Naked eye genetics: NMR assists development

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  • Published: Jul 15, 2016
  • Author: David Bradley
  • Channels: NMR Knowledge Base
thumbnail image: Naked eye genetics: NMR assists development

Simple setup

A new detection system for genotyping DNA and RNA that can be evaluated with the naked eye has been developed Credit: Angewandte Chemie/Wiley)

Nuclear magnetic resonance (NMR) spectroscopy has been used to validate and verify the development of a new naked-eye nucleic acid detection and diagnostics technology developed by researchers in Germany.

The DNA polymerase enzymes are like the photocopiers of the molecular biology world, making duplicates of the genetic material. However, unlike a photocopying machine, they have to make as exact a copy of the original as they possibly can, no half-tone monochrome facsimiles, only full colour in all possible hues at high resolution. But, it is possible to dupe the duplicators and trick them into accepting DNA building blocks that come with baggage, a large protein coupled to the base, for instance, as German scientists have now demonstrated. They report details in the journal Angewandte Chemie and point to possible applications for this exploitation of the polymerases in a detection system for genotyping the nucleic acids, DNA and RNA, that can be evaluated with the naked eye rather than requiring sophisticated analytical tools. Such a system could be used in medical diagnostics and environmental analysis in the field without the need for expensive and complicated laboratory procedures or detailed training programs for users. Moreover, the polymerases used in the process are active at room temperature and so thermocycler equipment is not needed, which simplifies setup still further.

Complementary approach

Chemists Moritz Welter, Daniela Verga, and Andreas Marx in the Konstanz Research School Chemical Biology, at the University of Konstanz, Germany, explain how DNA polymerase enzymes, read the information from an unzipped single strand of DNA and then assimilate the complementary bases to create the second strand and so generate a new double helix when a cell divides. This biochemical machinery has been widely utilised in the laboratory for many years in the amplification of DNA samples. The process takes place one nucleotide at a time and the enzyme must recognize the shape of the raw nucleotides to be included in the growing strand, but they can also accept nucleotides that have modifications. This has been used in antiviral and anticancer drugs, wherein a false nucleotide is incorporated by the enzyme but to which the next nucleotide cannot be added and so the duplication process is stymied and the virus cannot replicate nor the cancer continue growing. The same principle is used to add markers to DNA for biotechnology applications.

Now, Marx and colleagues have demonstrated that highly selective DNA polymerases can even incorporate nucleotides that have been attached to large proteins such as another enzyme, the horseradish peroxidase, for instance. The over-burdened nucleotide with its protein baggage may have a molecular mass more than 100 times larger than the nucleotide itself, but this does not disturb the recognition process given that the enzyme's active site can still accommodate the basic nucleotide unit with the protein baggage simply protruding into the molecular milieu. The only caveat is that the linking group between the nucleotide and its attached protein group must maintain a minimum distance between them to avoid interference.

The team points out that this astonishing result could then be used to develop a novel detection system that makes a specific nucleic acid detectable to the naked eye as incorporation only occurs if the right complement is present, and with coupling and a few additional steps comes an obvious and visible colour change in the sample. This neat molecular trick should make it possible to detect any specific gene fragment associated with a genetic disease, cancer or other factor.

Diagnostics and detection

To make such a test, the team would use a short DNA segment (the primer), complementary to the sequence they wish to detect. This is attached to a support. If the test sample contains the DNA segment in question, the corresponding single strand (known as the template) binds to the primer. A single incorrect base precludes this binding process. Next, a DNA polymerase and a chimera made from a nucleotide and horseradish peroxidase are added. If the template is bound to the primer, the polymerase is activated and couples the carefully selected nucleotide chimera to the template. Any unbound chimera is washed away and a colourless reagent is then added. This reagent is converted to a brown-coloured dye by the horseradish peroxidase visible to the naked eye. The measured intensity of the colour can be used to estimate the concentration of the DNA fragment. Indeed, in a proof of principle, the team was able to detect as little as 1 femtomolar of target DNA.

Using the same approach it was also possible to target a nucleic acid with a well-defined point mutation. In this modification of the test, the primer has to terminate immediately prior to the target site. The template then binds to the primer, but the nucleotide–enzyme chimera only couples only if the template has the target base at this precise location. Additionally, by using a different polymerase, the team was able to detect specific bacterial RNAs, which could be used to quickly and readily identify specific pathogens.

Related Links

Angew Chem Int Edn 2016, online: "Sequence-Specific Incorporation of Enzyme–Nucleotide Chimera by DNA Polymerases"

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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