Abstract
All currently available DNA sequencing protocols rest fundamentally upon the homogeneity of the template. In this paper we describe the parallel DNA sequencing of various templates in one sample by a combination of the Sanger method and MALDI-TOF mass spectrometric analysis of the products. PCR-amplified hypervariable 16S rDNA fragments of the bacterium Escherichia coli DF1020 and cDNA of the 6-phosphofructo-1-kinase isoenzymes (PFK-1, EC 2.7.1.11) in rat brain were chosen as model systems for essentially heterogeneous templates. Avoiding cloning of the inhomogeneous PCR products we were able to read three sequences for both the 16S rDNA fragment of E.coli DF1020 and the cDNA of 6-phosphofructo-1-kinase from the peak lists of the Sanger sequencing reactions. Short sequences with a length between 21 and 25 nt were sufficient to reflect the heterogeneity of the 16S rDNA genes in E.coli and the existence of three isoenzymes of PFK-1 in rat brain.
INTRODUCTION
DNA sequencing is a basic method in molecular biology with applications ranging from the verification of the structure of any cloned DNA to the elucidation of the genome of whole organisms. The sequence ladders can be generated by exonuclease digestion (1), chemical cleavage (Maxam Gilbert method; 2), chain termination (Sanger method; 3) and sequence-based hybridization (4). Irrespective of the applied method, homogeneity of the template is an indispensable prerequisite for obtaining unambiguous sequence information. The only exception to this is represented by single nucleotide polymorphism (SNP) genotyping (5,6), where two templates which differ at only one position are sequenced simultaneously. Nevertheless, due to the widespread application of PCR-based methods a heterogeneity of sequencing templates is far from being uncommon. For example, RT–PCR products generated from alternatively spliced mRNAs are essentially inhomogeneous, as are amplicons obtained from two different alleles of a eukaryotic gene of one organism. Furthermore, sequence variability often exists if an organism has several copies of a gene per haploid genome, e.g. the genes coding for histones or rRNA. On average, bacterial cells have 3.6 copies of the 16S rRNA gene, which serves as a signature molecule for identification of bacteria (7,8). The 16S rDNA sequences of one organism are often heterogeneous (9).
To analyze the sequences of multiple templates present in one sample a preceding separation step by cloning into suitable vectors is usually necessary. To reflect the underlying sequence heterogeneity adequately, the number of clones that have to be sequenced always exceeds the number of different templates. This is not only a time-consuming and laborious but also an error-prone strategy. Single molecules containing PCR errors may be present as clones and increase the template complexity (false positives). On the other hand, the cloning efficiencies of various templates might be different, leading to an under-representation or even drop-out of some sequences in the clone library (false negatives). For these reasons a rapid method that offers the opportunity to recognize and determine sequence variability avoiding cloning steps would be desirable.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows a rapid and accurate mass determination of DNA molecules generated by Sanger sequencing (10–14). The identity of an incorporated base can be deduced from the mass difference between neighboring peaks. Sequence determination relies on the natural molecular weight differences of DNA bases. The analysis of Sanger sequencing reactions by mass spectrometry offers several advantages over that by acrylamide gel or capillary electrophoresis. No modification of the primer or the nucleotides is necessary and absolute masses of the primer extension products are determined. The enhanced sequence information provided by the precision of the mass spectrometric data suggested the possibility of a parallel sequencing of heterogeneous templates.
The feasibility of the approach is proved in this paper by successful simultaneous sequencing of the multiple 16S rDNA fragments of the bacterium Escherichia coli DF1020, an E.coli K12 derivative, and the cDNA of the three 6-phosphofructo-1-kinase isoenzymes present in rat brain by the Sanger method and MALDI-TOF MS product analysis.
MATERIALS AND METHODS
Chemicals and enzymes
The dNTPs, ddNTPs and the ThermoSequenase™ DNA polymerase were purchased from Amersham Pharmacia Biotech (Freiburg, Germany). Ampli-Taq DNA polymerase was obtained from Applied Biosystems (Weiterstadt, Germany). 3-Hydroxypicolinic acid (3-HPA) and ammonium citrate were purchased from Sigma-Aldrich (Steinheim, Germany). Primers for PCR and DNA sequencing are summarized in Table 1 and were obtained from Metabion GmbH (Martinsried, Germany). The purity of the oligonucleotides was checked by MALDI-TOF MS.
