DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry

Abstract

Sets of RNA ladders can be synthesized by transcription of a bacteriophage-encoded RNA polymerase using 3'-deoxynucleotides as chain terminators. These ladders can be used for sequencing of DNA. Using a nicked form of phage SP6 RNA polymerase in this study substantially enhanced yields of transcriptional sequencing ladders. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of chain-terminated RNA ladders allowed DNA sequence determination of up to 56 nt. It is also demonstrated that A-->G and C-->T variations in heterozygous and homozygous samples can be unambiguously identified by the mass spectrometric analysis. As a step towards single-tube sequencing reactions, alpha-thiotriphosphate nucleotide analogs were used to overcome problems caused by chain terminator-independent, premature termination and by the small mass difference between natural pyrimidine nucleotides.

Figure 1

Transcriptional sequencing of DNA by gel electrophoresis. Transcriptional sequencing reactions of the previously described template 1w1 (15) containing a hairpin-independent terminator were carried out with intact (lanes 1–5) and nicked (lanes 6–10) SP6 RNA polymerases. Standard transcription (lanes 1 and 10) and sequencing reaction (lanes 2–9, G, A, U and C) products were labeled with [α-³²P]CTP and resolved in parallel using 12% polyacrylamide 8 M urea gel electrophoresis. The nicked RNA polymerase ignores the terminator signal (T), as previously reported (15), and produces only run-off product (R). The size of RNA is shown (in nucleotides) on the left.

Figure 2

Transcriptional sequencing of DNA by MS. Sequencing reactions were carried out with the previously described template 5q10 (15) using nicked SP6 RNA polymerase. The RNA fragments were (A) separated by gel electrophoresis as in Figure 1, and (B) analyzed by MALDI-TOF MS. Base-line corrected spectra are superimposed.

Figure 3

Mass spectra for the detection of A→G variation. Sequencing reactions of a region involving the MELAS syndrome mutation were performed using nicked SP6 RNA polymerase and analyzed by MALDI-TOF MS. Only a portion of mass spectra of (A) G-specific and (B) C-specific reactions around the mutation is shown. The top (N) and middle (M) spectra in both panels show normal (A-allele) and MELAS (G-allele) samples, respectively. The bottom (H) spectra show a 1:1 mixture of the two samples. The major dG and dC peaks of N, M and H samples in the range 5000–5750 Da are shown as cut off in height (to reduce wasted background space in the graphs), except for the dG peak of H sample near 5000 Da position.

Figure 4

Transcriptional sequencing with thio-analogs of UTP. Sequencing reactions of the template 1w1 (15) with intact SP6 RNA polymerase were carried out using (A) UTP, (B) S⁴UTP (4sU) or (C) UTPαS (αsU) and the other normal rNTPs. Multi-round transcription reactions of HindIII-digested 1w1 were run in parallel to the left of each panel. See Figure 1 legend.

Figure 5

Mass spectra for the detection of C→T variation. Transcriptional sequencing reactions were carried out with intact T7 RNA polymerase using UTPαS instead of UTP. The templates were derivatives of the previously described plasmid pKJ02 (21) containing a T7 promoter. The TTT sequence at the +16 to +18 positions of pKJ02 was changed to CCC and CTC, and the two resulting templates are different only at the +17 position. Only C-specific reactions analyzed by MALDI-TOF MS are shown. The top (N) and middle (M) spectra show the +17C- and +17T-carrying samples, respectively, and the bottom (H) shows a 1:1 mixture of the two samples.