title: Improved Multiplex Ligation-dependent Probe Amplification (i-MLPA) for rapid copy number variant (CNV) detection.
abstract: BACKGROUND
  In Multiplex Ligation-dependent Probe Amplification (MLPA), copy number variants (CNVs) for specific genes are identified after normalization of the amounts of PCR products from ligated reference probes hybridized to genomic regions that are ideally free from normal variation. However, we observed ambiguous calls for two reference probes in an investigation of the human 15q11.2 region by MLPA among 20 controls, due to the presence of single nucleotide polymorphisms (SNPs) in the probe-binding regions. Further in silico analysis revealed that 18 out of 19 reference probes hybridize to regions subject to variation, underlining the requirement for designing new reference probes against variation-free regions.
  
  
  METHODS
  An improved MLPA (i-MLPA) method was developed by generating a new set of reference probes to reduce the chances of ambiguous calls and new reagents that reduce hybridization times to 30 min from 16h to obtain MLPA ratio data within 6h. Using i-MLPA, we screened 240 schizophrenia patients for CNVs in 15q11.2 region.
  
  
  CONCLUSIONS
  Three deletions and two duplications were identified among the 240 schizophrenia patients. No variation was observed for the new reference probes. Taken together, i-MLPA procedure helps obtaining non-ambiguous CNV calls within 6h without compromising accuracy.
authors.affiliation: Department of Biological Sciences, Birla Institute of Technology and Science Pilani - Hyderabad Campus, Jawahar Nagar, Shameerpet Mandal, Hyderabad 500078, India.
authors.forename: Sonal
authors.initials: S
authors.lastname: Saxena
authors.affiliation: Department of Biological Sciences, Birla Institute of Technology and Science Pilani - Hyderabad Campus, Jawahar Nagar, Shameerpet Mandal, Hyderabad 500078, India.
authors.forename: Kavitha
authors.initials: K
authors.lastname: Gowdhaman
authors.affiliation: Department of Zoology, Thiagarajar College, Madurai 625009, India.
authors.forename: Poornima
authors.initials: P
authors.lastname: Kkani
authors.affiliation: Department of Biological Sciences, Birla Institute of Technology and Science Pilani - Hyderabad Campus, Jawahar Nagar, Shameerpet Mandal, Hyderabad 500078, India.
authors.forename: Bhavyasri
authors.initials: B
authors.lastname: Vennapusa
authors.affiliation: MS Chellamuthu Trust and Research Foundation, Madurai 625020, India.
authors.forename: Chellamuthu
authors.initials: C
authors.lastname: Rama Subramanian
authors.affiliation: MS Chellamuthu Trust and Research Foundation, Madurai 625020, India.
authors.forename: S
authors.initials: S
authors.lastname: Ganesh Kumar
authors.affiliation: Department of Biological Sciences, Birla Institute of Technology and Science Pilani - Hyderabad Campus, Jawahar Nagar, Shameerpet Mandal, Hyderabad 500078, India. Electronic address: MohanKN@hyderabad.bits-pilani.ac.in.
authors.forename: Kommu Naga
authors.initials: KN
authors.lastname: Mohan
mesh_terms: D002869:Chromosome Aberrations; D002884:Chromosomes, Human, Pair 15 / Q000235:genetics; D056915:DNA Copy Number Variations / Q000235:genetics*; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D013997:Time Factors
keywords: CNV detection; DGV; MLPA