Table 1. Sequences of oligonucleotide primers used in this study.
| Primer | Sequence |
|---|---|
| S-D-Bact-0006-S-18 | 5′-GAGAGTTTGATCCTGGCT-3′ |
| S-D-Bact-0785-A-20 | 5′-GACTACCAGGGTATCTAATC-3′ |
| Bio-S-D-Bact-0053-a-S-11 | 5′-Bio-ACATGCAAGTC-3′ |
| S-D-Bact-0341-b-A-17 | 5′-ACTGCWGCCWCCCGTAG-3′ |
| RQ-Rev | 5′-TTCAGCCACCACTG-3′ |
The first four primers are named according to Alm et al. (15).
PCR amplification of the 16S rDNA fragment of Escherichia coli DF1020
To achieve sufficient PCR specificity and high efficiency of the primer elongation in the following sequencing reactions, the 16S rDNA fragment was amplified in two steps. In a first PCR a 799 bp fragment of the 16S rRNA gene of E.coli was amplified with the primers S-D-Bact-0006-S-18 and S-D-Bact-0785-A-20. The reaction mixture contained 106 bacterial cells, 1× PCR buffer, 2.0 mM MgCl2, 200 µM dNTP, 25 pmol each primer, 1.25 U Ampli-Taq DNA polymerase and water to a final volume of 50 µl. The initial denaturation at 94°C for 7 min was followed by 35 amplification cycles (denaturation at 94°C for 30 s, primer annealing for 30 s and primer extension at 72°C for 30 s) and a final extension step (72°C for 10 min). The initial annealing temperature of 65°C was decreased by 0.5°C at each cycle until 55°C was reached. The PCR products were used as templates for a second PCR. An aliquot of 50 µl of the reaction mixture contained 1 µl of the first PCR product (104-fold diluted), 1× PCR buffer, 2.0 mM MgCl2, 200 µM dNTP, 25 pmol each primer Bio-S-D-Bact-0053-a-S-11 and S-D-Bact-0341-b-A-17 and 1.25 U Ampli-Taq DNA polymerase. The amplification profile was 94°C for 5 min, 35 amplification cycles (94°C for 30 s, 26°C for 30 s, 72°C for 30 s) and a final extension step (72°C for 10 min). The low annealing temperature is due to the unusually short primer Bio-S-Bact-0053-a-S-11. The primer has a melting point of 28.9°C.
Sanger cycle sequencing reaction
The Sanger cycle sequencing reactions were performed in separate vials for each dideoxynucleotide. The products of the second PCR and the PCR-amplified cDNA of the 6-phosphofructo-1-kinase isoenzymes were purified with Genopureds (Bruker, Bremen, Germany) according to the manufacturer’s instructions and used as template. The sequencing was carried out in 20 µl reactions consisting of 2 µl of template, 20 pmol Bio-S-D-Bact-0053-a-S-11 (for E.coli) or RQ-Rev (for the cDNA of the 6-phosphofructo-1-kinase isoenzymes), 100 µM dNTP, 10 µM ddATP, ddCTP, ddGTP or ddTTP and 2 U ThermoSequenase in reaction buffer [5 mM (NH4)2SO4, 1.5 mM (for E.coli) or 2.5 mM MgCl2 (for the cDNA of the 6-phosphofructo-1-kinase isoenzymes), 10 mM Tris–HCl, pH 9.5]. The cycling conditions for the 16S rDNA fragment were identical to those of the second PCR. The cycle sequencing conditions for the cDNA of PFK-1 as template were initial denaturation at 95°C for 3 min followed by 35 amplification cycles (95°C for 30 s, 49.4°C for 30 s, 72°C for 30 s) and a final extension step (72°C for 7 min).
Sample preparation for MALDI-TOF MS
Samples were desalted before analysis using ZipTipC18 (Millipore, Eschborn, Germany). The ZipTipC18 was prewetted with acetonitrile (ACN) and equilibrated with 50% (v/v) ACN in nanopure water and 0.1 M triethylammonium acetate (TEAA), pH 7.0. The probe was mixed 1:5 with 0.5 M TEAA, bound to the ZipTipC18 and washed three times with 0.1 M TEAA and nanopure water. The sequencing products were eluted in 10 µl of 40% (v/v) ACN in nanopure water and dried down with a SpeedVac concentrator. The purified sequencing reaction products were dissolved in 1 µl of water and spotted onto 1 µl of pre-crystallized matrix (50 mg/ml 3-HPA and 10 mg/ml ammonium citrate in water) on a 384-well polished stainless steel target. The mixture was allowed to dry at room temperature.
