title: The Human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection.
abstract: Somatic mitochondrial mutations are common in human cancers, and can be used as a tool for early detection of cancer. We have developed a mitochondrial Custom Reseq microarray as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The MitoChip contains oligonucleotide probes synthesized using standard photolithography and solid-phase synthesis, and is able to sequence >29 kb of double-stranded DNA in a single assay. Both strands of the entire human mitochondrial coding sequence (15,451 bp) are arrayed on the MitoChip; both strands of an additional 12,935 bp (84% of coding DNA) are arrayed in duplicate. We used 300 ng of genomic DNA to amplify the mitochondrial coding sequence in three overlapping long PCR fragments. We then sequenced >2 million base pairs of mitochondrial DNA, and successfully assigned base calls at 96.0% of nucleotide positions. Replicate experiments demonstrated >99.99% reproducibility. In matched fluid samples (urine and pancreatic juice, respectively) obtained from five patients with bladder cancer and four with pancreatic cancer, the MitoChip detected at least one cancer-associated mitochondrial mutation in six (66%) of nine samples. The MitoChip is a high-throughput sequencing tool for the reliable identification of mitochondrial DNA mutations from primary tumors in clinical samples.
authors.affiliation: Department of Pathology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Anirban
authors.initials: A
authors.lastname: Maitra
authors.forename: Yoram
authors.initials: Y
authors.lastname: Cohen
authors.forename: Susannah E D
authors.initials: SE
authors.lastname: Gillespie
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Mambo
authors.forename: Noriyoshi
authors.initials: N
authors.lastname: Fukushima
authors.forename: Mohammad O
authors.initials: MO
authors.lastname: Hoque
authors.forename: Nila
authors.initials: N
authors.lastname: Shah
authors.forename: Michael
authors.initials: M
authors.lastname: Goggins
authors.forename: Joseph
authors.initials: J
authors.lastname: Califano
authors.forename: David
authors.initials: D
authors.lastname: Sidransky
authors.forename: Aravinda
authors.initials: A
authors.lastname: Chakravarti
mesh_terms: D001826:Body Fluids / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D019295:Computational Biology / Q000379:methods / Q000458:organization & administration; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005838:Genotype; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000235:genetics; D008214:Lymphocytes / Q000737:chemistry; D016555:Matched-Pair Analysis; D008928:Mitochondria / Q000235:genetics* / Q000473:pathology; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D010189:Pancreatic Juice / Q000737:chemistry / Q000378:metabolism / Q000382:microbiology; D015203:Reproducibility of Results; D001749:Urinary Bladder Neoplasms / Q000737:chemistry / Q000235:genetics
chemical_list: D004272:DNA, Mitochondrial; D004273:DNA, Neoplasm; D004247:DNA