DE4141178A1 - New nucleic acid sequencing method - using one labelled nucleotide at one time in cycles comprising elongation, wash, label detection and removal of the label, then repeating - Google Patents

Abstract

Claimed is a nucleic acid (NA) sequencing method in which a double strand of an at least partially single stranded template NA is synthesised starting from a double stranded NA piece or a primer by (i) incubation of the NA with a DNA elongation mixt. including a polymerase and one of the four nucleotides which is labelled, (ii) sepg. the NA from the elongation mixt., (iii) detection of any labelled nucleotide insertions, (iv) removal of the label if there had been an insertion, and repeating (i)-(iv) for all four nucleotides separately. Pref., the NA is anchored to a fixed phase such as biotin/avidin. USE/ADVANTAGE - Very long NAs and small amts. of NAs can be detected with a high accuracy since each DNA mol. contributes to the labelling. The limited capacity of gel matrixes is overcome so that any length of NA can be sequenced. Even if a wrong nucleotide had been incorporated the wrong signal is removed so that it does not influence further sequencing.

Description

The invention relates to a method for sequencing Nucleic acids.

Sequencing of nucleic acids now counts in bio chemical laboratories for daily routine tasks. Sequencing is usually done according to one of the performed two standard sequencing techniques, namely either the chemical degradation method according to Maxam-Gilbert, or but the enzymatic complementary strand synthesis method according to Sanger. Currently known automated sequences adornment methods using the Sanger method for the Labeling either end-labeled primers or labeled Terminator ddNTPs. With standard sequencing techniques the highlighted fragments on a by size separating medium, e.g. B. a polyacrylamide gel separated and determined via the marker. Markings are here radioactive labels, fluorescent labels etc. The Separation capacity of the gel matrix limits the resolution and the length of the DNA sequence that can be determined.

There are clear limits to this with the previously known systems set. Because sequencing is so common leads, it is definitely desirable to improve enable over the known methods.

The object of the present invention is therefore a method to provide for the sequencing of nucleic acids which can also be used to determine very long nucleic acid sequences can and where already very small amounts of nu small acid lead to clear results.

This object is achieved by the method according to the invention ren for the sequencing of nucleic acids, in which one to be sequenced, at least partially single-stranded  Nucleic acid as a template strand using a suitable Polymerase and all four nucleotides, starting from one Primer or a double-stranded section, step by step Step synthesizes a complementary strand, whereby one labeled nucleotides used, and what the following steps includes:

• (1) Incubate partially or partially primed double-stranded nucleic acid with an incubation medium consisting of a polymerase, one of the four Nucleotide types in labeled form and others for the Chain polymerization required substances, being a labeled nucleotide in the growing complementary strand is installed in the event that the next on to use the nucleotide present in the template strand the labeled nucleotide is complementary,
• (2) separating the nucleic acid, optionally extended by one nucleotide, from the incubation mixture of step ( 1 ) and carrying out one or more washing steps in a manner known per se,
• (3) Determine the incorporation of a nucleotide based on its Marker, and
• (4) if a labeled nucleotide is incorporated is removing the mark,

wherein steps (1) to (4) are cyclic for all four Nucleotide types are performed.

The completely new approach to the method according to the invention has resulted in that here for the complementary stranding only one kind of nucleotide and that in marker ter form is offered. So here's the next one on the Template strand present complementary to that in the Incubation mixture of the labeled nucleotide present the incorporation of the nucleotide together with its labeling by the Polymerase. In the context of the invention, a those used, which is capable of the labeled nucleotide to be built into the growing complementary strand. Depending on  The type of nucleic acid to be determined can therefore be determined here known polymerases, such as DNA polymerase, e.g. B. T7 DNA poly merase. As nucleic acids to be determined are not only suitable for DNA fragments, but also for RNAs. For the case that a labeled nucleotide is now incorporated is determined via the marking and thus the conclusion on the complementary base in the template strand enables.

If the base on the template strand is not complementary to that offered labeled nucleotide, there is no incorporation and it no marker signal is received.