MALDI-TOF MS
The MALDI-TOF MS measurements were performed on a Bruker Biflex III MALDI-TOF mass spectrometer (Bremen, Germany), equipped with a nitrogen laser (337 nm) and operated in positive ion linear delayed extraction mode at a 19 kV acceleration voltage and 400 ns delay time. Initially the instrument was calibrated with a mix of oligonucleotides in the mass range 2522–9488 Da.
Each individual spectrum represents an average of at least 45 laser shots applied to at least two positions within one spot. For each chain termination reaction (A, T, C and G reaction) at least three individual spectra were recorded. The spectrum with the best signal-to-noise ratio was used as the source for the primary peak list. To read the sequences from this list the experimentally obtained mass differences of neighboring peaks were manually compared with the masses of dAMP (313.2 Da), dGMP (329.2 Da), dCMP (289.2 Da), dTMP (304.2 Da), ddAMP (297.2 Da), ddGMP (313.2 Da), ddCMP (273.2 Da) and ddTMP (288.2 Da). Those peaks that finally contributed to DNA sequences were included in the combined peak list. Peaks caused either by primer dimers or by chain termination due to incorporation of a deoxynucleotide (false stops, revealed by their occurrence in more than one of the four chain termination reactions) were not included in the combined peak list.
RESULTS
A 16S rDNA fragment of the bacterium E.coli DF1020 and cDNA of 6-phosphofructo-1-kinase isoenzymes, both amplified by PCR, served as model systems for the simultaneous sequencing of heterogeneous templates by a combination of the Sanger method and MALDI-TOF MS. The sets of sequencing reactions were performed in four separate vials and mass spectra were recorded from the products of each reaction. The MALDI-TOF mass spectra obtained from the sequencing reactions of E.coli DNA and the cDNA are shown in Figures 1 and 2. Peaks of primer extension products were found in the range m/z 3730.2–11521.1 (for E.coli DF1020) and m/z 4183.8–10708.0 (for PFK-1 isoenzymes). The combined peak lists of the four nucleobase-specific chain termination reactions are compiled in Table 2. Three sequences for E.coli (Table 2A) as well as for the cDNA of PFK-1 isoenzymes of rat brain (Table 2B) could be deduced from these lists. The maximum reading length was 25 nt (Table 2A).
Figure 1.
(Previous page and above) MALDI-TOF mass spectra of the sequencing reactions of a PCR-amplified 16S rDNA fragment from E.coli DF1020. (A) A reaction; (B) C reaction; (C) G reaction; (D) T reaction. The first peak (m/z 3730.2) represents the unextended primer. For better visualization, only those peaks were labeled that contributed to the combined peak list. In the subviews these peaks are indicated by asterisks.
Figure 2.
(Previous page and above) MALDI-TOF mass spectra of the sequencing reactions of PCR-amplified cDNA of the 6-phosphofructo-1-kinase isoenzymes in rat brain. (A) A reaction; (B) C reaction; (C) G reaction; (D) T reaction. The first peak (m/z 4183.8) represents the unextended primer. For better visualization, only those peaks were labeled that contributed to the combined peak list. In the subviews these peaks are indicated by asterisks.
Table 2. Combined peak lists obtained by MALDI-TOF MS analysis of Sanger sequencing reactions of a PCR-amplified 16S rDNA fragment of E.coli DF1020 (A) and the PCR-amplified cDNA of PFK-1 isoenzymes of rat brain (B).
