Tag Archives: genome

New nanomapping technology: CRISPR-CAS9 as a programmable nanoparticle

A November 21, 2017 news item on Nanowerk describes a rather extraordinary (to me, anyway) approach to using CRRISP ( Clustered Regularly Interspaced Short Palindromic Repeats)-CAS9 (Note: A link has been removed),

A team of scientists led by Virginia Commonwealth University physicist Jason Reed, Ph.D., have developed new nanomapping technology that could transform the way disease-causing genetic mutations are diagnosed and discovered. Described in a study published today [November 21, 2017] in the journal Nature Communications (“DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle”), this novel approach uses high-speed atomic force microscopy (AFM) combined with a CRISPR-based chemical barcoding technique to map DNA nearly as accurately as DNA sequencing while processing large sections of the genome at a much faster rate. What’s more–the technology can be powered by parts found in your run-of-the-mill DVD player.

A November 21, 2017 Virginia Commonwealth University news release by John Wallace, which originated the news item, provides more detail,

The human genome is made up of billions of DNA base pairs. Unraveled, it stretches to a length of nearly six feet long. When cells divide, they must make a copy of their DNA for the new cell. However, sometimes various sections of the DNA are copied incorrectly or pasted together at the wrong location, leading to genetic mutations that cause diseases such as cancer. DNA sequencing is so precise that it can analyze individual base pairs of DNA. But in order to analyze large sections of the genome to find genetic mutations, technicians must determine millions of tiny sequences and then piece them together with computer software. In contrast, biomedical imaging techniques such as fluorescence in situ hybridization, known as FISH, can only analyze DNA at a resolution of several hundred thousand base pairs.

Reed’s new high-speed AFM method can map DNA to a resolution of tens of base pairs while creating images up to a million base pairs in size. And it does it using a fraction of the amount of specimen required for DNA sequencing.

“DNA sequencing is a powerful tool, but it is still quite expensive and has several technological and functional limitations that make it difficult to map large areas of the genome efficiently and accurately,” said Reed, principal investigator on the study. Reed is a member of the Cancer Molecular Genetics research program at VCU Massey Cancer Center and an associate professor in the Department of Physics in the College of Humanities and Sciences.

“Our approach bridges the gap between DNA sequencing and other physical mapping techniques that lack resolution,” he said. “It can be used as a stand-alone method or it can complement DNA sequencing by reducing complexity and error when piecing together the small bits of genome analyzed during the sequencing process.”

IBM scientists made headlines in 1989 when they developed AFM technology and used a related technique to rearrange molecules at the atomic level to spell out “IBM.” AFM achieves this level of detail by using a microscopic stylus — similar to a needle on a record player — that barely makes contact with the surface of the material being studied. The interaction between the stylus and the molecules creates the image. However, traditional AFM is too slow for medical applications and so it is primarily used by engineers in materials science.

“Our device works in the same fashion as AFM but we move the sample past the stylus at a much greater velocity and use optical instruments to detect the interaction between the stylus and the molecules. We can achieve the same level of detail as traditional AFM but can process material more than a thousand times faster,” said Reed, whose team proved the technology can be mainstreamed by using optical equipment found in DVD players. “High-speed AFM is ideally suited for some medical applications as it can process materials quickly and provide hundreds of times more resolution than comparable imaging methods.”

Increasing the speed of AFM was just one hurdle Reed and his colleagues had to overcome. In order to actually identify genetic mutations in DNA, they had to develop a way to place markers or labels on the surface of the DNA molecules so they could recognize patterns and irregularities. An ingenious chemical barcoding solution was developed using a form of CRISPR technology.

CRISPR has made a lot of headlines recently in regard to gene editing. CRISPR is an enzyme that scientists have been able to “program” using targeting RNA in order to cut DNA at precise locations that the cell then repairs on its own. Reed’s team altered the chemical reaction conditions of the CRISPR enzyme so that it only sticks to the DNA and does not actually cut it.