The method according to the invention is further characterized by the the following removal of the marking in the case where the Incorporation of a labeled nucleotide has taken place draws. With "removal of the marking" is within the Invention means that the nucleic acid behaves in a way delt that the marker signal disappears. This be does not mean, however, that chemically the mar Kischen part of the molecule must take place, it is u. a. also one Bleaching with a strong laser in the event of fluorescence labeling conceivable (see e.g. Scalettar et al., Biophys. J. 53, 215 (1988) or Mathies, R.A. Stryer, L., single Molecule Fluorescence Detection: A Feasibility Study using Phycoerythrin. Applications of Fluorescence in the Biomedical Sciences, pp. 129-140 (1986) Alan R. Liss, Inc.). It is in in any case, it is essential that during step (4) by removing the label the labeled nucleotide is not chemically modified so that the polymerization of the next nucleotide complementary to the template strand is prevented.

Is in the method according to the invention, for example, at Determination of a DNA sequence as the next base after the primer or partial double-stranded dATP installed, he  is due to the marking signal, which of the Control signal of the DNA deviates without the label for which Templatestrang at this position that the base T exists is. Are several identical bases on the template strand, here that is, tymidine, are present in the complementary strand a large number of adenosine molecules built in correspondingly stronger marker signal results.

The same step as for the highlighted Nu Kleotid adenosine in the incubation mixture from step (1) has been described, is successively cyclic for all Nucleotide types performed. So for each base on the Ultimately, the template strand was the complementary marked Nu kleotid offered, built into the chain, due to the Remove the marking after each appropriate installation only the last built-in labeled nucleotide Signal generated. For this reason, the marker signal not accumulative and the accuracy of the process very much large.

Fig. 1 shows with the aid of a flow chart diagram of the course of the process.

In the context of the invention it is preferred to use a single strand to use ge nucleic acid for sequencing and in 5'-3'- An oligonucleotide in front of the sequence to be sequenced hybridize as a primer.

In a preferred embodiment of the invention, the nucleic acid to be sequenced to a solid prior to step (1) Phase bound. This makes it possible, especially in automa systems, the nucleic acid easily into the for the solutions and mixtures required in various steps immerse yourself and easily from these solutions again separate.  

It is important that the solid phase has no or only gives little background for the marker signal, e.g. B. must if fluorescence is used as the label, the solid Phase consist of a material that is not in the same world length emission range fluoresces like fluorescence labeling of the nucleotides used.

The nucleic acid can be anchored to the solid phase done according to known methods, preferably he follows the anchoring via a specific binding pair, the a partner is connected to the fixed phase and its other partner is bound to the nucleic acid. Especially is preferred here as a specific binding pair in the context of Invention the system biotin / avidin or biotin / streptavidin used, again preferably avidin or streptavi bound to the solid phase according to methods known per se is and the nucleic acid is modified by biotin.

All materials that are inert are suitable as the solid phase against the substances in the solutions used, allow fixation of the nucleic acid and, as above, he believes not to differ with the marking. As preferred Examples can be glass, a polymer membrane or polymer or Glass beads, so-called beads, are called.

In a further preferred embodiment of the invention is used instead of nucleic acids in a solid phase fixed form a flow system for steps (1) to (4) where the nucleic acid remains in solution and with the help of filters and / or capillaries of those used other substances is separated.

As a marker in the nucleotides, Erfin a fluorescent label is preferably used. Here in particular, fluorescent labels can be used be, as in the German patent application P 41 25 745  are described. Also the so-called "time resolved fluorescence "can advantageously be used as a marker the.

