The Human MitoChip: A High-Throughput Sequencing Microarray for Mitochondrial Mutation Detection
- Anirban Maitra1,3,
- Yoram Cohen2,
- Susannah E.D. Gillespie3,
- Elizabeth Mambo2,
- Noriyoshi Fukushima1,
- Mohammad O. Hoque2,
- Nila Shah4,
- Michael Goggins1,
- Joseph Califano2,
- David Sidransky1,2, and
- Aravinda Chakravarti3,5
- 1 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
- 2 Departments of Otolaryngology and Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
- 3 McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
- 4 Affymetrix, Inc., Santa Clara, California 95051, USA
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.
Footnotes
-
[Supplemental material is available online at www.genome.org. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: B. Vogelstein, V. Velculescu, J. Jones, and S. Kern.]
-
Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.2228504.
-
↵5 Corresponding author. E-MAIL aravinda{at}jhmi.edu; FAX (410) 502-7544.
-
- Accepted February 12, 2004.
- Received December 1, 2003.
- Cold Spring Harbor Laboratory Press