Method of DNA sequencing by polymerisation

Abstract

The present invention relates to a method for the determination of a nucleic acid sequence by physical manipulation. The method is based on the precise determination of the localization of the replicating fork on the template by measuring the physical distance between one end of the molecule and the fork. This allows the determination of the physical location of the site where a pause or a blockage of the replication occurs, and deducing therefrom information on the sequence of the nucleic acid.

Claims

1. A method for detecting blockage of the renaturation of a hairpin molecule, said method comprising: a) providing a nucleic acid hairpin molecule consisting of a double-stranded stem and a single-stranded loop, wherein 5 and 3 ends of the nucleic acid hairpin molecule are bound to different surfaces; b) applying a force to one of the surfaces such that it is moved away from another of the surfaces, thereby yielding a completely denatured hairpin molecule; c) hybridizing a known single-stranded nucleic acid primer to the completely denatured hairpin molecule; d) extending the primer with a polymerase, thereby generating a nucleic acid molecule complementary to the completely denatured hairpin molecule; e) reducing the force applied to allow renaturation of the completely denatured hairpin molecule in the presence of the nucleic acid molecule complementary to the completely denatured hairpin molecule; and f) detecting blockage of the renaturation of the completely denatured hairpin molecule in step e) due to generation of the nucleic acid molecule complementary to the completely denatured hairpin molecule.

2. The method of claim 1, wherein steps a)-f) are repeated.

3. The method of claim 1, wherein the force is reduced to less than or equal to 12 pN.

4. The method of claim 1, wherein the force is reduced to less than or equal to 11 pN.

5. The method of claim 1, wherein the force is reduced to less than or equal to 10 pN.

6. The method of claim 1, wherein the nucleic acid hairpin molecules are made by replicating RNA molecules.

7. The method of claim 1, wherein said one of the surfaces is a magnetic bead.

8. The method of claim 7, wherein the force is a magnetic force.

9. The method of claim 1, wherein the force in step b) is above or equal to 15 pN.

10. The method of claim 1, wherein said extending the primer with a polymerase occurs in a reaction mix comprising a pool of dNTPs, wherein one of the dNTPs in the pool is present at a low concentration as compared to the other dNTPs in the pool.

11. The method of claim 1, wherein said extending the primer with a polymerase occurs in a reaction mix comprising a pool of nucleotides comprising dNTPs and one ddNTP.

12. The method of claim 1, further comprising disassembling the nucleic acid molecule complementary to the completely denatured hairpin molecule after step f).

13. The method of claim 12, wherein steps a)-f) are repeated after said disassembling the nucleic acid molecule complementary to the completely denatured hairpin molecule.

Description

LEGENDS OF THE FIGURES

(1) FIG. 1 Principle of detection of the hybridization of oligo-nucleotides to their complementary sequence on a hairpin DNA. The hairpin DNA anchoring the bead to the surface (a) is momentarily unzipped by increasing the force pulling on the bead to a value above 16 pN. In that phase the complementary fragment in solution hybridizes to its target on the opened DNA hairpin, thus preventing the rezipping of the hairpin (b) when the force is reduced back to its initial value. The hairpin refolding presents four plateaus occuring at well defined extensions but with variable duration. The top plateau at 73.71 nm is associated with the 83 bp fully opened hairpin at F.sub.test, while the bottom one corresponds to the hairpin completely refolded. The two intermediate plateaus at 25.47 nm and 35.17 nm occur because two oligos have been placed in the solution. From these change in extension (z.sub.high-z) it is possible to deduce where along the hairpin the complementary sequence has paired. Here according to their positions the blocks coincide with location 28.66 bp and 39.60 bp in very good agreement with their expected positions at 29 bp and 40 bp. The plateau positions are better estimated by fitting Gaussian to the histogram obtained from several opening/closing cycles (here 20 cycles).

(2) FIG. 2: Illustration of single molecule Sanger sequencing. Real-time record of synthesis on a 1.2 kbps single molecule hairpin with T4 DNA polymerase (dTNP =dGNP =dCNP =500 M, dATP =5 M, ddATP =400 M, clamp and clamp loader) in a commercial buffer (5X Reaction Buffer: 335 mM Tris-HCI (pH 8.8 at 25 C.), 33 mM MgCl.sub.2, 5 mM DTT, 84 mM (NH.sub.4).sub.2SO.sub.4, Fermentas; fermentas.com/en/products/all/modifying-enzymes/mesophilic-polymerases/ep0064-t4-dna-polymerase). At high force, the peak of the curve shows the existence of hairpin sample. At middle force, the rising curve shows the processive synthesis of a new complementary strand by the T4 DNA polymerase, while the plateau represents the pause occurring when one ddNTP is incorporated. At low force, the falling edge of the curve shows the exonuclease activity removing the strand just synthesized. These synthesis and exonuclease phases may be repeated in cycles.

