A nanopore for detecting DNA damage

Scientists around the world are competing among themselves to see who will be able to perform an efficient floor of the DNA - the genetic material that makes us all - faster and cheaper with the help of nanometer pores. Now, scientists from the University of Utah have been able to apply this idea to detect DNA damage that can lead to mutations and diseases.

"We use this method as well as synthetic organic chemistry to locate the damaged sites in the DNA while the molecule passes through the nanopore," says researcher Henry White, professor of chemistry at the University of Utah and lead author of the article. Strands of the DNA molecule consist of nucleotide bases marked with the letters A, T, G and C. Chains of DNA strands make up the genes.

The new method searches for and locates the places where a nucleotide base is missing - one of the most common types of defects in the human genome, which includes 3 billion bases. This type of genetic defect occurs in a typical cell about eighteen thousand times a day in light of the fact that we are exposed to damaging factors, from the sun's rays to the gases emitted by cars. Most of this type of damage is repaired by the body itself, but sometimes it leads to a gene mutation that can eventually manifest itself in the disease.

By combining the method for detecting DNA damage with the help of nanopores and other methods to modify DNA, the researchers hope that their innovative technique, for which a commercial patent is sought, can be used to detect types of damage other than base missing. "Damages to the DNA bases contribute to most age-related diseases, including melanoma, lung, colon and breast cancer, Huntington's disease and atherosclerosis," says one of the researchers.

Base pairing is the process by which the order of the position and nature of the bases that make up each of the two strands that make up the double helix of DNA is determined. This is the basic method for deciphering the genome, or the genetic fingerprint, of living beings, and for locating mutations in the genes that cause diseases.

"Twenty years ago, the first sequence of the human genome cost a billion dollars, while today the cost ranges from only 5,000 to 20,000 dollars," says the researcher. "The U.S. National Institutes of Health is running a project to sequence a genome at a cost of only $1,000, and it is expected that this rate will also decrease."

"DNA sequencing is important in many fields: it is used by the police to incriminate or exonerate criminal suspects, by biology researchers to understand how a living creature works, and by farmers to improve the plants and crops essential to the modern world," says the researcher. Faster and cheaper acceptance of an individual's genome paves the safe way to the field called "personalized medicine" - medicine in which the medical treatments will be based on the genetic sensitivity of each and every patient.

The nanoporous floor is carried out by passing a strand of DNA through a nanometer hole when these two components are immersed in an electrically charged solution called an electrolyte. Some of the solution also flows through the hole. The researchers can measure varying levels of electric current when different bases of the DNA pass through the hole and thereby block the flow of the electrolytic solution. Unlike efforts to obtain DNA flooring with the help of nanopores, the University of Utah chemists do not "read" the sequence of DNA bases as the strand moves through the pore, but instead locate a single base defect. "It is important to know how a damaged base leads to a mutation since this is the first step in the development of a disease," the researchers explain. "Currently, we are able to see the damaged site and roughly determine its relative position in the DNA segment we are examining - at the level of 10-5 bases." Until now, the longest piece of DNA that the researchers passed through the nanopore was 100 bases long and the scientists were able to locate one or two damaged sites. "We still need to carry out a long study in order to find ways to improve this level," explains the researcher.

The pore used by many of the researchers in this field and the chemists from the University of Utah is called alpha-hemolysin - a protein derived from bacteria. In order to transfer a DNA strand through this pore, it is fixed into a glass membrane at the bottom of a dish. A soap solution (used as a fatty bilayer) is placed on the membrane and forms a sheath over the pore. This pore is shaped like a mushroom - wider at the top, where the DNA is trapped, and narrower at the bottom, where the strand must pass through a smaller passage. The size of this tiny transition is only 1.4 nanometers, while the size of the strand itself is only one nanometer.

The billions of bases that make up DNA are connected to a skeleton made up of a sugar and phosphorus unit. In order to detect DNA damage of the missing base type, the researchers apply an electric voltage that causes a current to pass through the solution. A positive electrode located in the solution outside the pore pulls the DNA through the pore due to the negatively charged phosphorus units in the DNA backbone. The researchers created intentional damage in the DNA of a sample by removing several bases. Where the bases are missing, the sugar unit in the DNA backbone becomes exposed. The researchers attached to this exposed unit a defined chemical molecule, a certain salt. The idea was to make this DNA, which had undergone a specific change, slowly cross the nanopore so that the location of the missing bases could be located. In order to optimize the method, the researchers tested several electrolytic solutions and arrived at the optimal solution so that the crossing rate of the strand would be appropriate.

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