|Mitochondrial DNA Sequencing Tool Updated
High-tech laboratory tools, like computers, are often updated
publicly as their analytical capabilities expand. In the September
issue of the Journal of Molecular Diagnostics, NIH grantees report
they have developed a second generation “lab on a silicon chip” called
the MitoChip v2.0 that for the first time rapidly and reliably
sequences all mitochondrial DNA. Mitochondria, the energy-producing
organelles that power our cells, are unique because they are equipped
with their own genetic instructions distinct from the DNA stored
in the cell nucleus.
The authors say their full-sequence chip will be a key tool in
accelerating research on mitochondrial DNA, a growing area of scientific
interest. This interest stems from data that suggests natural sequence
variations and/or mutations in each person’s mitochondrial DNA
could be biologically informative in fields as diverse as cancer
diagnostics, gerontology, and criminal forensics.
According to Dr. Joseph Califano, a scientist at Johns Hopkins
University School of Medicine in Baltimore and senior author on
the paper, the MitoChip v2.0 showed in his group’s hands better
sensitivity that its predecessor to sequence variations in head
and neck cancer samples. The v2.0 also detected nearly three dozen
variations in the non-coding D-loop, long considered to be a sequencing
no-man’s land and which the original MitoChip did not include.
“At this point, we don’t foresee a MitoChip v3.0,” said Califano,
whose research was supported by the NIH’s National Institute of
Dental and Craniofacial Research. “The v2.0 is a very good tool
in that we’ve also arrayed 500 of the most common haplotypes —
or grouped patterns of known DNA variations — banked in the mitochondrial
Mitochondria are oblong, thread-like structures dispersed throughout
the cell’s cytoplasm. Hundreds to thousands of mitochondria exist
in each human cell, occupying up to a quarter of their cytoplasm.
Sometimes informally described as “cellular power plants,” mitochondria
convert organic materials into ATP, the cell’s energy currency
and without which life would cease.
As early as the 1920s, scientists uncovered clues that mitochondria
might play a role in causing cancer. But like the other DNA in
the cell nucleus, scientists lacked the needed research tools throughout
most of the 20th century to systematically study the chemical composition
of the mitochondrial genome, or complete set of genes, and its
association to human disease.
In the early 1980s, scientists in England performed the then-Herculean
feat of sequencing the complete human mitochondrial genome. The
genome consisted of 16,568 base pair, or units, of DNA and encoded
37 contiguous genes.
By 1996, new technology brought new opportunity. Scientists with
the company Affymetrix in Santa Clara, Calif. developed the first
mitochondrial sequencing microarray. Roughly the size of a quarter,
the silicon chip had lithographically annealed to it up to 135,000
short, arrayed bits of DNA sequence that, collectively, spanned
most of a single strand of mitochondrial DNA.
The chip exploited the fact that DNA exists naturally as a double-stranded
molecule. By gathering mitochondrial DNA and breaking it into short,
single-stranded bits, the scientists showed that each bit would
pair, or hybridize, with its complementary sequence arrayed on
the chip. By crude analogy, each bit is like a unique magnet that
sticks to its mirror image.
But if the extracted DNA contains mutations or other variations
from the standard consensus sequence annealed to the chip, the
bits with those changes would appear abnormal to the specially
designed computer software programs that read the chip. The software
programs will read not only the identity of the expected bases
of DNA in a process called “base calling” but those of the variations.
The Affymatrix chip enabled laboratories to resequence the mitochondrial
genome much faster than the traditional manual and automated strategies.
Just as importantly, like an iPod to music lover, the chip served
as the broad technological platform for laboratories to customize
arrays more attuned to their research interests.
In 2004, Dr. Anirban Maitra and his colleagues at Johns Hopkins
did exactly that with the MitoChip v1.0. In addition to nitty-gritty
technical innovations that vastly improved the rate and speed of
the chip, the v1.0 marked the first mitochondrial resequencing
microarray designed as a potential screening tool for cancer. “With
mitochondrial DNA, there is a mass advantage,” said Dr. Anirban
Maitra, an author on this month’s paper whose research is supported
by the NIH’s National Cancer Institute. “Whereas nuclear DNA contains
just two copies of every gene, there are literally hundreds of
mitochondria in most cells. If you are screening saliva or other
bodily fluids with a limited number of cells to analyze, mitochondrial
DNA gives you more to work with and a better chance of detecting
mutations that might be associated with a developing cancer.”
Dr. Maitra said that despite the original Mitochip’s 96 percent
success rate assigning base calls, there was room for improvement.
Led by Drs. Shaoyu Zhou and Keyaunoosh Kassauei, the Hopkins group
cobbled together the MitoChip v2.0. reported in this month’s Journal
of Molecular Diagnostics. It yielded essentially the same base-call
success rate as its predecessor, showed near perfect reproducibility
in replicate experiments, and detected more variations than the
As a proof of principle, the Mitochip v2.0 also detected 31 variations
in the non-coding D-loop of 14 head and neck tumor samples. Included
in this tally were several mutations that possibly are informative
of the disease.
“The real interesting thing is nobody has been able to study these
D-loop alterations very well,” said Califano “They clearly occur
in tumor cells, and there is some type of selection process for
them. But their functional significance has been hard to know.
Now, you can sequence the D loop so readily and begin to look harder
for associations in certain cancers.”
The National Institute of Dental and Craniofacial Research
is the nation’s leading funder of research on oral, dental, and
Created in 1937 as the federal government's principal agency
for cancer research, NCI is the oldest of the 27 institutes and
centers that comprise the NIH in Bethesda, Md.
The National Institutes of Health (NIH) — The Nation's
Medical Research Agency — includes 27 Institutes and
Centers and is a component of the U.S. Department of Health and
Human Services. It is the primary federal agency for conducting
and supporting basic, clinical and translational medical research,
and it investigates the causes, treatments, and cures for both
common and rare diseases. For more information about NIH and
its programs, visit www.nih.gov.