“Because the CRISPR enzyme is a protein that’s physically bigger than the DNA molecule, it’s perfect for this barcoding application,” Reed said. “We were amazed to discover this method is nearly 90 percent efficient at bonding to the DNA molecules. And because it’s easy to see the CRISPR proteins, you can spot genetic mutations among the patterns in DNA.”

To demonstrate the technique’s effectiveness, the researchers mapped genetic translocations present in lymph node biopsies of lymphoma patients. Translocations occur when one section of the DNA gets copied and pasted to the wrong place in the genome. They are especially prevalent in blood cancers such as lymphoma but occur in other cancers as well.

While there are many potential uses for this technology, Reed and his team are focusing on medical applications. They are currently developing software based on existing algorithms that can analyze patterns in sections of DNA up to and over a million base pairs in size. Once completed, it would not be hard to imagine this shoebox-sized instrument in pathology labs assisting in the diagnosis and treatment of diseases linked to genetic mutations.

Here’s a link to and a citation for the paper,

DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle by Andrey Mikheikin, Anita Olsen, Kevin Leslie, Freddie Russell-Pavier, Andrew Yacoot, Loren Picco, Oliver Payton, Amir Toor, Alden Chesney, James K. Gimzewski, Bud Mishra, & Jason Reed. Nature Communications 8, Article number: 1665 (2017) doi:10.1038/s41467-017-01891-9 Published online: 21 November 2017

This paper is open access.

Authenticating chocolate and a bit about coffee

Apparently, not all premium chocolate is actually premium, like wine, expensive, premium product can be mixed with a more common variety to be sold at the higher, premium price.  Now, scientists in a collaboration which spans the US, China, and Trinidad and Tobago have found a way to authenticate premium chocolate according to a Jan. 15, 2014 news release on EurekAlert,

For some people, nothing can top a morsel of luxuriously rich, premium chocolate. But until now, other than depending on their taste buds, chocolate connoisseurs had no way of knowing whether they were getting what they paid for. In ACS’ Journal of Agricultural and Food Chemistry, scientists are reporting, for the first time, a method to authenticate the varietal purity and origin of cacao beans, the source of chocolate’s main ingredient, cocoa.

Dapeng Zhang and colleagues note that lower-quality cacao beans often get mixed in with premium varieties on their way to becoming chocolate bars, truffles, sauces and liqueurs. But the stakes for policing the chocolate industry are high. It’s a multi-billion dollar global enterprise, and in some places, it’s as much art as business. There’s also a conservation angle to knowing whether products are truly what confectioners claim them to be. The ability to authenticate premium and rare varieties would encourage growers to maintain cacao biodiversity rather than depend on the most abundant and easiest to grow trees. Researchers have found ways to verify through genetic testing the authenticity of many other crops, including cereals, fruits, olives, tea and coffee, but those methods aren’t suitable for cacao beans. Zhang’s team wanted to address this challenge.

Applying the most recent developments in cacao genomics, they were able to identify a small set of DNA markers called SNPs (pronounced “snips”) that make up unique fingerprints of different cacao species. The technique works on single cacao beans and can be scaled up to handle large samples quickly. “To our knowledge, this is the first authentication study in cacao using molecular markers,” the researchers state.

Here’s an image, provided by the researchers, illustrating their work,

Courtesy American Chemical Society [downloaded from http://pubs.acs.org/doi/abs/10.1021/jf404402v]

Courtesy American Chemical Society [downloaded from http://pubs.acs.org/doi/abs/10.1021/jf404402v]

Here’s a link to and a citation for the paper,

Accurate Determination of Genetic Identity for a Single Cacao Bean, Using Molecular Markers with a Nanofluidic System, Ensures Cocoa Authentication by Wanping Fang, Lyndel W. Meinhardt, Sue Mischke, Cláudia M. Bellato, Lambert Motilal, and Dapeng Zhang. J. Agric. Food Chem., 2014, 62 (2), pp 481–487 DOI: 10.1021/jf404402v Publication Date (Web): December 19, 2013
Copyright © 2013 American Chemical Society

