Methods for determining cross-hybridization

Details Methods for determining cross-hybridization Methods for determining cross-hybridization

1999-06-18

2001-01-09

Zitomer, Stephanie W. (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S287200, C536S023100, C536S024310

Reexamination Certificate

active

06171794

ABSTRACT:

TABLE OF CONTENTS
1. FIELD OF THE INVENTION
2. BACKGROUND
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE DRAWINGS
5. DETAILED DESCRIPTION
5.1. DETERMINING CROSS HYBRIDIZATION
5.1.1. DETERMINING HYBRIDIZATION LEVELS
5.1.2. OBTAINING DISSOCIATION PROFILES
5.1.3. COMPUTATIONAL METHODS
5.2. IMPLEMENTATION SYSTEMS AND METHODS
5.3. MEASUREMENT OF HYBRIDIZATION LEVELS
5.3.1. MICROARRAYS GENERALLY
5.3.2. PREPARING PROBES FOR MICROARRAYS
5.3.3. ATTACHING PROBES TO THE SOLID SURFACE
5.3.4. TARGET POLYNUCLEOTIDE MOLECULES
5.3.5. HYBRIDIZATION TO MICROARRAYS
6. EXAMPLES
6.1. MEASUREMENT OF DISSOCIATION CURVES
6.2. DETERMINATION OF DISSOCIATION PROFILES
6.3. FITTING A DISSOCIATION CURVE TO A PERFECT MATCH DISSOCIATION PROFILE
7. REFERENCES CITED
1. FIELD OF THE INVENTION
The field of this invention relates to methods for distinguishing the similarity of nucleic acid sequences by their hybridization properties, and particularly for distinguishing nucleic acid sequences in a sample which are completely complementary to a nucleic acid probe from sequences which are only partially complementary to the probe.
2. BACKGROUND
The ability to distinguish and compare nucleic acid molecules having varying levels of sequence similarity 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 several different nucleic acid sequences. For example, the nucleic acid sequence similarity of a pair of nucleic acid molecules can be distinguished by allowing the nucleic acid molecules to hybridize, 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).
In particular, 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 assays 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 expressed 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 that 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, depending on the temperature and salt conditions (i.e., the “stringency”) of the hybridization or post-hybridization washing conditions, detection of bound labeled nucleic acid species can be limited to sequences which are nearly identical to the labeled sequence, or can be extended to include many related sequences. For example, “highly stringent” wash conditions may be employed so as to destabilize all mismatched heteroduplexes such that hybridization signals are obtained only from sequences that are perfectly 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 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. 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 total or local concentrations.
Consequently, in assays such as those utilizing DNA microarrays, wherein both the immobilized nucleic acid probes and the labeled nucleic acid samples are complex mixtures of sequences, it is difficult to differentiate between true hybridization by identical sequences and cross-hybridization by partially related sequences. For example, the high concentration of some common sequences increases their rate of hybridization, as well as the fraction of such sequences which are bound to any other sequence. The high local concentration of probe at the surface of a microarry may also trap mismatch binding partners. Further, the overwhelming variety of possible binding partners to a given probe sequence reduces both the rate and extent of true match binding by sequences of average or less than average abundance.
In such situations, it is usually impossible to obtain hybridization conditions which limit binding to true matches for all, or even the majority of sequences. As a result, it is not possible to

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