Design and synthesis of cleavable biotinylated dideoxynucleotides for DNA sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Chunmei Qiu, Shiv Kumar, Jia Guo, Lin Yu, Wenjing Guo, Shundi Shi, James J. Russo, Jingyue Ju

Research output: Contribution to journalArticle

6 Scopus citations


Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based methods have been widely explored for DNA sequencing. We report here the design, synthesis, and evaluation of a novel set of chemically cleavable biotinylated dideoxynucleotides, ddNTPs-N 3-biotin, for the DNA polymerase extension reaction and its application in DNA sequencing by mass spectrometry (MS). These nucleotide analogs have a biotin moiety attached to the 5 position of the pyrimidines (C and U) or the 7 position of the purines (A and G) via a chemically cleavable azido-based linker, with different length linker arms serving as mass tags that contribute to large mass differences among the nucleotides. We demonstrate that these modified nucleotides are efficiently incorporated by DNA polymerase, and the DNA strand bearing biotinylated nucleotides is captured by streptavidin-coated beads and efficiently released using tris(2-carboxyethyl) phosphine in aqueous solution, which is compatible with DNA and downstream procedures. We performed Sanger sequencing reactions using these nucleotides to generate DNA fragments for MALDI-TOF MS analysis. Both synthetic DNA and polymerase chain reaction (PCR) products were accurately decoded, and a read length of approximately 37 bases was achieved using these nucleotides in MS sequencing.

Original languageEnglish (US)
Pages (from-to)193-201
Number of pages9
JournalAnalytical Biochemistry
Issue number2
StatePublished - Aug 15 2012
Externally publishedYes



  • Cleavable biotinylated nucleotides
  • DNA sequencing
  • Solid-phase capture

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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