Computational protocol: Analysis of Mitochondrial DNA Sequences in Childhood Encephalomyopathies Reveals New Disease-Associated Variants

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Protocol publication

[…] Excess primers and dNTPs were removed from the PCR amplified DNA fragments using 0.5 unit of shrimp alkaline phosphatase (Amersham pharamacia, USA) and 2 units of Exonuclease I (Amersham). The fragments were then cycle sequenced using Bigdye terminator cycle sequencing kit with a thermostable thermsequenase II DNA Polymerase. The products were precipitated using salt/ethanol to remove all unincorporated dye labeled terminators, and the pellet was diluted in formamide loading dye and analyzed with an ABI 3730 sequencing instrument (Applied Biosystems, USA). Both forward and reverse primers were used for sequencing.Sequence data was analyzed for mutations by ABI software (SeqScape version 2.1) and also confirmed by BLASTn program. All the nucleotide sequences were compared with the revised Cambridge reference sequence (CRS) and with those present in two mitochondrial databases; MITOMAP ( and Human mitochondrial genome database ( When the genomic change was located in an encoding region, we used the Mitoanalyzer programme (http://www.cstl.nist) to determine whether the mutation triggered any amino acid change in the polypeptide sequence. All the identified variants were also tested in patient's mothers to find out whether they are maternally inherited or are of de novo origin. […]

Pipeline specifications

Software tools SeqScape, BLASTN
Databases MITOMAP mtDB
Application Sanger sequencing
Organisms Homo sapiens
Diseases Brain Diseases, Mitochondrial Encephalomyopathies, Mitochondrial Diseases