In the context of the invention, Deoxyribonu kleotide used. In a further preferred embodiment However, the invention can also use dideoxyribonucleotides or so-called "caged" nucleotides (see e.g. Handbook of Fluorescent Probes and Research Chemicals, Richard P. Haugland, 1989, Molecular Probes, Inc., Eugene, USA or Matthews and Kricka, Analyt.Biochem. 169, 1 (1988)) be used. A nucleotide also appears to be suitable for which is coupled to the 3′OH group of the fluorescent dye (Krayevsky A., BBA, 1986, 868, p. 136). From this it follows that that only one dideoxyribonucleotide or the like is installed that can be, because the polymerase through these so-called termina toren no longer has the possibility to extend the chain further gladly, even if that of the next base on the template strand corresponding nucleotide is present. This means that even if the same bases appear several times on the template strand occur in each case, only in one incubation step a nucleotide is incorporated. This can affect the accuracy of the Method. After determining the mark will the dideoxynucleotide then into a "dNTP-like polymerisa extensionable nucleotide ". The termination of the Complementary strand synthesis is thereby canceled and the The next cycle of steps (1) to (4) can take place. It is, however, due to the use of dideoxyribonucleotides also enables a further simplified procedure in which all four types of nucleotides used as dideoxyribonucleotides be, however, the four different dideoxys each also have different fluorescent labels. Which one the dideoxynucleotides then actually growing in the Complementary strand was installed in step (3) of the The inventive method based on the existing markie tion can be determined. This preferred embodiment  the method according to the invention therefore means another significant work relief and acceleration of the Se accounting procedure.

In the context of the invention it is preferred to use the nucleic acid to denature before sequencing. Furthermore, it is preferred, in each case after step (3) or (4) with a correspond to the labeled nucleotide used in step (1) the unlabeled nucleotide gaps in the Complementary strand to fill. This measure should also Increase method accuracy even further. Since of course in the method according to the invention not only one molecule of DNA is determined, but many of them for the determination and the Generation of a detectable signal are necessary also occur that the correct installation in some strands of a nucleotide is incorrectly omitted. Then could also the next nucleotide was not included in the sequence are built, so these nucleic acids would be used for the further Signaling fail. With an unlabeled nucleotide possible defects are therefore filled in again, so that for all nucleic acids the same starting conditions for the next cycle of steps (1) to (4) with the next Nucleotide are given.

When using a solid phase-bound nucleic acid for sequencing, it is also possible to determine several, and indeed a very large number, nucleic acids at the same time. For this purpose, the nucleic acids to be sequenced are anchored to a solid phase in spatial separation and treated simultaneously with the various incubation mixtures and solutions. You can produce a type of microstructure on a smaller scale and sequence hundreds to thousands of DNA samples simultaneously on such a "DNA chip" on a micro scale (see Fig. 2). The DNA can be anchored to the solid phase by a kind of automated and computer-controlled cell microinjection. However, all other types of anchoring known per se are also possible here. Furthermore, the nucleic acid can also be anchored to the bottom of a microtiter plate and the solutions and incubation mixtures introduced into the wells and removed again.

As previously mentioned, it is within the scope of the invention sequencing in a machine is particularly preferred perform. It can be used to determine the marking specifically uses a Charge Coupled Device (CCD) camera will.

The inventive method, which is a completely new DNA sequencing technique without the need separation on agarose or polyacrylamide gels is a quick and extremely accurate way Sequencing nucleic acids. Already determining very small amounts of nucleic acids, many times less longer than with the previous methods, is possible because each DNA molecule contributes to the marker signal while at the previous methods a statistical distribution of Fragments of the nucleic acid and thus to determine for each de nucleotide only a fraction of the starting amount of nuclein acids is available.

The accuracy of the method is based on the fact that the Extension of the polymerized chain is interrupted as soon as the polymerase enzyme with a base on the template strand arrives that are not in the incubation mix with the mar Nucleotides with which the nucleic acid is incubated will exist. In theory, it can be wrong Install a base, which results in a low bake ground signal would result, however, after marking removal step (4) also removes the wrong marking, which is why the wrong signal cannot accumulate what the Resolution after a few cycles. Out  for this reason the background is in the process according to the invention ren very low and the accuracy of the sequence determination correspondingly high.

