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Epigenetic Sequencing Using Enzymatic Detection Method

2018-10-10
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Investigators at the University of Pennsylvania’s (UPenn) Perelman School of Medicine have provided a novel method for sequencing epigenetic modifications using 1,000-fold less DNA than the current gold standard technique. The UPenn researchers believe that their novel method could pave the way for better detection of cancer and other diseases in the blood. Findings from the new study were published recently in Nature Biotechnology through an article titled “Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase.”

 

 

The epigenetic chemical groups mark one of the four DNA “letters” in the genome, and it is differences in these marks along DNA that control which genes are expressed or silenced. To detect disease earlier and with increased precision, researchers have a growing interest in analyzing free-floating DNA in settings in which there is a limited amount, such as that extruded from tumors into the bloodstream.

 

“We’re hopeful that this method offers the ability to decode epigenetic marks on DNA from small and transient populations of cells that have previously been difficult to study, in order to determine whether the DNA is coming from a specific tissue or even a tumor,” explains senior study investigator Rahul Kohli, M.D., Ph.D., an assistant professor of biochemistry and biophysics, and medicine at UPenn.

 

Scientists have investigated these DNA modifications over the last two decades to understand better and diagnose an array of disorders, most notably cancer. In recent years, the major methods used to decipher the epigenetic code have relied on a chemical called bisulfite. While bisulfite has proven useful, it also presents major limitations: it is unable to differentiate the most common modifications on the DNA building block cytosine, and more significantly, it destroys much of the DNA it touches, leaving little material to sequence in the lab.

 

This newly described method – given the moniker ACE-seq – builds on the fact that a class of immune-defense enzymes, called APOBEC DNA deaminases, can be repurposed for biotech applications. Specifically, the deaminase-guided chemical reaction can achieve what bisulfite could do, but without harming DNA.

 

“This technological advance paves the way to better understand complex biological processes such as how the nervous system develops or how a tumor progresses,” notes co-senior study investigator Hao Wu, Ph.D., an assistant professor of genetics at UPenn.

 

Using this method, the team showed that determining the epigenetic code of one type of neuron used 1,000-times less DNA than required by the bisulfite-dependent methods. From this, the new method could also differentiate between the two most common epigenetic marks, methylation, and hydroxymethylation.

 

“We were able to show that sites along the genome that appear to be modified are in fact very different in terms of the distribution of these two marks,” Dr. Kohli concludes. “This finding suggests important and distinctive biological roles for the two marks on the genome.”

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