Genotyping G6PD SNPs from PEP DNA

Genomic DNA (gDNA) was extracted from blood samples using Nucleon BACC2 DNA extraction kits [Gen-Probe Life Sciences, Tepnel Research Products and Services, Manchester, UK]. Five nano-grammes of gDNA was whole-genome amplified by primer-extension pre-amplification (PEP) using N15 primers (Sigma, UK) and Biotaq (Bioline, UK) polymerase as previously described by Zhang et al. [17 (link)]. Sixty-eight single nucleotide polymorphisms (SNPs) located in the G6PD gene and its flanking regions were selected from the many hundreds of SNPs identified in the literature and on public databases based on the given criteria (1) previous associations with malaria, (2) predicted functional consequences with respect to G6PD enzyme activity (3) estimated minor allele frequency and (4) whether they made a viable Sequenom assay design [18 (link)]. Three gender-typing markers were also included. Assays were performed using the Sequenom® iPLEX platform according to manufacturer’s instruction using diluted PEP DNA (1:10). Genotype calls were made using the Sequenom® Typer v4.03 software [19 (link)].

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Dewasurendra R.L., Rockett K.A., Fernando S.D., Carter R., Kwiatkowski D.P, & Karunaweera N.D. (2015). G6PD gene variants and its association with malaria in a Sri Lankan population. Malaria Journal, 14, 93.

Publication 2015

N15 primers Biotaq iPLEX platform Typer v4.03 software

Assays Blood Enzyme activity G6pd Gender Gene Genome Genotype Iplex Malaria Primers Single nucleotide polymorphisms

Corresponding Organization :

Other organizations : University of Colombo, Wellcome Centre for Human Genetics, University of Oxford, University of Edinburgh

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Variable analysis

independent variables

Primer-extension pre-amplification (PEP) using N15 primers (Sigma, UK) and Biotaq (Bioline, UK) polymerase

dependent variables

Genotype calls for 68 single nucleotide polymorphisms (SNPs) located in the G6PD gene and its flanking regions

control variables

Genomic DNA (gDNA) extracted from blood samples using Nucleon BACC2 DNA extraction kits
Diluted PEP DNA (1:10) used for Sequenom® iPLEX platform assays

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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