Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-29
2004-01-06
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06673536
ABSTRACT:
1. FIELD OF THE INVENTION
This invention relates to the field of nucleic acid hybridization. In particular, the present invention relates to methods for ranking the relative specificity with which polynucleotide probes hybridize to a nucleic acid sequence. The invention also relates to methods of identifying and/or designing nucleic acid sequences which hybridize most specifically to a nucleotide sequence of interest.
2. BACKGROUND
The ability to measure abundances of different nucleic acid molecular species in a sample containing many different nucleic acid sequences is a matter of great interest to many researchers. Presently, assays involving hybridization of nucleic acid molecules to a complementary probe are the only way to detect the presence of a particular sequence or sequences in a complex sample comprising many different nucleic acid sequences. For example, the nucleotide sequence similarity of a pair of nucleic acid molecules can be distinguished by allowing the nucleic acid molecules to hybride, and following the kinetic and equilibrium properties of duplex formation (see, e.g., Sambrook, J. et al., eds., 1989
, Molecular Cloning: A Laboratory Manual
, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., at pp. 9.47-9.51 and 11.55-11.61; Ausubel et al., eds., 1989
, Current Protocols in Molecular Biology
, Vol I, Green Publishing Associates, Inc., John Wiley & Sons, Inc., New York, at pp. 2.10.1-2.10.16; Wetmur, J. G., 1991
, Critical Reviews in Biochemistry and Molecular Biology
26:227-259; Persson, B. et al., 1997
, Analytical Biochemistry
246:34-44; Albretsen, C. et al., 1988
, Analytical Biochemistry
170:193-202; Kajimura, Y. et al., 1990
, GATA
7:71-79; Young, S. and Wagner, R. W., 1991
, Nucleic Acids Research
19:2463-2470; Guo, Z. et al., 1997
, Nature Biotechnology
15:331-335; Wang, S. et al., 1995
, Biochemistry
34:9774-9784; Niemeyer, C. M. et al., 1998
, Bioconjugate Chemistry
9:168-175).
Some of the most widely used techniques employ oligonucleotide “probes,” (i.e., DNA molecules having a length up to about 100 bases and more typically fewer than about 50 bases) to selectively hybridize to, and thereby identify, nucleic acid sequences in a sample that contain complementary sequences. Many assays for detecting nucleic acid sequences in a sample comprise binding a set of nucleic acid probes to a solid support, permitting a labeled nucleic acid species to bind to the immobilized nucleic acid, washing off any unbound material, and detecting the bound, labeled sequence. For example, in blotting assays, such as dot or Southern Blotting, nucleic acid molecules may be first separated, e.g., according to size by gel electrophoresis, transferred and bound to a membrane filter such as a nitrocellulose or nylon membrane, and allowed to hybridize to a single labeled sequence (see, e.g., Nicoloso, M. et al., 1989
, Biochemical and Biophysical Research Communications
159:1233-1241; Vernier, P. et al., 1996
, Analytical Biochemistry
235:11-19). Other techniques have been developed to study the hybridization kinetics of polynucleotides immobilized in agarose or polyacrylamide gels (see, e.g., Ikuta S. et al., 1987
, Nucleic Acids Research
15:797-811; Kunitsyn, A. et al., 1996
, Journal of Biomolecular Structure and Dynamics
14:239-244; Day, I. N. M. et al., 1995
, Nucleic Acids Research
23:2404-2412), as well as hybridization to polynucleotide probes immobilized on glass plates (Beattie, W. G. et al., 1995
, Molecular Biotechnology
4:213-225) including oligonucleotide microarrays (Stimpson, D. I. et al., 1995
, Proc. Natl. Acad. Sci. U.S.A
. 92:6379-6383).
In DNA microarray expression assays, a complex mixture of labeled soluble sequences, derived, e.g., from genes in a population of cells, is analyzed by hybridization to another complex set of sequences which are separated into individual species, each bound separately to a solid support. The amount of labeled sequence bound to each sequence on the support is used as a measure of the level of expression of the species in the cells (see, e.g., Schena et al., 1995
, Science
270:467-470; Lockhart et al., 1996
, Nature Biotechnology
14
:
1675
-
1680
; Blanchard et al., 1996
, Nature Biotechnology
14:1649; Ashby et al., U.S. Pat. No. 5,569,588).
Equilibrium binding during hybridization of nucleic acids with complementary strands is related to (a) the similarity of the hybridizing sequences, (b) the concentration of the nucleic acid sequences, (c) the temperature, and (d) the salt concentration. Accordingly, it is well known that although hybridization is very selective for matching sequences, related sequences from other genes or gene fragments which are not perfectly complementary will still hybridize at some level. For oligonucleotide probes targeted at low-abundance species, or at species with closely related (i.e., homologous) molecular family members, such “cross-hybridization” can significantly contaminate and confuse the results of hybridization to the oligonucleotide probes. For example, cross-hybridization is a particularly significant concern in the detection of single nucleotide polymorphisms (SNP's) since the sequence to be detected (i.e., the particular SNP) must be distinguished from other sequences that differ by only a single nucleotide.
To some extent, cross-hybridization can be limited by regulating the temperature and salt conditions (i.e., the “stringency”) of the hybridization or post-hybridization washing conditions. For example, “highly stringent” wash conditions may be employed so as to destabilize all but the most stable duplexes such that hybridization signals are obtained only from the sequences that hybridize most specifically, and are therefore the most homologous, to the probe. Exemplary highly stringent conditions comprise, e.g., hybridization to filter-bound DNA in 5×SSC, 1% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., eds., 1989
, Current Protocols in Molecular Biology
, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, N.Y., at p. 2.10.3). Alternatively, “moderate-” or “low-stringency” wash conditions may be used to identify sequences which are related, not just identical, to the probe, such as members of a multi-gene family, or homologous genes in a different organism. Such conditions are well known in the art (see, e.g., Sambrook et al., supra; Ausubel, F. M. et al., supra). Exemplary moderately stringent wash conditions comprise, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra). Exemplary low-stringency washing conditions include, e.g., washing in 5×SSC or in 0.2×SSC/0.1% SDS at room temperature (Ausubel et al., 1989, supra).
However, the exact wash conditions that are optimal for any given assay will depend on the exact nucleic acid sequence or sequences of interest, and, in general, must be empirically determined. There is no single hybridization or washing condition which is optimal for all nucleic acid preparations and sequences. Indeed, even the most optimized conditions can only partially distinguish between competing sequences, especially when the competing sequences are quite similar, or when some of the competing sequences are present in excess amounts or at high concentrations.
Other existing techniques to minimize cross-hybridization involve the selection and use of particular oligonucleotide probes that are most specific for a particular target nucleic acid molecule of interest. For example, multiple different oligonucleotide probes which are complementary to different, distinct sequences of a target nucleic acid may be used (see, e.g., Lockhart et al. (1996)
Nature Biotechnology
14:1675-1680; Graves et al. (1999)
Trends in Biotechnology
17:127-134). In other techniques, the oligonucleotide probe is intentionally mismatched, and its hybridization to (or dissociation from) the target nucleic acid molecule is compared to that of the perfect match oligonucleotide probe so that a cross
Burchard Julja
Friend Stephen H.
Stoughton Roland
Kim Young
Rosetta Inpharmatics LLC.
Siew Jeffrey
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