 |
Description  |
|
|
FIELD OF THE INVENTION
The invention relates to a simple method for immobilizing synthetic nucleic
acid molecules onto a solid support. The invention further concerns the
use of such immobilized molecules in nucleic acid hybridization assays,
nucleic acid sequencing, and in the analysis of genomic polymorphisms.
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 0.1M MgCl.sub.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, J. A. L. 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 916:76-79
(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.TM. 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.
An alternative approach, the "Oligonucleotide Ligation Assay" ("OLA")
(Landegren, U. et al., Science 241:1077-1080 (1988)) has also been
described as capable of detecting single nucleotide polymorphisms. The OLA
protocol uses two oligonucleotides which are designed to be capable of
hybridizing to abutting sequences of a single strand of a target. One of
the oligonucleotides is biotinylated, and the other is detectably labeled.
If the precise complementary sequence is found in a target molecule, the
oligonucleotides will hybridize such that their termini abut, and create a
ligation substrate. Ligation then permits the labeled oligonucleotide to
be recovered using avidin, or another biotin ligand. Nickerson, D. A. et
al. have described a nucleic acid detection assay that combines attributes
of PCR and OLA (Nickerson, D. A. et al., Proc. Natl. Acad. Sci. (U.S.A.)
87:8923-8927 (1990). In this method, PCR is used to achieve the
exponential amplification of target DNA, which is then detected using OLA.
In addition to requiring multiple, and separate, processing steps, one
problem associated with such combinations is that they inherit all of the
problems associated with PCR and OLA.
Recently, several primer-guided nucleotide incorporation procedures for
assaying polymorphic sites in DNA have been described (Komher, J. S. et
al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids
Res. 18:3671 (1990); Syv anen, A. -C., et al., Genomics 8:684-692 (1990);
Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147
(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L.
et al., GATA 9:107-112 (1992); Nyr en, P. et al., Anal. Biochem.
208:171-175 (1993)). These methods differ from GBA.TM. in that they all
rely on the incorporation of labeled deoxynucleotides to discriminate
between bases at a polymorphic site. In such a format, since the signal is
proportional to the number of deoxynucleotides incorporated, polymorphisms
that occur in runs of the same nucleotide can result in signals that are
proportional to the length of the run (Syv anen, A. -C., et al., Amer. J.
Hum. Genet. 52:46-59 (1993)). Such a range of locus-specific signals could
be more complex to interpret, especially for heterozygotes, compared to
the simple, ternary (2:0, 1:1, or 0:2) class of signals produced by the
GBA.TM. method. In addition, for some loci, incorporation of an incorrect
deoxynucleotide can occur even in the presence of the correct
dideoxynucleotide (Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784
(1989)). Such deoxynucleotide misincorporation events may be due to the Km
of the DNA polymerase for the mispaired deoxy-substrate being comparable,
in some sequence contexts, to the relatively poor Km of even a correctly
base paired dideoxy-substrate (Kornberg, A., et al., In: DNA Replication,
Second Edition (1992), W. H. Freeman and Company, New York; Tabor, S. et
al., Proc. Natl. Acad. Sci. (U.S.A.) 86:4076-4080 (1989)). This effect
would contribute to the background noise in the polymorphic site
interrogation.
III. Oligonucleotide Immobilization On Plastic And Glass
Several of the above-described methods involve procedures in which one or
more of the nucleic acid reactants are immobilized to a solid support.
Currently, 96-well polystyrene plates are widely used in solid-phase
immunoassays, and several PCR product detection methods that use plates as
a solid support have been described. The most specific of these methods
require the immobilization of a suitable oligonucleotide probe into the
microtiter wells followed by the capture of the PCR product by
hybridization and colorimetric detection of a suitable hapten. It would be
desirable to have an improved immobilization method that could be used to
bind oligonucleotides to polystyrene such that their capacity to be used
for hybridization, sequencing, or polymorphic analysis would be retained,
and which would be rapid, convenient to use and inexpensive. The present
invention provides such an improved method.
The means by which macromolecules bind non-covalently to polystyrene and
glass surfaces is not well understood. Nevertheless, these adsorption
phenomena have proven to be important in the development and manufacturing
of immunoassays and other types of diagnostic tests where one component
needs to be immobilized.
Polystyrene is a very hydrophobic material because it normally contains no
hydrophilic groups. Microtiter plate manufacturers have developed methods
of introducing such groups (hydroxyl, carboxyl, carbonyl and others) onto
the surface of microwells to increase the hydrophilic nature of the
surface. Theoretically, this allows macromolecules to bind through a
combination of hydrophobic and hydrophilic interactions (Baler et al.,
Science 162:1360-1368 (1968); Baler et al., J. Biomed. Mater. Res.
