Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-13
2001-05-01
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C422S068100, C422S082070, C422S082050, C422S082080, C422S082110, C436S164000, C436S172000, C436S532000, C436S800000, C436S805000
Reexamination Certificate
active
06225068
ABSTRACT:
The invention relates to methods in the sequencing of individual macromolecules, particularly individual DNA and RNA molecules.
For several years there have been attempts worldwide to sequence individual DNA molecules or strands. The sequencing of an individual DNA strand comprises the following steps:
a) Starting from the DNA molecule to be sequenced, the so-called template, which is present in a large number, DNA molecules are synthesised as complementary copies, in which dye molecules are coupled to at least some of the nucleotides. These DNA molecules are frequently designated as marked DNA. The synthesis takes place with the aid of a polymerase. As a result marked double strands are obtained or single strands, if required, after denaturing.
b) From the marked DNA strands one individual one is bonded onto a carrier.
c) The carrier is transferred into a detection apparatus; in the detection apparatus the individual partially marked nucleotides are broken down successively by an exonuclease, i.e. they are split off from the DNA strand.
d) The split-off and marked mononucleotides are detected and identified in the detection apparatus. This can be done with the aid of spectrally resolved or time-resolved fluorescence spectroscopy or other nucleotide-specific methods, such as for example mass spectrometry.
In the past step b) was carried out in such a way that microspherules with a diameter between 0.5 and 5 &mgr;m are coated with avidin or streptavidin. Biotin is coupled onto the 5′ ends of the DNA strand. If the coated microspherules are dipped into a solution containing the biotinylated DNA strands, then the DNA strands bond onto the microspherules. If the concentration of the DNA strands in the solution is very small, then statistically only 0, 1, 2 or more DNA strands bond onto the spherules, but on average it is only between 0 and 1 DNA strand. With the aid of optical tweezers a microspherule is then sought which shows a fluorescence signal due to the bonded DNA strand. In this way a microspherule onto which precisely one DNA strand is bonded is found with a certain probability.
In order that the fluorescence of the DNA strand bonded onto the microspherule can be seen in spite of the Brownian movement of the microspherules, the DNA strand must be irradiated with a high-power excitation light. This can lead to a destruction of those dyes with which the DNA strand is dyed. In addition, the dye molecules in a highly-marked DNA strand fluoresce only poorly because of various fluorescence extinguishing processes.
The splitting off and detection of the mononucleotides in step d) frequently takes place in flow systems (J. Biomolecular Struc. & Dynamics, Volume 7 (1989) page 301). Due to a suitable construction of a flow system, the marked and split-off mononucleotides flow past a detection apparatus. This operates with a relatively large detection volume in the range of picoliters. The background signal is correspondingly great due to contaminants and Raman scattering. Furthermore, the flow system necessitates a relatively large distance between the collecting optical unit and the detection volume, as a result of which the collection efficiency drops.
The object of the invention is to improve the method for sequencing an individual DNA molecule.
In order to achieve this object, for the step b) of individual strand sequencing referred to in the introduction there is provided a method for extracting an individual fluorescible macromolecule from a fluid, this method being characterised in
that a region of a tip of a fibre having a diameter of at most a few micrometers is coated with molecules, the molecules being selected so that they can form a bond with the material of the fibre tip and the fluorescible macromolecule;
that the coated fibre tip is dipped into the fluid containing the fluorescible macromolecule, so that the region of the fibre tip is irradiated with light of the excitation wavelength of the fluorescible macromolecule;
that the fluorescent light from the surroundings of the fibre tip is detected; and
that the fibre tip is removed from the fluid as soon as the detected fluorescent light exceeds a predetermined intensity.
Due to the fact that a fibre with an extremely small tip is used, and also only a region of this tip is coated with molecules which can produce a bond between the fibre tip and the fluorescible macromolecule, there is only a very small surface onto which the fluorescible macromolecule can bond. Because the surface is so small, the probability that only one single molecule will bond onto the fibre tip is substantially increased.
Furthermore, on-line monitoring of the bonding of a fluorescible macromolecule onto the fibre tip is achieve. As soon as a predetermined increase in the intensity of the detected light is established, bonding of a fluorescible macromolecule onto the fibre tip can be assumed with a very high degree of probability. The fibre tip is then immediately removed from the fluid.
In the on-line monitoring a relatively long time is available for the fluorescence detection of the fluorescible macromolecule, which can no longer diffuse and which is bonded onto the fibre tip. Therefore the excitation intensity can be chosen to be correspondingly low. As a result, in turn, the probability of a photochemical destruction of the fluorescibility of the macromolecule is reduced.
If the concentration of the fluorescible macromolecule in the fluid is chosen in such a way that bonding onto the fibre tip occurs only at large time intervals, then the probability of two fluorescible macromolecules bonding simultaneously onto the fibre tip is very small. Consequently, when the fluorescent light exceeds a predetermined intensity the probability is very great that only one single fluorescible macromolecule has bonded onto the fibre tip.
The fluorescible macromolecule can be removed with the aid of the fibre tip from the fluid and transported to any other apparatus.
The given method for extracting an individual fluorescible macromolecule from a fluid is not restricted to DNA molecules. It can be used quite generally for any fluorescible macromolecule so long as a bond can be made between the macromolecules and the fibre tip. Also the method can be used both in gases and in fluids.
In an advantageous embodiment of the invention the coating of the fibre tip with molecules is achieved in that the surface of the fibre tip is coated with photobiotin molecules; that a spatially delimited region of the surface of the fibre tip is exposed to light in the wavelength range from approximately 300 to 360 nm in such a way that the photobiotin molecules are bonded onto the fibre tip in the irradiated region; that after this the unbonded photobiotin molecules are washed off from the fibre tip; and that the fibre tip is thereafter brought into contact with a solution containing avidin or streptavidin.
If biotin is bonded on the fluorescible macromolecule, then because of the strong bonding between biotin and avidin or streptavidin a coating of the fibre tip with these molecules suggests itself. This method constitutes a particularly elegant possibility of achieving a coating with avidin or streptavidin in the smallest possible region of the fibre tip. The coating of the fibre tip can be achieved by the use of focussed light in a region limited to fractions of a &mgr; m
2
terms of surface area. A fibre tip produced from glass or PMMA is pretreated in a known manner for the coating with photobiotin.
In an advantageous embodiment of the invention a dye-marked double-strand DNA molecule is extracted from an aqueous solution, wherein the aqueous solution has added to it intercalation dye molecules, the excitation wavelength of which lies in a different wavelength range from the excitation wavelength of the dye molecules. Light of the excitation wavelength of the intercalation dye molecules is input into the fibre and passed to the fibre tip in such a way that it has a small depth of penetration into the aqueous solution. The intercalation dye molecules are excited to f
Blakely & Sokoloff, Taylor & Zafman
Jones W. Gary
Taylor Janell E.
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