DNA sequencing by MALDI-TOF MS using alkali cleavage of RNA/DNA chimeras

Abstract

Approaches developed for sequencing DNA with detection by mass spectrometry use strategies that deviate from the Sanger-type methods. Procedures demonstrated so far used the sequence specificity of RNA endonucleases, as unfortunately equivalent enzymes for DNA do not exist and therefore require transcription of DNA into RNA prior to fragmentation. We have developed a novel, rapid and accurate concept for DNA sequencing using mass spectrometry and RNA/DNA chimeras and applied it to sequence mitochondrial DNA. Our method is based on the preparation of a chimeric RNA/DNA with a DNA polymerase that also incorporates ribonucleotides. Sequencing is carried out with one ribonucleotide (ATP, CTP or GTP) and the other three nucleotides in their deoxyribo-form. The product is treated with alkali, which cleaves 3' of all ribonucleotides to form a terminal 3' phosphate. Conditions have been streamlined so that molecular, biological and alkali cleavage conditions are compatible with matrix-assisted laser desorption/ionization time-of-flight (MALDI) mass spectrometric analysis. Fragment analysis by MALDI MS provides a sequence-specific fingerprint, which allows the identification of differences between a reference and another sequence. Due to the mass profile, the position and kind of the mutation can be assigned. These differences between signatures are indicative of known, unidentified, rare and private mutations. This novel DNA sequencing protocol was applied to sequence the hypervariable region 1 (HV1) of mitochondrial DNA in 22 individuals.

Figure 1.

Capillary electropherograms of HVI reverse ribo extension products of sample C207 prior to fragmentation: (A) with ATP; (B) with CTP and (C) with GTP show that extension is full length (440 bases). Mobility is influenced by ribonucleotide content.

Figure 2.

Spectra of the reverse ATP trace of HVI of sample C004 (a) and sample C018 (b) which corresponds to the Cambridge Reference Sequence. (A) In the 7-mer region two peaks change intensity. A decrease of the relative intensity from 3 to 2 of the peak at mass 2272.5 Da due to the loss of the fragment GGGTTGA is observed together with an increase of relative intensity of another peak at 2207.4 Da from 1 to 2 due to the appearance of the fragment TGTGCTA. These spectra were recorded on a Bruker Ultraflex II in reflectron mode which achieves isotopic resolution in this mass range. (B) In the 12-mer region, a new peak with a mass of 3868.5 Da appears which corresponds to the fragment GTTGGGGGTTGA. These spectra were recorded on a Bruker Autoflex in linear mode.

Figure 3.

Spectra of the 9-mer region of the reverse CTP trace of HVI of sample C004 (a) and C018 (b) Cambridge Reference Sequence. The polymorphism 16311 of sample C004 results in a shift of the peak at 2799.8 Da (TTTATGTAC) to 2815.8 Da (TTTATGTGC) due to an A to G base change in the fragment. These spectra were recorded on a Bruker Autoflex in linear mode.

Figure 4.

Spectra of the 7-mer region of the reverse GTP trace of HVI of sample C004 (a) and C018 (b) Cambridge Reference Sequence. A fragment at 2191.4 Da which corresponds to TACTATG disappeared. These spectra were recorded on a Bruker Ultraflex II in reflectron mode which achieves isotopic resolution in this mass range.