18:335-355 (1984); Good et al., in L. H. Lee (ed.) Fundamentals of
Adhesion, Plenum, N.Y., chapter 4 (1989)) (FIG. 1). In practice, some
proteins do bind more efficiently to the treated hydrophilic polystyrene
than to the untreated material. Covalent binding to polystyrene,
especially microtiter wells, has proven to be difficult, so passive
adsorption remains the most commonly used method of binding macromolecules
to such wells. The term "polystyrene" may also be used to describe
styrene-containing copolymers such as: styrene/divinyl benzene,
styrene/butadiene, styrene/vinyl benzyl chloride and others.
While polystyrene is an organic hydrophobic substrate, glass provides an
inorganic hydrophobic surface with hydrophilic islands. The most common
glass format in immunoassays is the microscope slide. Laboratory-grade
glasses are predominantly composed of SiO.sub.2, but they also may contain
B.sub.2 O.sub.3, Na.sub.2 O, Al.sub.2 O.sub.3 as well as other oxides
(FIG. 2).
SUMMARY OF THE INVENTION
The present invention provides an improved immobilization method that
permits the rapid, and inexpensive immobilization of nucleic acid
molecules to a solid support. The invention is extremely simple, allowing
immobilization of oligonucleotides by incubation with a salt or a cationic
detergent. The immobilized molecules can be used for hybridization,
sequencing, or polymorphic analysis.
In detail, the invention provides a method for immobilizing a nucleic acid
molecule to a polystyrene or glass support, the method comprising the
steps:
(A) incubating the nucleic acid molecule in the presence of the solid
support; the incubation being in the presence of a reagent selected from
the group consisting of the inorganic salt sodium chloride (NaCl), the
organic salt tetramethylammonium chloride ((CH.sub.3).sub.4 NCl) (both
preferably provided at a concentration of at least about 50 mM) and a
cationic detergent (preferably provided at a concentration of 0.03 to 100
mM)]; and
(B) subsequently the washing support with an aqueous solution of a
non-ionic detergent.
The invention particularly concerns the embodiments of the above method
wherein, in step A, the cationic detergent is a water-soluble carbodiimide
(preferably EDC, provided at a concentration of from about 30 mM to about
100 mM) or wherein the cationic detergent is selected from the group
consisting of octyldimethylamine (provided at a concentration of from
about 50 mM to about 150 mM) and cetyl triethyl ammonium bromide (provided
at a concentration of from about 0.03 mM to about 0.25 mM).
The invention further concerns the embodiments of the above methods
wherein, in step B, the non-ionic detergent is Tween, preferably provided
in a solution that additionally contains buffered saline.
The invention is additionally directed to the embodiments of the above
methods wherein the nucleic acid molecule has a length of at least 12
nucleotide residues, up to 100 residues and a 3' and a 5'-terminus, and
wherein such molecule is immobilized to the support by non-covalent
interactions at the oligonucleotide's 3'-terminus, an internal region or,
at its 5'-terminus.
The invention further concerns the embodiments of the above methods wherein
the oligonucleotides are applied to the support in a specific pattern or
grid by microdeposition methods such as inkjet printing. Another
embodiment involves the immobilization of oligonucleotides to polystyrene
pins, arranged in an array matching a standard 96-well plate.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the binding of a generic macromolecule to a hydrophilic
polystyrene surface.
FIG. 2 illustrates the binding of a generic macromolecule to a typical
glass surface.
FIG. 3 is a diagram of a GBA.TM. genetic bit analysis protocol.
FIG. 4 illustrates the effect of TMAC concentration on the binding of an
oligonucleotide to polystyrene.
FIG. 5 illustrates the effect of CTAB concentration on the binding of an
oligonucleotide to polystyrene.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. The Immobilization of Nucleic Acid Molecules
The present invention concerns a method for immobilizing a synthetic
nucleic acid molecule onto a solid support. Recently, several methods have
been proposed as suitable for immobilizing an oligonucleotide to a solid
support. Holmstrom, K. et al., for example, exploit the affinity of biotin
for avidin and streptavidin, and immobilize biotinylated nucleic acid
molecules to avidin/streptavidin coated supports (Holmstrom, K. et al.,
Anal. Biochem. 209:278-283 (1993)). Another recent method requires the
precoating of the polystyrene or glass solid phases with poly-L-Lys or
poly L-Lys, Phe, followed by the covalent attachment of either amino- or
sulfhydryl-modified oligonucleotides using bifunctional crosslinking
reagents. Both methods have the disadvantage of requiring the use of
modified oligonucleotides as well as a pretreatment of the solid phase
(Running. J. A. et .al., BioTechniques 8:276-277 (1990); Newton, C. R. et
al. Nucl. Acids Res. 21:1155-1162 (1993)).
Kawai, S. et..al. describe an alternative method in which short
oligonucleotide probes were ligated together to form multimers and these
were ligated into a phagemid vector (Kawai, S. et al., Anal. Biochem.
209:63-69 (1993)). The oligonucleotides were immobilized onto polystyrene
plates and fixed by UV irradiation at 254 nm. A method for the direct
covalent attachment of short, 5'-phosphorylated primers to chemically
modified polystyrene plates ("Covalink" plates, Nunc) has also been
proposed by Rasmussen, S. R. et al., (Anal. Biochem. 196:138-142 (1991)).
