Matrix-assisted Laser Desorption/Ionization for Sequencing Single-stranded and Double-stranded DNA

Abstract

The DNA sequence of a single-stranded and double-stranded template was determined. The templates were sequenced using the chain termination method and cycle sequencing method and detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The sequencing products were analyzed successfully without the laborious and expensive methods for removal of the template. Direct sequencing of the double-stranded template was achieved with minimal post-reaction purifications, which could be extremely important for mutation analysis and clinical diagnosis. A systematic study of the mechanisms and kinetics of sequencing reactions was also performed. The details of this analysis and directions for future improvements of the quality of sequencing are presented.

Chemical Cleavage Sequencing of DNA Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…Synthetic oligonucleotide (5′-biotin-tac tcc cct gcc ctc cac aag atg ttt tgc) designed from exon 5 of human p53 gene and a 60 mer containing 5′-biotin-(gaa) 20 were used as templates. The chemical cleavage sequencing reactions were performed as described 2 with minor modifications.…”

Nucleic Acid Analysis

“…DNA sequences were recovered from immobilized biotin-streptavidin complexes by treatment with NH 4 OH as a purification step prior to sequencing by MALDI-TOF mass spectrometry (125). A MALDI mass spectrometric method was reported for sequencing of single-and double-stranded DNA (126). Oligonucleotide segments up to 35 mer were sequenced by fragmentation in MALDI-TOF mass spectrometry (127 was applied to the detection of mutations (including substitutions and deletions) involving single nucleotides (128).…”

MALDI-ToF/MS as a diagnostic tool for the confirmation of sulfur mustard exposure†**

“…Matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-ToF/MS) is a relatively new mass spectrometric technique that has been used in molecular weight determination as well as mixture analysis of biomolecules such as proteins and peptides, 1-5 nucleotides, [6][7][8][9][10][11][12] glycolipids, 13 carbohydrates 14 and oligosacharides. 15 This technique has been proven to be a comparatively advantageous analytical technique that allows for fast mass analysis time and requires little sample preparation of biomacromolecules.…”