Damage to the genome is implicated in the progression of cancer and stress-induced diseases. DNA lesions exist in low levels, and cannot be amplified by standard PCR because they are frequently strong blocks to polymerases. Here, we describe a method for PCR amplification of lesion-containing DNA in which the site and identity could be marked, copied and sequenced. 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. These marker nucleotides constitute an unnatural base pair, allowing 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. 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.
Modifications to nucleotides in the genome can lead to mutations or are involved in regulation of gene expression, and therefore, finding the site of modification is a worthy goal. Robust methods for sequencing modification sites on commercial sequencers have not been developed beyond the epigenetic marks on cytosine. Herein, a method to sequence DNA modification sites was developed that utilizes DNA glycosylases found in the base excision repair pathway to excise the modification. This approach yields a gap at the modification site that is sealed by T4-DNA ligase yielding a product strand missing the modification. Upon sequencing, the modified nucleotide is reported as a deletion mutation, identifying its location. This approach was used to detect a uracil (U) or 8-oxo-7,8-dihydroguanine (OG) in codon 12 of the KRAS gene in synthetic oligodeoxynucleotides. Additionally, an OG modification site was placed in the VEGF promoter in a plasmid and sequenced. This method requires only commercially available materials and can be put into practice on any sequencing platform, allowing this method to have broad potential for finding modifications in DNA.
GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC(MBI)TP and dC(FBI)TP) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The MBI/FBI-labeled DNA probes exerted low fluorescence that was increased 2-3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The FBI label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent(exo-) polymerase.
The design of four new fluorinated biaryl fluorescent labels and their attachment to nucleosides and nucleoside triphosphates (dNTPs) by the aqueous cross-coupling reactions of biarylboronates is reported. The modified dNTPs were good substrates for KOD XL polymerase and were enzymatically incorporated into DNA probes. The photophysical properties of the biaryl-modified nucleosides, dNTPs, and DNA were studied systematically. The different substitution pattern of the biaryls was used for tuning of emission maxima in the broad range of 366-565 nm. Using methods of computational chemistry the emission maxima were reproduced with a satisfactory degree of accuracy, and it was shown that the large solvatochromic shifts observed for the studied probes are proportional to the differences in dipole moments of the ground (S(0)) and excited (S(1)) states that add on top of smaller shifts predicted already for these systems in vacuo. Thus, we present a set of compounds that may serve as multipurpose base-discriminating fluorophores for sensing of hairpins, deletions, and mismatches by the change of emission maxima and intensities of fluorescence and that can be also conviently studied by (19)F NMR spectroscopy. In addition, aminobenzoxazolyl-fluorophenyl-labeled nucleotides and DNA also exert dual pH-sensitive and solvatochromic fluorescence, which may imply diverse applications.
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