (MLPA) (4), which has been found to afford cost-effective, reliable, and rapid screening of multiple loci for copy number changes. During MLPA, two sequence-tagged half-probes (one small, synthetic half-probe and one large, M13-derived half-probe) are hybridized to their genomic target sequence and ligated together at 54°C using thermostable DNA ligase. The ligated probes are then amplified by fluorescently labeled universal primers that correspond to the probes’ sequence tags. Each probe is designed to be uniquely sized, resulting in a ladder of amplified products that can be visualized and quantified by automated fluorescent electrophoretic analysis. MLPA can currently screen at least 40 loci in one reaction with as little as 20 ng template DNA (4). Both MLPA and the related oligonucleotide ligation assay (OLA) techniques (5,6) utilize DNA ligase, which is exquisitely sensitive to DNA mismatches, to obtain the target specificity and discrimination difficult to achieve by PCR alone. Both techniques have been used to genotype multiple single nucleotide polymorphisms (SNPs) (4,6,7), whereas MLPA can also be used to screen for dosage variation such as the identification of large deletions in the BRCA1 gene (8) and expression profiling of up to 45 signaling pathway transcripts by reverse transcription MLPA (RT-MLPA) (9). However, for assays in target region(s) for which there are no existing probe sets, the main disadvantage of MLPA is the time-consuming process of creating a library of half-probes by cloning into a family of M13 phage vectors.
A recent publication described two individuals with deletions in the region 3q26–q28, which is associated with anophthalmia and microphthalmia (10). These two individuals had overlapping deletions in the same region of chromo-some 3, with a common deleted region estimated to be 6.7 Mb in size at the time. In addition to eye abnormalities, both patients displayed craniofacial malformations and other dysmorphic features.
To investigate such deletions, without the laborious probe-cloning process required for conventional MLPA, we created a completely synthetic MLPA probe set up to 156 nucleotides in size that corresponded to multiple loci in this region and tested them on DNA from these patients. The use of synthetic probes has been successfully demonstrated in a similar multiplex technique that couples ligase detection reaction and PCR (LDR/PCR) for mutation detection and to assess gene copy number in tumor cell line DNA (11,12). This study represents a novel extension of synthetic probe use in the context of MLPA analysis to assess target copy number.Materials and Methods Probes and Oligonucleotides
All patient and control DNA were donated or obtained from Guy's Hospital. Fifteen uniquely sized synthetic probes were designed to investigate the deleted region ((Table 1)). Nine synthetic probes (3–5, 7–9, 11, 13, and 16) were designed to lie between 182 and 189 Mb on chromosome 3 from the ENSEMBL database (http://www.ensembl.org/datasearch.html), covering the largest known deletion, that in patient 1. Two probes (6 and 19) were designed to be distal to the deletion, one of which (probe 19) was known to be present by FISH analysis using bacterial artificial chromosome (BAC) clone 135A1 (GenBank® accession no. AC080129). The two probes proximal to the deletion (1 and 2) had an unknown copy number because the position of BAC clone 134F2, which had defined the proximal side of the deletion, had been revised. Finally, two control probes, 17 and 18, from chromosomes 2 and 17, respectively, were also included.Table 1. Probes Used for Multiplex Ligation-Dependent Probe Amplification (MLPA) Analysis of 3q Region
The 5′ half-probes are preceded by the 5′ universal primer tag (see Materials and Methods). The 3′ half-probes are preceded by a 5′ phosphate group and followed by the 3′ universal primer tag as described in Materials and Methods. Probe sizes (in nucleotides) show the total length, inclusive of universal primer tags. Nucleotides adjacent to the ligation site are indicated in bold type. Minus and plus signs indicate the deleted and nondeleted loci of the patients (P1 and P2), respectively; and a question mark indicates that the deletion status was unknown before this study.