(3) FIG. 3: Illustration of sequencing based on unbalanced dNTP concentration. Real-time record of synthesis on a 1.2 kbps single molecule hairpin with T4 DNA polymerase (dTNP =dGNP =dCNP =33M, dATP =20 nM, clamp and clamp loader) in commercial buffer (5X Reaction Buffer: 335 mM Tris-HCl (pH 8.8 at 25 C.), 33 mM MgCl.sub.2, 5 mM DTT, 84 mM (NH.sub.4).sub.2SO.sub.4, Fermentas; fermentas.com/en/products/all/modifying-enzymes/mesophilic-polymerases/ep0064-t4-dna-polymerase). At high force (21 pN), the peak of the curve shows the existence of hairpin sample and could be used to hybridize a primer if needed. At middle force (11.7 pN), the rising edge of the curve shows the processive synthesis of a new complementary strand by the T4 DNA polymerase, while the transient pause corresponds to the presents when a dATP is needed. At low force (1.6 pN), the falling shows the cleavage of the newly-synthesized strand, due to the activation of the exonuclease activity of the T4 DNA polymerase. This synthesis and cleavage cycle is repeatable.

(4) FIG. 4A-B: Detection of the sequencing based on unbalanced dNTP concentration to their complementary sequence on a hairpin. FIG. 4A: Real-time record of synthesis on a 83 bp single molecule hairpin with T4 DNA polymerase (dTNP =dGNP =dCNP =33 M, dATP =20 nM, clamp and clamp loader) in commercial buffer (5X Reaction Buffer: 335 mM Tris-HCl (pH 8.8 at 25 C.), 33 mM MgCl.sub.2, 5 mM DTT, 84 mM (NH4)2SO4, Fermentas; fermentas.com/en/products/all/modifying-enzymes/mesophilic-polymerases/ep0064-t4-dna-polymerase). At high force (21pN), the peak of the curve shows the existence of hairpin sample and could be used to hybridize a primer if needed. At middle force (11.7 pN), the rising edge of the curve shows the processive synthesis of a new complementary strand by the T4 DNA polymerase, while the transient pause corresponds to the position where a dATP is needed. At low force (1.6 pN), the falling shows the cleavage of the newly-synthesized strand, due to the activation of the exonuclease activity of the T4 DNA polymerase. This synthesis and cleavage cycle is repeatable. The position histogram of these transient pauses is deduced similarly as previously described in FIG. 1 and base calls are indicated (arrows). The true sequence of the template is shown on the right side of the figure. FIG. 4B: There are two ways to measure the extension change. First, we can use the Zzip as reference; alternatively, we can also use the position of the hairpin loop as reference.

(5) FIG. 5. Illustration of hairpin design. The DNA fragment of interest is ligated with a DNA loop (with abasic and LNA bases) and two partially complementary DNA fragments (one ssDNA with Biotin on the end, one dsDNA with Dig at extremity), which eventually forms a hairpin that can be bound to a super-paramagnetic bead covered with streptavidin (DYNAL) while the other extremity to a glass coverslip treated with anti-dig.

EXPERIMENTAL EXAMPLES

(6) DNA Preparation

(7) A double-strand (ds)DNA fragment of unknown sequence and of a size comprised between a few tens and a few thousands base pairs, is ligated at one of its extremities to a DNA loop. Its other extremity is ligated to a dsDNA fragment allowing for the binding of its two strands to differently coated surfaces. For example, the free 3 end of one strand can be labeled with biotin allowing binding to streptavidin coated beads, whereas the 5 end on the opposite strand can be labelled with digoxigenine allowing its binding to surfaces coated with an anti-Dig antibody. This end-labelling can be done by various ways known to the man of the art, such as the use of terminal transferase to add biotin (or dig) modified nucleotides or hybridization with suitably labelled oligo-nucleotides.

(8) Force Stretching Apparatus

(9) This DNA construct is incubated for a few minutes in a solution of adequate beads (for example streptavidin coated ones) to which it binds by one of its labelled (for example biotin) ends. The beads can be transparent if optical tweezers are later used for manipulation or magnetic if one uses magnetic traps or tweezers for manipulation.

(10) The bead-DNA assembly is injected in a fluidic chamber the surface of which has been treated such as to bind the other labelled end of the molecule (for example a surface coated with anti-Dig to bind the Dig-labelled end of the DNA). The beads are thus anchored to the surface via a DNA-hairpin, see FIG. 1a. The distance of the bead to the surface is then monitored by various means known to the man of the art: for example the diffraction rings of their image on a camera can be used to deduce their distance, or the light intensity they scatter (or emit by fluorescence) when illuminated in an evanescent mode can be used to measure their distance. Alternatively, the magnetic field they generate can be measured (using a magnetic sensor such as GMR or Hall sensors) to deduce their distance to a sensor on the anchoring surface.

