Computational protocol: New MT-ND6 and NDUFA1 mutations in mitochondrial respiratory chain disorders

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Protocol publication

[…] Genomic DNA (gDNA) was extracted from skin fibroblasts (Data S1), blood, liver, and cardiac muscle using either phenol/chloroform- or column-based extraction. Whole mtDNA was first polymerase chain reaction (PCR)-amplified as two separate large amplicons (LA1 and LA2) avoiding the nonspecific amplifications from nDNA. Second-round PCR was performed using 46 primer pairs (mitoSEQrTM; Applied Biosystems, Carlsbad, CA) and the LA1 and LA2 amplicon mixture from first-round PCR as a template. PCR conditions were as follows: first-round PCR was performed in a reaction mixture containing 0.2 mmol/L of each dNTP, 0.25 U of Takara Ex Taq (Takara Bio, Shiga, Japan), 1× Ex Taq Buffer, 0.3 μmol/L of each primer, and extracted gDNA in a total volume of 50 μL. Initial denaturation was performed at 94°C for 2 min, followed by 30 cycles of 94°C for 20 sec, 60°C for 20 sec, and 72°C for 5 min, with a final extension at 72°C for 11 min. Second-round PCR was performed in a reaction mixture as above except with a 10,000-fold dilution of LA1 amplicon and a 100-fold dilution of LA2 amplicon (total volume of the PCR reaction, 10 μL). Initial denaturation was performed at 96°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 60°C for 45 sec, and 72°C for 45 sec, with a final extension at 72°C for 10 min.First- and second-round PCR products were separated by 1% and 2% agarose gels, respectively, then 10 μL of second-round PCR products were incubated with 1 μL of ExoSAP-IT reagent (GE Healthcare UK Ltd., Bucks, U.K.) at 37°C for 30 min to degrade remaining primers and nucleotides. The ExoSAP-IT reagent was then inactivated by incubating at 75°C for 15 min. PCR products were sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and an ABI3130xl Genetic Analyzer (Applied Biosystems). Sequence data were compared with the revised Cambridge sequence (GenBank Accession No. NC_012920.1) and sequences present in MITOMAP (http://mitomap.org/MITOMAP) and mtSNP (http://mtsnp.tmig.or.jp/mtsnp/index_e.shtml) using SeqScape software (Applied Biosystems). Whole mtDNA sequencing of seven samples was obtained using an Ion PGM™ sequencer (Life Technologies Corporation, Carlsbad, CA). […]

Pipeline specifications

Software tools MUSCLE, SeqScape
Databases MITOMAP
Applications Sanger sequencing, Nucleotide sequence alignment
Organisms Homo sapiens
Diseases Leigh Disease, Mitochondrial Diseases