Artificial DNA Base Pair Used as Amplifiable Marker of Genomic Damage

저자：   업로드：2015-11-10  조회수：

    The genome sustains so much wear and tear, much of it hardly noticeable, that even the small portion of DNA damage that stays unrepaired far exceeds our inventory-taking abilities. Taking stock of DNA damage, however, may become a lot easier now that scientists at the University of Utah have developed a new method that can register formerly imperceptible nicks and dings.

    The method combines several existing techniques, the most notable of which is the use of a so-called third base pair. This third base pair, an artificial addition to nature’s A-T and C-G base pairs, was developed by chemists at the Scripps Research Institute. It has now found what may be its first practical use.

    Details of the method appeared November 6 in the journal Nature Communications, in an article entitled, “Identification of DNA lesions using a third base pair for amplification and nanopore sequencing.” The article’s title, the alert reader will notice, hints at two of the other technologies that are incorporated in the DNA-flaw-finding method. The last piece of the puzzle is base excision repair, a means of cutting out DNA.

    By combining all these techniques, the University of Utah scientists found a way to overcome a pair of long-standing challenges: first, DNA lesions exist in low levels; second, DNA lesions cannot be amplified by standard PCR because they are frequently strong blocks to polymerases. Both of these challenges became tractable thanks to the introduction of the third base pair.

    “Here, we describe a method for PCR amplification of lesion-containing DNA in which the site and identity could be marked, copied, and sequenced,” wrote the authors of the Nature Communiations article. “Critical for this method is installation of either the dNaM or d5SICS nucleotides at the lesion site after processing via the base excision repair process.”

    The dNaM or d5SICS nucleotides constitute the artificial base pair, and they serve as markers. Moreover, these nucleotides allow large quantities of marked DNA to be made by PCR amplification.

    “Sanger sequencing confirms the potential for this method to locate lesions by marking, amplifying and sequencing a lesion in the KRAS gene,” the authors continued. “Detection using the α-hemolysin nanopore is also developed to analyse the markers in individual DNA strands with the potential to identify multiple lesions per strand.”

    In summary, the method has four steps:

    1. Find the damage and cut it out of the DNA the same way a cell does naturally, using base

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