Pyrosequencing: Applicability for studying DNA damage-induced mutagenesis

Irina G. Minko, Lauriel F. Earley, Kimberly E. Larlee, Ying Chih Lin, R. Stephen Lloyd

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

Site-specifically modified DNAs are routinely used in the study of DNA damage-induced mutagenesis. These analyses involve the creation of DNA vectors containing a lesion at a pre-determined position, DNA replication, and detection of mutations at the target site. The final step has previously required the isolation of individual DNA clones, hybridization with radioactively labeled probes, and verification of mutations by Sanger sequencing. In the search for an alternative procedure that would allow direct quantification of sequence variants in a mixed population of DNA molecules, we evaluated the applicability of pyrosequencing to site-specific mutagenesis assays. The progeny DNAs were analyzed that originated from replication of N6-(deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG)-containing vectors in primate cells, with the lesion being positioned in the 5′-GCNGG-3′ sequence context. Pyrosequencing detected ∼8% G to T transversions and ∼3.5% G to A transitions, a result that was in excellent agreement with frequencies previously measured by the standard procedure (Earley LF et al. [2013]: Chem Res Toxicol 26:1108-1114). However, ∼3.5% G to C transversions and ∼2.0% deletions could not be detected by pyrosequencing. Consistent with these observations, the sensitivity of pyrosequencing for measuring the single deoxynucleotide variants differed depending on the deoxynucleotide identity, and in the given sequence contexts, was determined to be ∼1-2% for A and T and ∼5% for C. Pyrosequencing of other DNA isolates that were obtained following replication of MeFapy-dG-containing vectors in primate cells or Escherichia coli, identified several additional limitations. Collectively, our data demonstrated that pyrosequencing can be used for studying DNA damage-induced mutagenesis as an effective complementary experimental approach to current protocols.

Original languageEnglish (US)
Pages (from-to)601-608
Number of pages8
JournalEnvironmental and Molecular Mutagenesis
Volume55
Issue number8
DOIs
StatePublished - Oct 1 2014

    Fingerprint

Keywords

  • COS7
  • DNA polymerases
  • Escherichia coli
  • MeFapy-deoxyguanosine
  • Translesion DNA synthesis

ASJC Scopus subject areas

  • Epidemiology
  • Genetics(clinical)
  • Health, Toxicology and Mutagenesis

Cite this