| Chain termination reaction | Sequence | |||||
|---|---|---|---|---|---|---|
| A | C | G | T | 1 | 2 | 3 |
| (A) | ||||||
| 3730.2 | 3730.2 | 3730.2 | 3730.2 | Unextended primer | Unextended primer | Unextended primer |
| 4043.8 | G | G | G | |||
| 4356.1 | A | A | A | |||
| 4669.1 | A | A | A | |||
| 4956.7 | C | C | C | |||
| 5286.8 | G | G | G | |||
| 5615.6 | G | G | G | |||
| 5919.5 | T | T | T | |||
| 6232.1 | A | A | A | |||
| 6545.1 | A | A | A | |||
| 6831.6 | C | C | C | |||
| 7146.5 | A | A | A | |||
| 7472.3 | G | G | G | |||
| 7803.5 | G | G | G | |||
| 8116.9 | A | A | A | |||
| 8429.5 | A | A | A | |||
| 8743.8 | A | |||||
| 8756.3 | G | G | ||||
| 9029.8 | C | |||||
| 9044.8 | C | |||||
| 9068.9 | A | |||||
| 9343.5 | A | |||||
| 9358.4 | A | |||||
| 9383.8 | A | |||||
| 9668.7 | G | |||||
| 9685.9 | G | |||||
| 9711.3 | G | |||||
| 9961.2 | C | |||||
| 9975.2 | C | |||||
| 10000.0 | C | |||||
| 10268.5 | T | |||||
| 10274.8 | T | |||||
| 10304.9 | T | |||||
| 10578.4 | T | T | ||||
| 10609.9 | T | |||||
| 10900.1 | G | G | ||||
| 10930.5 | G | |||||
| 11195.9 | C | C | ||||
| 11224.0 | C | |||||
| 11503.3 | T | T | ||||
| 11521.1 | T | |||||
| (B) | ||||||
| 4183.8 | 4183.8 | 4183.8 | 4183.8 | Unextended primer | Unextended primer | Unextended primer |
| 4458.9 | C | |||||
| 4473.1 | T | T | ||||
| 4762.4 | T | |||||
| 4777.6 | T | T | ||||
| 5052.0 | C | |||||
| 5066.3 | C | |||||
| 5106.2 | G | |||||
| 5341.3 | C | |||||
| 5370.4 | T | |||||
| 5395.8 | C | |||||
| 5630.1 | C | |||||
| 5677.9 | T | |||||
| 5699.1 | T | |||||
| 5958.7 | G | |||||
| 5980.7 | T | |||||
| 6002.9 | T | |||||
| 6248.0 | C | |||||
| 6308.9 | G | |||||
| 6332.6 | G | |||||
| 6576.6 | G | |||||
| 6638.1 | G | |||||
| 6662.2 | G | |||||
| 6906.9 | G | |||||
| 6968.4 | G | |||||
| 6990.8 | G | |||||
| 7195.1 | C | |||||
| 7295.0 | G | |||||
| 7320.7 | G | |||||
| 7506.4 | A | |||||
| 7605.6 | A | |||||
| 7630.6 | A | |||||
| 7810.7 | T | |||||
| 7910.2 | T | |||||
| 7934.7 | T | |||||
| 8122.7 | A | |||||
| 8214.1 | T | |||||
| 8247.0 | A | |||||
| 8411.7 | C | |||||
| 8505.5 | C | |||||
| 8537.4 | C | |||||
| 8737.5 | G | |||||
| 8830.9 | G | |||||
| 8866.8 | G | |||||
| 9063.3 | G | |||||
| 9163.4 | G | |||||
| 9192.8 | G | |||||
| 9371.4 | T | |||||
| 9463.2 | T | |||||
| 9498.6 | T | |||||
| 9699.2 | G | |||||
| 9789.4 | G | |||||
| 9825.7 | G | |||||
| 9989.7 | C | |||||
| 10080.7 | C | |||||
| 10115.4 | C | |||||
| 10296.0 | T | |||||
| 10388.2 | T | |||||
| 10419.7 | T | |||||
| 10671.3 | C | |||||
| 10708.0 | C | |||||
The incorporated nucleotides were deduced from the mass differences between neighboring peaks.
The experimentally determined sequences are identical to the sequences of the 16S rDNA operons of E.coli K12 and the three PFK-1 isoenzymes, which are published in the NCBI GenBank. Table 3 shows a list of the corresponding accession numbers. The sequences of the 16S rDNA fragment correspond to the sequence of two (sequence 1), one (sequence 2) and four (sequence 3) 16S rDNA operons of E.coli K12.
Table 3. The experimentally determined sequences and their corresponding accession numbers in the NCBI GenBank database for (A) 16S rDNA fragment of E.coli DF1020a and (B) cDNA of PFK-1 isoenzymes.
| Sequence | Identity to the NCBI GenBank accession numbers |
|---|---|
| (A) | |
| 1 | U00096 (rrsC, 3939431–3940971; rrsD, 3424858–3426399) |
| 2 | U00096 (rrsG, 2727636–2729178) |
| 3 | U00096 (rrsA, 4033120–4034661; rrsB, 4164238–4165779; rrsE, 4205725–4207266; rrsH, 223771–225312) |
| (B) | |
| 1 | U25651 (M-type of PFK-1) |
| 2 | X58865 (L-type of PFK-1) |
| 3 | AA819266 [cDNA clone UI-R-A0-al-d-10–0-UI.s2, putative identity to C-type of PFK-1 (GenBank accession no. L25387)] |
aThe operons and their locations in the genome are given in parentheses.
