Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-14
2002-05-14
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C436S094000, C536S025400
Reexamination Certificate
active
06387626
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a simple, and preferably cost effective, method for immobilizing nucleic acid molecules onto a solid support. The invention further concerns the use of such immobilized molecules in nucleic acid hybridization assays, sequencing by hybridization assays, and genetic analyses and combinatorial analyses involving nucleic acids or proteins for screening applications.
BACKGROUND OF THE INVENTION
The analysis of the structure, organization and sequence of nucleic acid molecules is of profound importance in the prediction, diagnosis and treatment of human and animal disease, in forensics, in epidemiology and public health, and in the elucidation of the factors that control gene expression and development. Methods for immobilizing nucleic acids are often important in these types of analyses. Three areas of particular importance involve hybridization assays, nucleic acid sequencing, and the analysis of genomic polymorphisms.
I. Nucleic Acid Hybridization
The capacity of a nucleic acid “probe” molecule to hybridize (i.e. base pair) to a complementary nucleic acid “target” molecule forms the cornerstone for a wide array of diagnostic and therapeutic procedures.
Hybridization assays are extensively used in molecular biology and medicine. Methods of performing such hybridization reactions are disclosed by, for example, Sambrook, J. et al. (In:
Molecular Cloning: A Laboratory Manual
, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), Haymes, B. D., et al. (In:
Nucleic Acid Hybridization, A Practical Approach
, IRL Press, Washington, D.C. (1985)) and Keller, G. H. and Manak, M. M. (In:
DNA Probes, Second Edition
, Stockton Press, New York, N.Y. (1993)) which references are incorporated herein by reference.
Many hybridization assays require the immobilization of one component. Nagata et al. described a method for quantifying DNA which involved binding unknown amounts of cloned DNA to microtiter wells in the presence of 0.1 M MgCl
2
(Nagata et al.,
FEBS Letters
183: 379-382, 1985). A complementary biotinylated probe was then hybridized to the DNA in each well and the bound probe measured colorimetrically. Dahlen, P. et al. have discussed sandwich hybridization in microtiter wells using cloned capture DNA adsorbed to the wells (Dahlen, P. et al.,
Mol. Cell. Probes
1: 159-168, 1987). An assay for the detection of HIV-1 DNA using PCR amplification and capture hybridization in microtiter wells has also been discussed (Keller, G. H. et al.,
J. Clin. Microbiol
. 29: 638-641, 1991). The NaCl-mediated binding of oligomers to polystyrene wells has been discussed by Cros et al. (French patent no. 2,663,040) and very recently by Nikiforov et al. (
PCR Methods Applic
. 3: 285-291, 1994). The cationic detergent-mediated binding of oligomers to polystyrene wells has very recently been described by Nikiforov et al.,
Nucleic Acids Res
. 22: 4167-4175.
II. Analysis of Single Nucleotide DNA Polymorphisms
Many genetic diseases and traits (i.e. hemophilia, sickle-cell anemia, cystic fibrosis, etc.) reflect the consequences of mutations that have arisen in the genomes of some members of a species through mutation or evolution (Gusella, J. F.,
Ann. Rev. Biochem
. 55:831-854 (1986)). In some cases, such polymorphisms are linked to a genetic locus responsible for the disease or trait; in other cases, the polymorphisms are the determinative characteristic of the condition.
Such single nucleotide polymorphisms differ significantly from the variable nucleotide type polymorphisms (“VNTRs”), that arise from spontaneous tandem duplications of di- or tri-nucleotide repeated motifs of nucleotides (Weber, J. L., U.S. Pat. No. 5,075,217; Armour, A. J. et al.,
FEBS Lett
. 307:113-115 (1992); Jones, L. et al.,
Eur. J. Haematol
. 39:144-147 (1987); Horn, G. T. et al. PCT Application WO91/14003; Jeffreys, A. J., U.S. Pat. No. 5,175,082); Jeffreys. A. J. et al.,
Amer. J. Hum. Genet
. 39:11-24 (1986); Jeffreys. A. J. et al.,
Nature
316:76-79 (1985); Gray, I. C. et al.,
Proc. R. Acad. Soc. Lond
. 243:241-253 (1991); Moore, S. S. et al.,
Genomics
10:654-660 (1991); Jeffreys, A. J. et al.,
Anim. Genet
. 18:1-15 (1987); Hillel, J. et al.,
Anim. Genet
. 20:145-155 (1989); Hillel, J. et al.,
Genet
. 124:783-789 (1990)), and from the restriction fragment length polymorphisms (“RFLPs”) that comprise variations which alter the lengths of the fragments that are generated by restriction endonuclease cleavage (Glassberg, J., UK patent application 2135774; Skolnick, M. H. et al.,
Cytogen. Cell Genet
. 32:58-67 (1982); Botstein, D. et al.,
Ann. J. Hum. Genet
. 32:314-331 (1980); Fischer, S. G. et al. (PCT Application WO90/13668); Uhlen, M., PCT Application WO90/11369)).
Because single nucleotide polymorphisms constitute sites of variation flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single nucleotide present at the site of variation; it is unnecessary to determine a complete gene sequence for each patient. Several methods have been developed to facilitate the analysis of such single nucleotide polymorphisms.
Mundy, C. R. (U.S. Pat. No. 4,656,127), for example, discusses a method for determining the identity of the nucleotide present at a particular polymorphic site that employs a specialized exonuclease-resistant nucleotide derivative. A primer complementary to the allelic sequence immediately 3′ to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. The Mundy method has the advantage that it does not require the determination of large amounts of extraneous sequence data. It has the disadvantages of destroying the amplified target sequences, and unmodified primer and of being extremely sensitive to the rate of polymerase incorporation of the specific exonuclease-resistant nucleotide being used.
Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087) discuss a solution-based method for determining the identity of the nucleotide of a polymorphic site. As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3′ to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.
An alternative method, known as Genetic Bit Analysis™ or GBA™ is described by Goelet, P. et al. (PCT Appln. No. 92/15712). The method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3′ to a polymorphic site. The labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087) the method of Goelet, P. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase. It is thus easier to perform, and more accurate than the method discussed by Cohen.
Cheesman, P. (U.S. Pat. No. 5,302,509) describes a method for sequencing a single stranded DNA molecule using fluorescently labeled 3′-blocked nucleotide triphosp
Boyce-Jacino Michael T.
Shi Jufang
Horlick Kenneth R.
Kalow & Springut LLP
Kalow, Esq. David A.
Orchid BioSciences, Inc.
Schmidt William D.
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