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During methylation, healthy genes can be switched on or off
potentially causing cancer without any changes in the
underlying DNA sequence. The current methods for methylation
screening, have significant drawbacks, explains lead study
author Vasudev Bailey, a biomedical engineering Ph.D.
candidate at Hopkins.
Methylation
specific PCR, which copies specific DNA sequences millions
of times within a few hours, may not be sensitive enough to
detect small amounts of methylation, and real time PCR,
which allows scientists to view increases in the amount of
DNA as it is copied, needs to be run several times and can
be expensive, he says.
The Hopkins-developed test makes PCR technology more
sensitive and efficient, Bailey said. The work was presented
at the American Association for Cancer Research's third
International Conference on Molecular Diagnostics in Cancer
Therapeutic Development being held September 22-25, 2008, in
Philadelphia.
"The impact of detecting DNA methylation is profound, as it
has been demonstrated that a larger number of tumor
suppressor genes become inactivated through DNA methylation
than by mutations," Bailey said. "Our method of methylation
screening provides an easy, cost-effective and valuable tool
for the early diagnosis of cancer, monitoring tumor behavior
and measuring the response of tumors to targeted cancer
therapies."
To test the technique, Bailey and colleagues treated
segments of DNA with the chemical compound sodium bisulfate.
This automatically converted unmethylated cytosines (one of
the bases of DNA) to uracils (one of the bases of
ribonucleic acid or RNA, which works with DNA to synthesize
proteins), while leaving the methylated cytosines untouched.
Then the scientists used PCR with labeled primers to copy
and label these DNA segments with the vitamin biotin. Next,
they added quantum dots (molecules about a billionth of a
meter in size with electrical properties) to the samples
that had been coated with the protein streptavidin. Like a
magnetic force, the biotin-coated methylated segments of DNA
were attracted to the streptavidin coating the quantum dots,
highlighting and quantifying DNA methylation.
The new test was sensitive enough to detect as little as 15
picograms of methylated DNA in the presence of a 10,000-fold
excess of unmethylated coding sequences, or the equivalent
of five cells. In addition, they demonstrated detection
capability in as few as eight PCR cycles. In collaboration
with his colleague Yi Zhang, also a PhD candidate at Johns
Hopkins school of medicine, they were able to see results
using very small samples (an average of 800 billionth of a
liter per reaction and more than fifty times less sample and
reagent as used currently) using a novel lab-on-chip system.
This system allows for minimal handing of samples from the
researcher, while allowing for simultaneous processing and
analysis of multiple samples. Researchers have a provisional
patent on the test.
In additional experiments, the researchers used the
technology to accurately detect methylation for the gene
ASC/TMS1, which promotes programmed cell death, in low
concentrations of DNA from human sputum. This was
accomplished with fewer steps and fewer PCR cycles.
Scientists also used the test to quantify the amount of
methylation reversal in bone marrow fluid samples taken from
patients with myelodysplastic syndrome – a disorder in which
bone marrow cells don't function normally – before and after
they had been treated with medications.
Bailey said the new test allows scientists to detect
methylation of multiple genes at the same time, or view
methylated and unmethylated DNA at the same time. It also
reveals the percentage of methylation at any given time. |