(11) To pull on the DNA molecule anchoring the beads to the surface various techniques have been described. The preferred embodiment uses a magnetic trap to pull on super-paramagnetic beads anchored to a surface by a DNA hairpin as described above. In this configuration, small magnets placed above the sample are used to apply a constant force on the anchored bead, whose position can be determined with <1 nm accuracy (depending on the pulling force and the dissipation due to hydrodynamic drag). In this series of experiments, the apparatus described in U.S. Pat. Nos. 7,052,650 and 7,244,391 was used. In addition, unless otherwise indicated, the experiments reported here were performed in 25 mM Tris pH 7.5, 150 mM KAc, 10 mM MgCl.sub.2, 0.2% BSA. In every case, the tethering hairpin can be mechanically fully unzipped by pulling on the beads with a force larger than about 16 pN. Reducing the tension on the molecule to below about 11 pN allows the hairpin to re-zip spontaneously (the unzipping transition is reversible though hysteretic). If, during the unzipped phase, binding of a molecule in solution (such as a protein or complementary oligo-nucleotides of DNA, RNA, LNA or PNA) to the stretched single stranded (ss)DNA occurred, this molecule will transiently block the rezipping of the hairpin when the force is lowered to below 11 pN. The principle of the assay is to switch between two forces: a large one F.sub.open to open the hairpin and a smaller one F.sub.test used to allow re-zipping and to measure the extension of the molecule at transient blockages. The blocking position is related to the sequence by a linear relation between full extension and the blocked one. For best accuracy, the full extension is preferably measured at the test force F.sub.test. This is achieved by designing the hairpin loop such that it requires a fraction of a second to refold once the force is reduced from F.sub.open to F.sub.test.

(12) The Hybridization Position of an Oligo-nucleotide Can Be Measured with a Basepair Resolution

(13) By measuring the extension of the DNA molecule (the distance of the bead to the surface) during one of these rezipping pauses, it is possible to determine the position of the blockage with a nanometer precision (1 nm corresponds to the distance spanned by two nucleotides (1 bp) in a ssDNA under a 10 pN force). The unzipping configuration displays the largest ratio of extension to basepair (in dsDNA the ratio is only 0.34 nm per bp).

(14) The accuracy of this measurement is limited by two noise contributions: The accuracy of the measuring method, The brownian motion of the bead.

(15) Different techniques can be used to measure the vertical position of the bead. One of the simplest relies on video microscopy (U.S. Pat. Nos. 7,052,650 and 7,244,391). The results in FIG. 1 where obtained with this method, typical resolution reaches 1 nm for a 1 second averaging. Other methods with better resolution have been demonstrated, such as laser illumination with PSD sensors that reaches 0.1 nm in resolution (Greenleaf and Block, Science, 313: 801, 2006) and evanescent wave illumination (Singh-Zocchi et al., Proc Natl Acad Sci USA., 100(13): 7605-7610, 2003 , Liu et al., Biophys J., 96(9): 3810-3821, 2009).

(16) The intrinsic limitation in resolution is given by the brownian fluctuations of the bead pulling on a ssDNA molecule. <x.sup.2>=4k.sub.BT f (6r)/k.sup.2.sub.ssDNA(F) where k.sub.ssDNA(F) is the stiffness of a ssDNA molecule, k.sub.B is Boltzman constant, T the absolute temperature, the viscosity of water, r the bead's radius and f is the frequency range of the measurement. k.sub.ssDNA(F=10 pN)=0.05/Nb (N/m), where Nb is the number of bases of the ssDNA. For the 84 bp hairpin this leads to 0.04 nm of noise over 1 second (f=1 Hz) averaging. The larger noise in FIG. 1 (1 nm) is essentially due to the measuring device, not the intrinsic fluctuations. The intrinsic brownian noise increases with the size of the hairpin: a 1200 bp hairpin leads to a noise of 0.6 nm when averaging over 1 second.

(17) Diagnostics and Sequencing By Mechanical Detection of Polymerization.

(18) We have shown that the T4 DNA polymerase can replicate a DNA hairpin when the force is high enough to sufficiently destabilize the fork (F.sub.test). Like in the classical Sanger sequencing, the incorporation of a specific ddNTP will prevent further elongation of the nascent strand by the T4 DNA polymerase. In our method, this blockage can be easily identified, as shown in FIG. 6. Upon lowering the force, the exonuclease activity of the T4 DNA polymerase is activated and the enzyme excises the newly-synthesized strand. Thus, by repeated cycles of synthesis and cleavage, the molecule (hairpin) can be sequenced by identifying the blockage positions first in the presence of ddATP, and then of each of the other ddNTPs, i.e. ddTTP, ddCTP, and ddGTP.

(19) Similarly, the double-stranded hairpin molecule can be sequenced in a buffer comprising a deficit of one of the four dNTPs compared to the others, i.e. this dNTP is present at a very low concentration as compared to the others. Thus, whenever the T4 DNA polymerase, during polymerization, reaches a position requiring the addition of the limiting nucleotide, a transient pause occurs, as exemplified on FIG. 7. As described hereabove, the newly-synthesized brand can be excised by lowering the force, which results in the activation of the exonuclease activity of the enzyme. Thus, by repeated cycles of synthesis and cleavage, the molecule (hairpin) can be sequenced by identifying the pause positions first in the presence of low concentrations of dATP (for example), and then in turn of each of the other dNTPs, i.e. dTTP, dCTP, and dGTP.