Computational protocol: Clinical and Mutational Analysis of the GCDH Gene in Malaysian Patients with Glutaric Aciduria Type 1

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Protocol publication

[…] Approximately 5 to 10 mL of peripheral blood was collected from all seven patients in EDTA tubes and genomic DNA was isolated using the QIAcube (Qiagen, Hilden, Germany). Both the quantity and quality of extracted DNAs were measured using NanoDrop ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE, USA). Primers were designed in-house to amplify all coding exons and flanking intronic sequences of GCDH gene. PCR was performed in a volume of 50 μL containing 100 ng genomic DNA, 0.1 U Taq DNA polymerase (Fermentas), 1x of Pfu PCR buffer with MgSO4, 1 μmol of each of the primers, and 0.2 mM of dNTP mix. Amplification was performed using touchdown PCR protocol as described previously [].The PCR products were purified using the QIAquick PCR Purification kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. Cycle sequencing was performed using the BigDye® Terminator cycle sequencing v3.1 chemistry (Applied Biosystems, Foster City, CA, USA) followed by purification using DyeEx 2.0 Spin Kit (Qiagen). Purified cycle sequencing products were dried on vacuum concentrator, resuspended in 10 μL of Hi-Di formamide, and analyzed on the Applied Biosystems 3500 Genetic Analyzer. Sequencing results were aligned to the reference sequence of the GCDH gene (GenBank NM_000159.3) using SeqScape Software v3.0 (Applied Biosystems) to identify any DNA variants. All variants identified were annotated against publicly available databases such as the HGMD and dbSNP (http://www.ncbi.nlm.nih.gov/SNP/). The pathogenicity of novel DNA variants was evaluated by using MutationTaster2 software (http://www.mutationtaster.org/). DNA samples from 50 healthy individuals were tested for the presence of novel DNA variants identified in our patients. […]

Pipeline specifications

Software tools SeqScape, MutationTaster
Databases dbSNP HGMD
Application Sanger sequencing
Organisms Homo sapiens
Diseases Genetic Diseases, Inborn, Multiple Acyl Coenzyme A Dehydrogenase Deficiency