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Stuffer-free multiplex ligation-dependent probe amplification based on conformation-sensitive capillary electrophoresis: a novel technology for robust multiplex determination of copy number variation.

Authors
  • Shin, Gi Won
  • Jung, Seung-Hyun
  • Yim, Seon-Hee
  • Chung, Boram
  • Yeol Jung, Gyoo
  • Chung, Yeun-Jun
Type
Published Article
Journal
Electrophoresis
Publisher
Wiley (John Wiley & Sons)
Publication Date
Oct 01, 2012
Volume
33
Issue
19-20
Pages
3052–3061
Identifiers
DOI: 10.1002/elps.201200334
PMID: 22965760
Source
Medline
License
Unknown

Abstract

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 without 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.

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