All three sequences of the PFK-1 isoenzymes expressed in rat brain were found. Sequence 1 and 2 are identical to the M- and L-type PFK-1, respectively, and sequence 3 to a sequence putatively assigned to rat C-type phosphofructokinase. Another database entry for rat brain C-PFK mRNA is almost identical to the latter but differs exclusively at the fourth base after the 3′ end of the primer RQ-Rev (T instead of C). The C at this position was found in sequence 3 and was confirmed by conventional Sanger sequencing of the cloned cDNA.
To validate the developed method of simultaneous sequencing we cloned the PCR products obtained from E.coli and PFK and sequenced them individually by the classical Sanger method evaluating the reactions by PAGE. In both cases all three sequences were found.
DISCUSSION
Using a 16S rDNA fragment of the bacterium E.coli and the cDNA of PFK-1 as paradigms we showed that the evaluation of Sanger sequencing reactions by MALDI-TOF MS offers the opportunity to recognize sequence heterogeneity and to read the sequences of several templates in parallel. This goes far beyond the determination of SNPs by primer extension reactions and MALDI-TOF MS, which was established in recent years (16–20).
MALDI-TOF MS analysis of Sanger sequencing products is superior to established gel- or capillary-based methods by determining absolute fragment masses with high precision. This allows the reading of the sequence of a homogeneous DNA template from a Sanger reaction performed in a single tube without a modification of the primer or the nucleotides (12). However, with respect to the maximum sequence reading length mass spectrometry is limited, since the currently available UV-MALDI-TOF mass spectrometers allow the accurate analysis of DNA molecules up to a length of about 40 bases (∼12 300 Da) (18). For the time being, MALDI-TOF MS evaluation of Sanger sequencing products is not suitable for genome-wide sequencing projects, but may be useful in applications for which short sequence information is sufficient, e.g. the control of the orientation of cloned DNA fragments or the identification of DNA or RNA molecules. The sequence reading lengths achieved in this paper are comparable to that published by others (12) and were sufficient to verify the sequence heterogeneity of the 16S rDNA of E.coli and the cDNA of PFK-1 in rat brain.
The ultimate advantage of the MALDI-TOF MS-based evaluation of Sanger sequencing products over all existing DNA sequencing approaches lies in the opportunity to simultaneously analyze more than one template per sample. At present, no upper limit to the number of sequences which can be read in parallel can be given. A sequencing of 16S rDNA fragments of different Streptococcus species by the method described in this paper showed that simultaneous detection of more than three sequences is possible. In any case, simultaneous analysis of several templates requires performance of the Sanger sequencing in four individual vials and recording of the mass spectra of the series of extension products generated by each of the dideoxynucleotides separately. However, this represents no significant restriction, since MALDI-TOF MS analysis is fast and can be easily automated. The resulting unambiguous correlation of each peak with one of the four nucleotides in the combined peak list supports the correct attribution of nucleotides to those observed mass differences that do not fit straightforwardly to one of the expected alternatives. The templates analyzed in parallel need not be present in equimolar concentrations. Within the rat brain PFK-1 cDNA the sequence of the L-type isoform could be detected although the respective message represents only 5% of the PFK-1 mRNA (21). Recently, Sun et al. demonstrated that MALDI-TOF analysis has the potential to detect mutant alleles in the presence of a 10 000-fold excess of the normal alleles (22).
The concurrently analyzed templates may be related to each other, as in the examples studied in this paper, but completely unrelated sequences (as long as they share the binding site for the sequencing primer) could also be analyzable by the same method. This is in contrast to multiplex pyrosequencing, which is restricted to SNP analysis and the verification of closely related sequences (23).
For the convenient analysis of the sequences of complex template mixtures the manual evaluation of the peak list performed in this paper should be replaced by an automated approach. The corresponding computer program is currently under development. Furthermore, the use of a cleavable primer for sequencing would enable the analysis of the Sanger products in a lower mass range and thus with improved resolution (24,25).
Acknowledgments
ACKNOWLEDGEMENTS
We thank Dr M. Bigl (Institute of Biochemistry, Medical Faculty, University of Leipzig, Germany) for providing PCR-amplified cDNA of the 6-phosphofructo-1-kinase isoenzymes from rat brain. This work was funded by the Sächsisches Staatsministerium für Umwelt und Landwirtschaft (no. 13-8802.3527).
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