Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-06-29
2003-02-25
Whisenant, Ethan C. (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C436S094000, C536S023100, C536S025300, C536S025320
Reexamination Certificate
active
06524829
ABSTRACT:
The invention relates to a method for the base sequencing of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
Today, the base sequencing of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) belongs to the most important analysis techniques in biotechnology, the pharmaceutical industry, food industry, medical diagnostics and other fields of application. The decipherment of the genomes of organisms offers the possibility of diagnosis, therapy and prevention of illnesses as well as the targeted modification of the human genome to generate organisms having modified characteristics. Sufficiently rapid sequencing methods are required to allow the use of this potential.
The classical sequencing methods according to Sanger et al (Proceedings of the National Academy of Science, USA, 74, 5463-7; 1997) as well as Maxam and Gilbert (Proceedings of the National Academy of Science, U.S.A., 74, 560-564; 1977), which are still today the basis for the standard sequencing methods, need 1 to 3 days for sequencing 200 nucleotides. This appears to be too slow, for example, for the problem to sequence the human genome with approximately 3·10
9
base pairs.
More recent attempts at accelerating sequencing methods have concentrated on methods in which individual nucleotides were detected using fluorescence spectroscopy. U.S. Pat. No. 4,962,037 discloses a sequencing method, according to which a complementary nucleic acid strand, where a fluorescence dye molecule characteristic for the base is bonded covalently to each base, is synthesized on a single strand. This nucleic acid fluorescence-tagged molecule is bonded to a particle surface, with the individual particles being held, for example, in a liquid flow with a micro-injection pipette. Each fluorescence-tagged base is then successively cleaved from the nucleic acid strand by use of an exonuclease, and is guided in the liquid flow in the focus of a laser beam, where, after excitation, the fluorescence specific for the base is detected. The velocity of this sequencing method is limited theoretically only by the cutting rate of the exonuclease so that a sequencing velocity of 100 to 1,000 bases/seconds is assumed.
One precondition for the performance of the method disclosed in U.S. Pat. No. 4,962,037 is that only one single nucleic acid molecule is held to one single particle. The manipulation of a single particle with a single nucleic acid molecule is, however, technically very difficult and complicated and has proven not to be suited for practical applications. Furthermore, the use of an exonuclease is necessary which is able to cleave the dye-tagged nucleotides. This makes the development of this method more complicated and the use for this of modified exonucleases causes in addition as a rule an increased inexactness in the determination of the base sequence.
It is therefore the object of the invention to provide a method for base sequencing of DNA or RNA in which the advantages as regards the high velocity of a sequencing method with individual molecule detection, as is described in the prior art, is utilized but where at the same time the above-mentioned disadvantages are overcome.
To solve this object the invention provides a first method for base sequencing of DNA or RNA, comprising the steps:
(1) immobilizing DNA or RNA single strands on a surface;
(2) focussing a laser beam on a single, immobilized single strand;
(3) producing a DNA or RNA complementary strand of said immobilized, focussed single strand by adding a solution containing (i) a mixture of nucleotides of the bases adenine, cytosine, guanine and thymine for producing a DNA complementary strand or a mixture of nucleotides of the bases adenine, cytosine, guanine and uracil for producing a RNA complementary strand and (ii) a polymerase, with
3a) at least two of the four nucleotides of the bases adenine, cytosine, guanine and thymine or at least two of the four nucleotides of the bases adenine, cytosine, guanine and uracil being differently luminescence-tagged in part or in full,
3b) each insertion of a luminescence-tagged nucleotide into the complementary strand being detected with a single-molecule detector, and
3c) the luminescence signal of the previous luminescence-tagged nucleotide being deleted prior to the insertion of the respective next luminescence-tagged nucleotide.
The invention further provides a second method for base sequencing of DNA or RNA, comprising the steps:
(1) immobilizing DNA or RNA single strands on a surface;
(2) focussing a laser beam on a single, immobilized single strand;
(3′) producing a DNA or RNA complementary strand of said immobilized, focussed single strand by sequential addition of solutions containing respectively (i) one nucleotide of the bases adenine, cytosine, guanine and thymine for producing a DNA complementary strand or one nucleotide of the bases adenine, cytosine, guanine and uracil for producing an RNA complementary strand and (ii) a polymerase, with
3a′) the nucleotide contained in the solution being luminescence-tagged,
3b) each insertion of a luminescence-tagged nucleotide into the complementary strand being detected with a single-molecule detector, and
3c) upon detection of the insertion of a luminescence-tagged nucleotide in the complementary strand, the luminescence signal of the inserted nucleotide being deleted, and
3d′) rinsing occurring prior to the addition of the respective next solution.
The term DNA or RNA single strand designates according to the invention a non-hybridized DNA or RNA molecule. Such a single strand can be obtained by direct isolation from an organism including gene technology methods as well as by the treatment of such molecules with restriction enzymes. Oligonucleotides, PCR products and c-DNA count as these single strands. The production of the single strands from the double strands is known to the person skilled in the art, e.g. from J. Sambrook et al., “Molecular Cloning”, 2
nd
edition, Cold Spring Harbor Laboratory Press, 1989. The treatment with restriction enzymes can be performed directly before immobilization, which causes an immobilization of molecules with different base sequences. The single strands have preferably 5 to 2,000 bases, especially preferred is 100 to 1,000 bases. In principle, however, base lengths of up to 100 kilo-bases come under consideration.
In step (1) of the method according to the invention, the DNA or RNA single strands are immobilized on a surface. The surface is preferably the surface of a planar support which comprises the optical transparency necessary for the single molecule detection described below. A glass support is especially preferred, in particular a quartz glass support. In a preferred embodiment, the surface of the support, on which the single strands are immobilized, is chemically modified by application of a Langmuir-Blodgett film. A Langmuir-Blodgett film of a cellulose derivative is especially preferred, in particular trimethyl silylether cellulose cinnamate (TMSCC) and aminoalkyl trimethyl silylether cellulose (ATMSC).
The single strands can be be immobilized on the surface by adsorption, via a covalent bond as well as via a scavenger molecule. Scavenger molecules are in particular nucleotide oligomeres which are immobilized on the surface and can bind the single strands by hybridization. The immobilization of the oligomer on the surface can take place by adsorption or by covalent binding to a chemically reactive group on the surface. The immobilization with the (strept-)avidin biotin technique is particularly preferred, with the oligomer being derived with biotine and binding to a (strept-)avidin molecule immobilized on the surface. The immobilization of the (strept-)avidin molecule is not restricted. In a preferred embodiment, the (strept-)avidin molecules are immobilized on the surface via a Langmuir-Blodgett film of a cellulose derivative. It is in particular preferred to coat the surface first with 1 to 8 mono-layers of aminoalkyl trimethylsilylether cellulose (ATMSC) and thereafter with 1 to 8 mono-layers trimethyl si
Lu Frank
Molecular Machines & Industries GmbH
Whisenant Ethan C.
LandOfFree
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