Poster abstracts

Poster number 36 submitted by Tabatha Giesler

Optimizing and Validation Of a Massively Multiplexed Bead Array For Discovery (MMBAD) To Be Used In Identifying Functional Variants Wchich Affect Transcription In Artificial Systems.

Tabatha R. Giesler (Camera of Molecular and Behavioral Neurogenetics in The Battelle Center for Mathematical Medicine, The Research Institute at Nationwide Childrens Hospital & The Ohio State University.), Christopher W. Bartlett (Camera of Molecular and Behavioral Neurogenetics in The Battelle Center for Mathematical Medicine, The Research Institute at Nationwide Childrens Hospital & The Ohio State University.)

Abstract:
A panel comprising of 47 Single Nucleotide Polymorphisms (SNPs), also known as Cognitive Panel 1 (CP1), the first of a series of panels to be developed in the future, was optimized as a Massively Multiplexed Bead Array for Discovery (MMBAD). All SNPs in CP1 have some evidence of functional effects in either brain or cell line systems. The panel utilizes 47 DNA primer sets in a single polymerase chain reaction (PCR). Next, a Ligase Detection Reaction (LDR) is performed on the amplicons to interrogate the specific alleles, each coupled to a unique fluorophore. Finally, a dual laser multidetermined flow cytometry device visualizes all members of the panel in the multiplex reaction and quantifies the signal intensity of each allele. To validate the CP1 SNPs themselves, we genotyped a series of 4 large extended pedigrees from the anonymous, publicly available Centre d'Etude du Polymorphisme Humain (CEPH) collection. This comparison allowed for error checking (via missegregation of alleles in the pedigrees) as a measure of which assays could be successfully multiplexed together. Consequently, several of the SNPs were removed from CP1 due to these conflicts, leaving 30 SNPs in the final CP1 panel. Once optimized and validated, the final panel will be used to examine the gene expression levels in the CEPH lymphoblast cell lines to asses if these variants functionally affect transcription in this artificial system.

References:
Myers AJ, Gibbs JR, Webster JA, Rohrer K, Zhao A, Marlowe L, Kaleem M, Leung D, Bryden L, Nath P, Zismann VL, Joshipura K, Huentelman MJ, Hu-Lince D, Coon KD, Craig DW, Pearson JV, Holmans P, Heward CB, Reiman EM, Stephan D, Hardy J. (2007) A survey of genetic human cortical gene expression. Nat Genet 39:1494-9

Keywords: Gene Expression, Polymorphism, Multiplex