Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-03
2003-12-16
Benzion, Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S174000, C536S023100
Reexamination Certificate
active
06664051
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a solid carrier to which nucleotide derivatives (e.g., oligonucleotides, polynucleotides, and peptide-nucleotides) are attached, which is generally named DNA chip and which is favorably employable for detecting, with high sensitivity, complementary nucleic acid fragments.
BACKGROUND OF THE INVENTION
Detection of a nucleic acid fragment is generally performed using a probe DNA which is complementary to the nucleic acid fragment to be detected, by way of hybridization. The probe DNA is generally fixed to a solid carrier (e.g., solid substrate) to produce a DNA chip. In the detection, a nucleic acid fragment in a sample liquid is labelled with a fluorescent label or a radio-isotope label, and then the sample liquid is brought into contact with the probe DNA of the DNA chip. If the labelled nucleic acid fragment in the sample liquid is complementary to the probe DNA, the labelled nucleic acid fragment is combined with the probe DNA by hybridization. The labelled nucleic acid fragment fixed to the DNA chip by hybridization with the probe DNA is then detected by an appropriate detection method such as fluorometry or autoradiography. The DNA chip is widely employed in the gene technology, for instance, for detecting a complementary nucleic acid fragment and sequencing a nucleic acid.
The DNA chip is described, for instance, in Fodor S.P.A., Science, 251, 767(1991) and Schena M., Science, 270, 467(1995). The DNA chip is understood to efficiently detect a small amount of complementary nucleic acid fragments in a small amount of a sample liquid.
Detection of nucleic acid fragment using an electrochemical label is also known (Japanese Patent Provisional Publication No. 9-288080, and a preprint of the 57th Analytical Chemistry Conference pp. 137-138 (1996)),
The electrochemical label such as N-hydroxysuccinimide ester of ferrocenecarboxylic acid is attached to a probe DNA. The probe DNA is fixed onto an electroconductive substrate having an output terminal. In the detection procedure, a sample liquid containing target nucleic acid fragments is brought into contact with the probe DNA having the ferrocene derivative label in the presence of an electrochemically active thread intercalator. The target nucleic acid fragment, if it is complementary to the probe DNA, is hybridized with the probe DNA. Into the formed hybrid structure, the electrochemically active thread intercalator is intercalated. Thereafter, a potential is applied to the electroconductive substrate to measure an electric current flowing through the ferrocene derivative label and the thread intercalator.
P. E. Nielsen et al., Science, 254, 1497-1500(1991) and P. E. Nielsen et al., Biochemistry, 36, pp.5072-5077 (1997) describe PNA (Peptide Nucleic Acid or Polyamide Nucleic Acid) which has no negative charge and functions in the same manner as DNA does. PNA has a polyamide skeleton of N-(2-aminoethyl) glycine units and has neither glucose units nor phosphate groups.
Since PNA is electrically neutral and is not charged in the absence of an electrolytic salt, PNA is able to hybridize with a complementary nucleic acid fragment to form a hybrid which is more stable than the hybrid structure giver by a DNA prove and its complementary nucleic acid fragment (Preprint of the 74th Spring Conference of Japan Chemical Society, pp. 1287, reported by Naomi Sugimoto).
Japanese Patent Provisional Publication No. 11-332595 describes a PNA probe fixed on a solid carrier at its one end and a detection method utilizing the PNA probe. The PNA probe is fixed onto the solid carrier by the avidinbiotin method.
The aforementioned P. E. Nielsen et al., Science, 254, 1497-1500(1991) also describes a PNA probe labelled with an isotope element and a detection method of a complementary nucleic acid fragment.
Since the PNA probe shows no electric repulsion to a target nucleic acid fragment in a sample liquid, an improved high detection sensitivity is expected.
At present, two methods are known for preparing a DNA chip having a solid carrier and oligonucleotide or polynucleotide fixed onto the carrier. One preparation method comprises preparing oligonucleotide or polynucleotide step by step on the carrier. This method is named “on-chip method”. A typical on-chip method is described in Foder, S.P.A., Science, 251, page 767 (1991)
Another preparation method comprises fixing a separately prepared oligonucleotide or polynucleotide onto a solid carrier. Various methods are known for various oligonucleotides and polynucleotides.
In the case that the complementary derivative (which is synthesized using ni as meld) or a PCR product (which is a DNA fragment prepared by multiplying cDNA by PCR method), an aqueous solution of the prepared DNA fragment is spotted onto a solid carrier having a polycationic coat in a DNA chip-preparing device to attach the DNA fragment to the carrier via electrostatic bonding, and then blocking a free surface of the polycationic coats.
In the case that the oligonucleotide is synthetically prepared and has a functional group, an aqueous solution of the synthetic oligonucleotide is spotted onto an activated solid carrier to produce covalent bonding between the oligonucleotide and the carrier surface. See Lamture, J. B., et al., Nucl. Acids Res., 22, 2121-2125, 1994, and Guo, Z., et al., Nucl. Aids Res., 22, 5456-5465, 1994. Generally, the oligonucleotide is covalently bonded to the surface activated carrier via a spacer or a cross-linker.
Also known is a process comprising the steps of aligning sell polyacrylamide gels on a glass plate and fixing synthethic oligonucleotides onto the glass plate by making a covalent bond between the polyacrylamide and the oligonucleotide (Yershov, G., et al., Proc. Natl. Acad. Sci. USA, 94, 4913(1996)). Sosnowski, R. G., et al., Proc. Natl. Acad. Sci. USA, 94, 1119-1123 (1997) discloses a process comprising the steps of an array of microelectrodes on a silica chip, forming on the microelectrode a streptoavidin-comprising agarose layer, and attaching biotin-modified DNA fragment to the agarose layer by positively charging the agarose layer. Schena, M., et al., Proc. Natl. Acadl Sci. USA, 93, 10614-10619 (1996) teaches a process comprising the steps of preparing a suspension of an amino group-modified PCR product in SSC (i.e., standard sodium chloride-citric acid buffer solution), spotting the suspension onto a slide glass, incubating the spotted glass slide, treating the incubated slide glass with sodium borohydride, and heating thus treated slide glass.
As is explained above, most of the known methods of fixing a separately prepared DNA fragment onto a solid carrier utilize an electrostatic bonding or a covalent bonding such as described above.
In any DNA chip having a separately prepared DNA fragment on its solid carrier, the DNA, fragment should be firmly fixed onto the carrier, so as to perform smoothly hybridization between the fixed DNA fragment and a target DNA fragment complementary to the fixed DNA fragment.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an improved method for preparing a nucleic acid fragment-detecting means by fixing separately produced nucleotide derivatives to a solid carrier by covalent bonding
The present invention resides in a method for fixing a plurality of nucleotide derivatives to a solid carrier which comprises bringing nucleotide derivatives having a reactive group at each one terminal into contact with a solid carrier having thereon reactive groups in an aqueous phase in the presence of a transferase which is capable of producing a covalent bond by rearrangement of the reactive group of the nucleotide derivative and the reactive group of the solid carrier.
REFERENCES:
patent: 5403711 (1995-04-01), Walder et al.
patent: 6344316 (2002-02-01), Lockhart et al.
patent: 6420112 (2002-07-01), Balhorn et al.
Zubay. Biochemistry, 3rdEdition, 1993. Wm. C. Brown Publishers, p. 200.
Seshimoto Osamu
Shinoki Hiroshi
Sudo Yukio
Benzion Gary
Fuji Photo Film Co. , Ltd.
Goldberg Jeanine
Reed Smith LLP
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