This story reminded me that coffee too is sold at premium prices. Billed as the most expensive coffee in the world, Kopi Luwak, is harvested, so they say, from civet excrement and I have to wonder how anyone could authenticate that a bean had actually passed through a civet’s gastrointestinal tract and out the other end. I’ve also wondered how the practice of plucking coffee beans from civet excrement started (from the Kopi Luwak Wikipedia essay; Note: Links have been removed) here’s an answer to the second question,

The origin of kopi luwak is closely connected with the history of coffee production in Indonesia. In the early 18th century the Dutch established the cash-crop coffee plantations in their colony in the Dutch East Indies islands of Java and Sumatra, including Arabica coffee introduced from Yemen. During the era of Cultuurstelsel (1830—1870), the Dutch prohibited the native farmers and plantation workers from picking coffee fruits for their own use. Still, the native farmers wanted to have a taste of the famed coffee beverage. Soon, the natives learned that certain species of musang or luwak (Asian Palm Civet) consumed the coffee fruits, yet they left the coffee seeds undigested in their droppings. The natives collected these luwaks’ coffee seed droppings, then cleaned, roasted and ground them to make their own coffee beverage.[11] The fame of aromatic civet coffee spread from locals to Dutch plantation owners and soon became their favourite, yet because of its rarity and unusual process, the civet coffee was expensive even during the colonial era.[citation needed]

I guess that in the future when you eat premium chocolate you can be sure that you’ve gotten what you paid for. As for coffee, I’m sure that industry is working on its authentication processes too and in the meantime, you’ll have to rely on your palate.

Revising the view on genomes: mouse and human

Researchers in Canada and the US have resolved a question about DNA and structural protein. From the Oct. 4, 2012 news release on EurekAlert,

Scientists in Canada and the United States have used three-dimensional imaging techniques to settle a long-standing debate about how DNA and structural proteins are packaged into chromatin fibres. The researchers, whose findings are published in EMBO [European Molecular Biology Organization] reports, reveal that the mouse genome consists of 10-nm chromatin fibres but did not find evidence for the wider 30-nm fibres that were previously thought to be important components of the DNA architecture.

Scientists were trying to understand how DNA can be packed into a cell,

“DNA is an exceptionally long molecule that can reach several metres in length. This means it needs to be packaged into a highly compact state to fit within the limited space of the cell nucleus,” said David Bazett-Jones, Senior Scientist at the Hospital for Sick Children, Toronto, and Professor at the University of Toronto, Canada. “For the past few decades, scientists have favoured structural models for chromatin organization where DNA is first wrapped around proteins in nucleosomes. In one possible model, the strand of repeating nucleosomes is wrapped further into a higher-order thick 30-nm fibre. In a second model, the 30-nm fibre is not required to compact the DNA. Differences between these models have implications for the way the cell regulates the transcription of genes.”

Scientists offer reasons for why they concluded Previous studies have suggested for a 30-nm fibre model in earlier studies,

The researchers offer several reasons for the observation of wider fibres in earlier studies. In some cases, the conditions outside of the cell, including those used in earlier studies where chromatin was extracted from the cell, may have given rise to structural artifacts. For some of the earlier spectroscopic studies, it may even be a question of poor resolution of existing 10-nm fibres.

Here’s what the scientists found,

“Our results revealed that the 30-nm chromatin fibre model is not consistent with the structure we found in our three-dimensional spectroscopic images,” said Bazett-Jones. “It was previously thought that the transition between thinner and thicker fibres represented a change from an active to repressed state of chromatin. However, our inability to detect 30-nm fibres in the mouse genome leads us to conclude that the transcriptional machinery has widespread access to the DNA packaged into chromatin fibres.”

The results are consistent with recent studies of the human genome which suggest that approximately 80% of the genome contains elements that are linked to biological function. Access to enhancers, promoters and other regulatory sequences on such a wide region of the genome means that all of these sites must be accessible. The 10-nm model of chromatin fibres provides sufficient access to DNA to allow potential target sites to be reached. The 30-nm model would not accommodate such widespread access.

You can read more about the research both in the EurekAlert news release or in the Oct. 5, 2012 news itemon Azonano. Or, you can read the article, Open and closed domains in the mouse genome are configured as 10-nm chromatin fibres, if you can get behind the paywall.