Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-04
2002-01-29
Whisenaut, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S287200, C436S094000, C536S023100
Reexamination Certificate
active
06342353
ABSTRACT:
The present invention relates to a method allowing the identification of signatures corresponding to a specific sequence of double-stranded nucleic acid.
In the field of DNA sequence analysis, the present invention relates to a method which makes it possible to accelerate or bypass the process of genomic DNA sequencing, a problem which is of considerable industrial interest. The method according to the present invention allows the identification of specific signatures of a sequence on a long DNA fragment. This method makes it possible in particular to rapidly search for the presence of particular structures in the base sequence and/or the quality of the pairings which may be partial and, in particular, have mismatches. This method also allows analysis and comparison of the genomic differences among various patients.
It is understood that the invention also applies to single-stranded DNA-single-stranded DNA duplexes, perfectly paired or not perfectly paired, or alternatively to single-stranded DNA-single-stranded RNA duplexes, perfectly paired or not perfectly paired, or alternatively to single-stranded RNA-single-stranded RNA duplexes, perfectly paired or not perfectly paired. Furthermore, the duplex may consist of the at least partial re-pairing of two single strands obtained from samples of different origins. Finally, the invention also applies to the secondary structures of a sole single-stranded DNA or of a sole single-stranded RNA.
To clarify the disclosure, there is used in the text which follows the specific example of a double-stranded DNA, it being understood that the above generalizations can be easily achieved by simply developing the methods described.
A “signature” makes it possible to “characterize” a DNA sequence.
By way of example, the pairing of the G and C bases involves three hydrogen bonds, whereas the pairing of the T and A bases involves only two bonds; under these conditions, if the DNA double helix is mechanically opened by separately pulling the ends of two strands on the same side of an isolated DNA molecule, it can be expected that the signal corresponding to the forces exerted depends on the sequence.
Thus, the implementation of the present invention has made it possible to demonstrate that the separation of two paired strands essentially results in the obtaining of a “signature”, that is to say a set of specific and local information which is linked to the sequence and/or to the state of pairing.
Unexpectedly, during the opening of a duplex, the signal in force may exhibit in particular serrated structures, with a gentle rise and a more abrupt descent, where the opening occurs, in part, in short bursts. The forces measured during the opening therefore exhibit a set of particular characteristics, or signature, a complex combination of the sequence where the regions rich in GC are harder to open than the regions rich in AT, and of the mechanical stiffness of the system (molecular construction and system of measurement), which come into play to induce events where several contiguous bases open more rapidly.
By modifying the mechanical stiffness of the system, it is possible to increase (at low stiffness), or decrease (at high stiffness) the effects of instability and to obtain a set of signatures for the same sequence.
Thus, a “signature” does not constitute a base-to-base sequencing of an entire sequence, but makes it possible to obtain a set of specific and local information, linked to the sequence and/or to the state of pairing.
Measurements of force on isolated molecules of DNA currently constitute a very active field (see references 1 to 11). For a range of forces extending from one subpicoNewton to tens of picoNewtons, which forces are typically involved in weak molecular interactions, sensitive devices for the measurement of forces such as optical tweezers (Svoboda et al., 1993; Yin et al., 1996) or flexible microneedles are being increasingly used.
In a typical configuration intended to open DNA, the molecules may be specifically anchored on two solid substrates (microscope slide, micropipette, microparticle). One of the ends being attached and the other being connected, for example via a particle, to a device for measuring force.
Patent WO 94/23065 describes a device for measuring force, base-to-base, during mechanical opening of DNA using an atomic force microscope.
However, the base-to-base resolution is fraught with basic limitations linked to thermal noise (Thompson al., 1995; Viovy et al., 1994).
Moreover, carrying out the opening in practice exhibits operational difficulties, in particular:
the design of the molecular construction,
the chemistry of the surfaces and their preparation,
the manner of selecting the place where the measurement is carried out.
In particular, it is crucial, in order to carry out these experiments, to reduce:
the level of adsorption of the study molecule on the surfaces (microscope slide or particle);
the adhesive interactions between the particles and between the particles and the surfaces.
Finally, since a single measurement can take several tens of minutes, it is necessary to have a very efficient selection mechanism in order to macroscopically identify the most useful points so as to carry out the measurements despite the fact that the chemistry of the surfaces is imperfect, and of course, despite the fact that the constructions themselves may be imperfect.
The present invention includes the aspects relating to the chemistry of the surfaces and the specific constructions so as to obtain satisfactory results.
More particularly, the present invention relates to a method for characterizing a nucleic acid duplex comprising two at least partially paired nucleic acid sequences, characterized in that at least one “signature” of said duplexes, which is linked to the variation in force necessary to unpair, respectively re-pair, said two sequences is recorded, and in that the signature obtained or some characteristics thereof is compared with references.
It is understood, by way of example, that a reference may consist of another measurement, a combination of several measurements, or alternatively of a numerical calculation, for example such as that which will be described in the text which follows, or alternatively a combination of the preceding cases.
“Nucleic acid duplex” is understood to designate any type of DNA/RNA duplex as mentioned above.
More particularly, the present invention relates to a method characterized in that the 5′ end of one of the strands of the duplex is attached to a support 1 and the 3′ end of the other strand of the duplex is attached to a support 2, the variation in force necessary to unpair, respectively pair, said sequences being measured by moving apart, respectively bringing closer, said supports 1 and 2, characterized in that, during the attachment, the point of attachment of the 3′ end on the support 2 and the point of attachment of the 5′ end on the support 1 are linked together by a molecular string having a length of at least 0.03 &mgr;m, and preferably of between 0.5 and 30 &mgr;m.
Within the framework of the present invention, and to clarify the notations, the 5′ end of one of the strands of the duplex will be called “5′ end of the duplex” and the 3′ end of the other strand of the duplex will be called “3′ end of the duplex”, and, in this definition, the strands can of course be reversed.
“Molecular string” is understood to designate both the distance calculated on the molecule (it being possible for the latter to be folded), for example the spacer arm (mode I), and the spatial distance (measured in particular along the strands between the two points of attachment (mode II).
Supports 1 and 2 may also be interchanged in the developments which follow.
Advantageously, the method in accordance with the invention is characterized in that the structure of the duplex is locally modified in particular by the creation of a triplet, a complex or the attachment of a protein.
A few terms which will later be used in accordance with their respe
Bockelmann Ulrich
Essevaz-Roulet Baptiste
Heslot François
Centre National de la Recherche Scientifique "CNRS"
Lu Frank
Whisenaut Ethan
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