Electrophoresis 2012, 00, 1–10 1 Gi Won Shin 1* Seung-Hyun Jung 2* Seon-Hee Yim 2,3 Boram Chung 4 Gyoo Yeol Jung 4,5** Yeun-Jun Chung 2 1 Institute of Environmental and Energy Technology, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea 2 Department of Microbiology, Integrated Research Center for Genome Polymorphism, The Catholic University of Korea School of Medicine, Seoul, Korea 3 Department of Medical Humanities and Social Sciences, The Catholic University of Korea School of Medicine, Seoul, Korea 4 School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea 5 Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea Received June 23, 2012 Revised July 18, 2012 Accepted July 23, 2012 Research Article Stuffer-free multiplex ligation-dependent probe amplification based on conformation-sensitive capillary electrophoresis: A novel technology for robust multiplex determination of copy number variation Developing diagnostic tools based on the application of known disease/phenotype- associated copy number variations (CNVs) requires the capacity to measure CNVs in a multiplex format with sufficient reliability and methodological simplicity. In this study, we developed a reliable and user-friendly multiplex CNV detection method, termed stuffer- free MLPA-CE-SSCP, that combines a variation of multiplex ligation-dependent probe amplification (MLPA) with CE-SSCP. In this variation, MLPA probes were designed with- out the conventionally required stuffer sequences. To separate the similar-sized stuffer- free MLPA products, we adopted CE-SSCP rather than length-dependent conventional CE analysis. An examination of the genomic DNA from five cell lines known to vary in X-chromosome copy number (1–5) revealed that copy number determinations using stuffer-free MLPA-CE-SSCP were more accurate than those of conventional MLPA, and the CV of the measured copy numbers was significantly lower. Applying our system to measure the CNVs on autosomes between two HapMap individuals, we found that all peaks for CNV targets showed the expected copy number changes. Taken together, our results indicate that this new strategy can overcome the limitations of conventional MLPA, which are mainly related to long probe length and difficulties of probe preparation. Keywords: Capillary electrophoresis-single-strand conformation polymorphism / Copy number variation / Multiplex ligation-dependent probe amplification / Stuffer sequence DOI 10.1002/elps.201200334 1 Introduction In addition to single-base variations, referred to as SNPs, large-scale deletions and amplifications have been implicated as causes of human diseases and phenotypic variation, as well as evolutionary drivers [1–3]. Numerous quantitative as- says have been developed to detect these structural varia- tions, generally termed copy number variations (CNVs) [4], Correspondence: Professor Yeun-Jun Chung, Department of Mi- crobiology, Integrated Research Center for Genome Polymor- phism, The Catholic University of Korea, 505 Banpo-dong, Socho- gu, Seoul 137-701, Korea E-mail: yejun@catholic.ac.kr Fax: +82-2-596-8969 Abbreviations: CNV, copy number variation; MLPA, mul- tiplex ligation-dependent probe amplification; NGS, next- generation sequencing; qPCR, real-time quantitative PCR but population genetic and disease association studies have predominantly employed array-based genomic screening and PCR-based validation assays. Hybridization-based copy number detection tools, such as array-comparative genomic hybridization and SNP geno- typing array analysis, are now the major technologies for genome-wide CNV analysis [5]. However, because of the possibility of nonspecific hybridizations, some subtle CNVs cannot be reliably determined using these approaches; ac- cordingly, CNVs determined by hybridization-based tools should be validated by a quantitative genetic assay [6]. Next-generation sequencing (NGS) technology has recently emerged as a new method for CNV detection as well as ∗ These authors contributed equally to this article. ∗∗ Additional corresponding author: Professor Gyoo Yeol Jung, E-mail: gyjung@postech.ac.kr Colour Online: See the article online to view Figs. 2, 3 and 5 in colour. C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com