To further increase the accuracy of the sequence determination can increase the nucleic acid sample to be determined in four Partial samples are separated and then all steps of the method according to the invention, each with the four nu small acid aliquots are carried out. The sequence will then not just about the positive signal from one sample true, in which the incorporation of a labeled nucleotide takes place found, but also about the lack of a signal in the samples, which are labeled with the other three nucleos were incubated.

In summary, the method according to the invention offers one fast and easily automatable possibility with great Accuracy of the sequence of nucleic acids even when available its reliably only very small amounts put.

The following figures further explain the invention:

Fig. 1 shows a flow scheme for the inventive Ver drive,

Fig. 2 shows the arrangement of many different nucleic acids, here DNA molecules, on a solid phase on a microscale for simultaneous processing.

Fig. 3 shows the detection of incorporation of fluorescein-12-dUTP by Klenow DNA polymerase.

Claims (17)

1. A method for the sequencing of nucleic acids, in which one step by step a complementary strand to an at least partially single-stranded nucleic acid to be sequenced as a template strand using a suitable polymerase and all four nucleotides, starting from a primer or a double-stranded section synthesized using labeled nucleotides and comprising the following steps:

• (1) Incubating the primer-provided or partially double-stranded nucleic acid with an incubation mixture consisting of a polymerase, one of the four nucleotide types in labeled form and other substances required for chain polymerization, with a labeled nucleotide being incorporated into the growing complementary strand in the event that the next nucleotide present on the template strand is complementary to the labeled nucleotide used,
• (2) separating the nucleic acid, which may have been extended by one nucleotide, from the incubation mixture of step (1) and carrying out one or more washing steps in a manner known per se,
• (3) determining the incorporation of a nucleotide based on its label, and
• (4) if the insertion of a labeled nucleotide has taken place, removal of the label,

wherein steps (1) to (4) are cyclic for all four types of nucleotides can be performed. 2. The method according to claim 1, characterized, that the nucleic acid to be sequenced before step (1) is anchored to a fixed phase.   3. The method according to claim 2, characterized, that anchoring through a specific tie pair one partner with the fixed phase ver is bound and its other partner to the nucleic acid re bound. 4. The method according to claim 3, characterized, that as a specific binding pair the Bio tin / avidin or biotin / streptavidin used. 5. The method according to any one of claims 2 to 4, characterized, that as a solid phase glass, a polymer membrane or Polymer or glass beads are used. 6. The method according to any one of the preceding claims, characterized, that as an primer an oligonucleotide before the se hybridizing sequence. 7. The method according to claim 1 or 6, characterized, that for steps (1) to (4) a flow system used in which the nucleic acid remains in solution and with the help of filters and / or capillaries from others Substances is separated. 8. The method according to any one of the preceding claims, characterized, that one marks a fluorescence as the labeling of the nucleotides marker used.   9. The method according to any one of the preceding claims, characterized, that one uses deoxyribonucleotides as nucleotides. 10. The method according to any one of claims 1 to 8, characterized, that one uses dideoxyribonucleotides as nucleotides and after determining the marking in Step (3) or optionally after step (4) in De oxyribonucleotide-like polymerization extendable Transferred nucleotides. 11. The method according to claim 11, characterized, that all four nucleotide types are different fluorescent labels together in one inkuba tion mixture in step (1) and the sub Separation of the built-in nucleotides via their markie tion takes place. 12. The method according to any one of the preceding claims, characterized, that you denatu the nucleic acid before sequencing riert. 13. The method according to any one of the preceding claims, characterized, that after step (3) or (4) use one with the correspond to the labeled nucleotide from step (1) the unlabeled nucleotide, if any Fills in gaps in the complementary strand. 14. The method according to any one of claims 1 to 6 and 8 to 13, characterized, that you have several to be sequenced on the solid phase Arrange nucleic acids in spatial separation from each other core and sequenced them simultaneously.   15. The method according to any one of the preceding claims, characterized, that steps (1) to (3) and optionally (4) in a machine. 16. The method according to any one of the preceding claims, characterized, that to determine the mark a batch Coupled Device (CCD) Camera used.