Real-time dna sequencing using detection of pyrophosphate release pdf

An increased signal-to-noise ratio was obtained minisequencing where dideoxynucleotide incorpora- by substitution of deoxyadenosine a-thiotriphosphate tion was detected after adding all four nucleotides. As a model, Here, we investigated if real-time sequencing by syn- 15 bases of a single-stranded PCR product were se- thesis can be achieved by a proper choice of enzyme quenced.

The possibility for parallel processing of and substrate in a solid-phase format. We also show many samples in an automated manner is discussed. The need for simple, fast, and automated methods for sequence-based analysis is evident.

All rights of reproduction in any form reserved. Washing of the immobilized single- stranded DNA and hybridization to sequencing prim- ers was carried out as described earlier The four different nucleotides are added stepwise to the PCR product were used as templates for real-time DNA immobilized template hybridized to a primer. The PPi released in sequencing. The height of the signal lized onto streptavidin-coated super paramagnetic is proportional to the number of bases which have been incorporated.

These steps tavidin as described above, and a primer was hybrid- are repeated in a cycle and the sequence of the template is deduced. The immobilized See the text for further details. The of mV for 0. One picomole of the immobi- sequencing procedure was carried out by stepwise elon- lized DNA fragment and 3 pmol DNA polymerase were gation of the primer strand upon sequential addition added to the solution described above.

The sequencing of the different deoxynucleoside triphosphates Phar- reaction was started by adding 40 pmol of one of the macia, Biotech. The reaction was ments between each nucleotide addition was performed carried out at room temperature. The PPi released the assay. The luminometer was calibrated to give a re- The sequence data obtained from the real-time DNA sponse of 10 mV for the internal light standard.

The sequencing were confirmed by semiautomated solid- luminescence output was calibrated by the addition of phase sequencing The standard assay volume was 0. From Fig. According to these results there is therefore a great advantage in using dATPaS instead of dATP if a DNA polymerase that can accept this nucleotide is used in the sequencing by synthesis protocol.

Solid-Phase Technique Several different parameters important for the suc- cess of the real-time DNA sequencing approach were optimized in a model system using two different syn- thetic DNA templates.

To simplify sequencing of sev- eral bases, the DNA was immobilized on a solid-phase. Here we have used two types of streptavidin-coated FIG. Both types of beads have a high binding capac- the luminescence output was detected. The experimental conditions were as described under Materials and Methods.

We found that the larger beads M allow a faster washing procedure due to their higher sedimen- tation rate data not shown.

The template were chosen. In Fig. The reactions were started by addi- Thereby, real-time signals are obtained by the ELIDA tion of the next correct base dCTP and the traces show only when complementary bases are incorporated. In the release of PPi during the incorporation of the base. In the subsequent mined by the luciferase assay Fig. However, we also observed that dATP interfered with the detection system.

This interference is a major problem when the method is used to detect a single- base incorporation event. Several approaches to de- crease this background activity were tested data not shown and among those the largest positive effect was achieved by replacing the natural dATP with dATPaS.

An addition of 0. The extent of PPi synthesis as a function of template con- of the emission from an equivalent addition of ATP. The experimental conditions were as described under instead be started by addition of DNA polymerase. The Materials and Methods. The sequencing procedure was started by addition of dATPaS. The small signal observed is due to PPi contamination in the nucleotide solution. After a washing step the poly- merase reaction was allowed to proceed about 1 min before the washing was started , dGTP was added; a signal corresponding to incorporation of one residue FIG.

Real-time detection of one base incorporation on three dif- was observed. The next base added was dCTP; a signal ferent templates. The 1. The reactions were started by addition of 40 pmol of the indicated deoxynucleotide and the PPi was now detected. The experimental conditions gave no signal. Thereafter, dATPaS was added again. This time the incorporation of two identical residues was noted. The latter detected incorporation confirmed earlier observations 16 that dATPaS is efficiently in- the relevant signal difference was recorded Fig.

A signal corresponding to incorporation of one the ELIDA are proportional to the DNA concentration residue was obtained after the next addition which was within a broad interval The upper limit for the dGTP. By continuing this cyclic procedure further in- assay is pmol PPi formed 1 mM The lower formation about the sequence was obtained. It is im- limit is mainly determined by the volume used, and by portant to note that enough nucleotides must be added contamination of PPi and ATP in the different solu- to allow longer extensions when there is a stretch of tions.

The sequencing procedures were re- peated several times on the same template with the same result. The decrease in signal due to loss and Effect of DNA Polymerase Concentration aggregation of beads during the washing procedure In the next series of experiments the effect of poly- measured by the decrease in optical density has been merase concentration on the sequencing procedure was compensated for in Fig.

The loss was lower for the studied. At lower polymerase firmed by semiautomated solid-phase Sanger sequenc- concentrations biphasic kinetics a fast phase followed ing data not shown. The sequencing reac- plate and the subsequent binding to a nonextended tions are continuously monitored in real-time. The incorporation rate as a function procedure for rapid detection of pyrophosphate re- of nucleotide concentration was also studied. We ob- lease can be envisioned. The rate- mM for Klenow and Sequenase 2.

The latter results are in accordance with data sulfurylase, while the luciferase reaction is fast and from the literature The incorporation rate for the Klenow DNA polymerase has been estimated by dif- ferent methods 17 — In this ity of the new approach.

Genome Res. Google Scholar. Nordstrom, T, Ronaghi, M. Nordstrom, T, Gharizadeh, B. CrossRef Google Scholar. Gharizadeh, B. Garcia, C. Gene , — Monstein, H. FEMS Microbiol. BioTechniques 25 , — PubMed Google Scholar. Elahe Elahi 1 Mostafa Ronaghi 1 1. Personalised recommendations. Cite protocol How to cite? ENW EndNote.

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