DNA sequencing: nanopore with motor
New DNA sequencing method promises a revolution in genetic engineering
Alexander Malianov, Informnauka based on ScienceNOW and PhysOrg materialsResearchers from the University of Washington, led by physics professor Jens Gundlach, have developed a sensor that allows you to quickly and inexpensively determine the genetic code by changing the current level when passing a DNA chain through a nanopore.
The work promising revolutionary changes in the genome sequencing procedure is published in the latest issue of Nature Biotechnology (Manrao et al., Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase).
The idea of determining the sequence of nucleotides in DNA by measuring the changes in the ion current created by each nucleotide during the sequential passage of a single-stranded macromolecule through an ion channel (nanopore) has been discussed among molecular biologists and biophysicists for a long time. However, a working system of this type has been created only now. (In fact, "systems of this type" have been on sale for a long time, and within a year at least two more DNA sequencers based on nanopore–VM should appear on the market.)
Earlier, Gundlach's group experimented with dragging DNA through the ion channel of a mutant form of porin A, one of the membrane protein channels of the bacterium Mycobacterium smegmatis (MspA). A small potential difference in an aqueous solution of potassium chloride on different sides of the membrane pushes ions through the nanopore penetrating it. When entering the ion channel, the DNA molecule partially blocks the flow of ions, and by the magnitude of the current decrease, it is easy to understand which of the four types of nucleotides – adenine, guanine, thymine or cytosine – is currently passing through the nanopore.
However, the problem was passing the DNA chain through the channel at a more or less constant speed - fast enough for such a sequencing method to make sense, but at the same time so that the ion current had time to change and these changes could be recorded using modern electronic equipment.
It was possible to solve this difficult task by attaching the DNA polymerase of phage (bacterial virus) phi29, a genetic parasite of Bacillus subtilis bacteria, to the porin. This component ensured that DNA was dragged through the channel evenly and at the right speed, as it does during DNA replication (building a complementary DNA chain on an existing matrix).
"We have supplemented the protein nanopores we have developed with a motor that is able to carry a DNA molecule through the nanopore," says Professor Gundlach. Such an engine was first used by researchers from the University of California at Santa Cruz, but the ion channel they used was too wide to reliably distinguish the types of nucleotides by changes in the ion current.
In an article in Nature Biotechnology (scientists report a successful demonstration of a new technology using six different DNA strands ranging in length from 42 to 53 nucleotides. The DNA polymerase motor provides passage through the pore of each nucleotide of the DNA chain in an average of 28 ms. The change in the ion current through the channel during the passage of a nucleotide is up to 40 pA and significantly differs for each of the four types of nucleotides that make up the hereditary molecule. These parameters make it possible to successfully determine the nucleotide sequence of DNA, and much faster than in the currently used sequencing methods.
The work of American scientists opens up prospects for fantastically accelerating and reducing the cost of the procedure for determining genetic sequences, which will allow sequencing the genome of each person in the near future, as well as determining DNA changes in real time for specific diseases. This will greatly accelerate and increase the possibilities of genetic engineering – from the creation of completely new species that do not exist in nature to the complete cure of hereditary diseases and victory over death.
The work was funded by the National Human Genome Research Institute (National Human Genome Research Institute) as part of a technology search program that allows for individual sequencing for less than $1,000. According to Professor Gundlach, until now the cost of one study has been measured in hundreds of thousands of dollars, but with the new technology it can be carried out in just $ 10 and 15 minutes.
In addition, the developed sequencing method will solve another important task facing modern molecular biology: rapid detection of methylation patterns and other chemical modifications of nucleotides in the body. As it has been established by recent studies, such changes, called epigenetic modifications, provide the transmission of a large stream of hereditary information in addition to the encoded sequence of four nucleotides and can cause some diseases, in particular cancer, diabetes and addiction. The new sensor will allow timely detection of predisposition to these diseases.
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28.03.2012