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  • yaximik
    Senior Member
    • Apr 2011
    • 199
    #1

    unnatural bases recognition

    Hi,

    I know that PacBio analysis pipeline can distinguish modified bases by analyzing incorporation kinetics, such as 5mC. Does anybody know about deamination products, like dU? Some polymerases just cannot get over it. In general I am interested whether this methodology can be applied to analysis of ancient DNA, obviously if long enough molecules have survived. Crazy idea, but still...
    Tags: None
  • rhall
    Senior Member
    • Aug 2012
    • 324
    #2
    Sounds like a great experiment. Other forms of DNA damage have already been shown to be detectable with PacBio:
    Direct Detection and Sequencing of Damaged DNA Bases - Genome Integrity
    http://www.genomeintegrity.com/content/2/1/10
    Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.

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