Review New applications and developments in the use of multiplex ligation-dependent probe amplification Multiplex ligation-dependent probe amplification (MLPA) is a commonly used technique for determining relative DNA sequence dosage (or copy number) in a complex DNA sample. Originally MLPA was designed as a copy number analysis tool for detecting disease-causing genomic mutations and has been successfully applied in the testing and identification of hundreds of genomic mutations in numerous genes including DMD, BRCA1, NF1, and TSC2. More recently, several modifications of the original technique have been implemented. Arguably the most important enhancement of MLPA has been probe generation by chemical synthesis, enabling the facile creation of novel probe sets for any desired application. Other newer applications of MLPA include methylation status determination, copy number analysis in segmentally duplicated regions, expression profiling, and transgene genotyping. MLPA has a potential major role in the analysis of common copy number variation in genome-wide association analyses, which may be enhanced by future improvements to increase throughput and lower costs, such as array-MLPA. Keywords: Copy number variation / Expression profiling / Multiplex ligation-dependent probe amplification / Mutation detection / Transgene genotyping DOI 10.1002/elps.200800126 1 Introduction Copy number variants (CNV), defined as heritable losses and gains of DNA segments of a size greater than 1 kb [1] within the human genome, have been known for over 2 decades. Recently, CNV have become of much greater interest as their frequency in the human genome and importance for human phenotypes have both been demon- strated. CNV have been shown to commonly affect at least 12% of the human genome [2]. Since they can affect coding sequences and/or regulatory elements, and thereby result in gene dosage effects, their biological effects range from neutral to modest to mutation-like. Although the great majority of CNV have an uncertain significance, increasing numbers are associated with clinical disease or significant effects on the human phenotype [3–8]. Two dramatic recent examples include the association of the copy number of a 300 kb region on chromosome 8p that consists of several b-defensin genes with a risk of psoriasis [9] and a positive correlation between the copy number of the AMY1 gene with the level of salivary amylase and high-starch diets [10]. Although a number of methods have been developed for assessment of CNV at the genome-wide level, such as array comparative genome hybridization (CGH) or high-density SNP arrays (reviewed in [11]), a major laboratory tool for analysis of CNV over small regions of the genome is multiplex ligation-dependent probe amplification (MLPA). Owing to its simplicity and robustness, MLPA is now widely used in many clinical genetic and research laboratories for the detection of disease-causing partial or complete gene deletions or amplifications. In fact, two methods for detec- tion of copy number variation involving multiple exons were developed; each of them takes advantage of the multiplex amplification of probes hybridized to specific genomic targets. Multiplex amplifiable probe hybridization (MAPH) was first described in 2000 [12], while MLPA was described two years later [13]. These methods have been formally compared [14]. Although the methods are based on similar principles, MLPA has become widely used due to its more limited DNA consumption (50 ng per assay) and its single-tube assay Piotr Kozlowski 1 Anna J. Jasinska 2 David J. Kwiatkowski 3 1 Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland 2 Center for Neurobehavioral Genetics, University of California, Los Angeles, CA, USA 3 Division of Translational Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Received February 22, 2008 Revised May 30, 2008 Accepted June 11, 2008 Abbreviations: AS, Angelman Syndrome; CGH, comparative genome hybridization; CNV, copy number variant; gDNA, genomic DNA; MAPH, multiplex amplifiable probe hybridization; MLPA, multiplex ligation-dependent probe amplification; MS-MLPA, methylation-specific MLPA; PWS, Prader–Willi Syndrome; RT-MLPA, reverse transcription- MLPA; UPD, uniparental disomy Correspondence: Dr. Piotr Kozlowski, Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, PAS, Noskowskiego 12/14, 61-704 Poznan, Poland E-mail: kozlowp@rose.man.poznan.pl Fax: 148-061-8528919 & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2008, 29, 4627–4636 4627