The covalent bond between the modified oligonucleotide and the solid phase
surface is introduced by condensation with a water-soluble carbodiimide.
This method is claimed to assure a predominantly 5'-attachment of the
oligonucleotides via their 5'-phosphates; however, it requires the use of
specially prepared, expensive plates.
The methods of the present invention depart from such methods, in not
requiring either the presence of specially modified nucleotides in the
molecule to be immobilized, or the use of expensively modified supports.
Any nucleic acid molecule (RNA or DNA) may be immobilized to such supports
using the methods of the present invention. The nucleic acid molecules may
ideally be 12-100 nucleotides long, and may be immobilized to the support
at either their 3'-terminus, their 5'-terminus, or at an internal (i.e.
non-terminal) region.
A nucleic acid molecule is said to be "immobilized" to a solid support if
it is either adsorbed to the support, or bonded thereto, with sufficient
strength that it cannot be removed from the support by washing with water
or an aqueous buffer. Knowledge of the precise chemical mechanism through
which such immobilization occurs is not needed for use of the present
invention.
Although any of a variety of glass or plastic solid supports can be used in
accordance with the methods of the present invention, polystyrene is the
preferred support. The support can be fashioned as a bead, dipstick, test
tube, etc. The conventional 96-well polystyrene microtiter dishes used in
diagnostic laboratories and in tissue culture are, however, an especially
preferred support. Any of a number of commercially available polystyrene
plates can be used directly for the immobilization, provided that they
have hydrophilic groups on the plastic surface. Examples of suitable
plates include the Immulon 4 plates (Dynatech) and the Maxisorp plates
(Nunc). Methods for synthesizing polystyrene are known in the art; such
methods are disclosed in, for example, treatises on plastics and polymers
such as Byrdson, J. A., Plastics Materials, Fifth Edition, Butterworth
Heinemann, London (1991), herein incorporated by reference.
Remarkably, in the method of the present invention, unmodified
oligonucleotides can be efficiently immobilized onto the surface of a
hydrophilic polystyrene plate simply by incubation in the presence of one
of two different groups of reagents that can be characterized as either
salts or cationic detergents. A hydrophilic polystyrene plate is defined
as one treated by the manufacturer or user to increase the number of
hydrophilic groups (i.e., --OH, --C.dbd.O, --COOH) on the surface of the
plastic. No immobilization takes place in the absence of a salt or
cationic detergent, i.e., when the oligonucleotide is present in a
salt-free or cationic detergent-free water solution.
The first group consists of chemicals like NaCl and (CH.sub.3).sub.4 NCl,
which work best when used at relatively high concentrations, generally
higher than 50 mM, and best at 250 to 500 mM. Even concentrations as high
as 1M can be used without any noticeable adverse effect on the
immobilization. The second group of immobilization reagents consists of
chemicals that are characterized by the presence of two structural
features: a positively charged "head" and a relatively hydrophobic "tail".
These are the typical features of cationic detergents. Representatives of
this group include the cationic detergent cetyltrimethyl ammonium bromide
(CTAB), octyldimethylamine hydrochloride, and
1-ethyl-3-(3'-dimethylaminopropyl)-1,3-carbodiimide hydrochloride (EDC).
These compounds can be used for oligonucleotide immobilization at very low
concentrations, as low as 0.03 mM for CTAB, but they inhibit the
immobilization when used at higher concentrations. The inhibitory
concentration differs between the reagents of this group. For CTAB, it is
as low as 0.5 mM, whereas for EDC it is about 500 mM. It should be noted
that the critical micelle concentration, cmc, for CTAB is about 1 mM.
Thus, it seems that once micelles are formed, the immobilization is
inhibited. Compounds of a similar structure, but with a negatively charged
"head" (or nonionic detergents) are completely inactive as oligonucleotide
immobilization reagents. A typical representative here is the anionic
detergent SDS (sodium dodecyl sulfate), which was found inactive over a
very large range of different concentrations (0.025 mM to 100 mM).
It is reasonable to assume that the two groups of reagents mentioned above
promote the immobilization of oligonucleotides to polystyrene plates by
different mechanisms. In the presence of NaCl and other salts, the
hydrophobic interactions between the oligonucleotide molecule and
hydrophobic regions at the polystyrene surface are enhanced to a degree
that allows the immobilization of the former. The presence of a salt
(increased ionic strength of the solution) decreases electrostatic
repulsion between the phosphates of the oligonucleotide backbone and
negatively charged groups on the polystyrene surface. This reduction
should enhance the hydrophobic binding of the oligonucleotide molecules.
The mechanism of binding in the presence of cationic detergents is probably
quite different. Here, initially there is association in solution between
the negatively charged oligonucleotides and the positively charged
detergent-like molecules. The number of detergent molecules that associate
with each oligonucleotide molecule will be dependent on the
oligonucleotide length, but should be significantly higher than one in the
case of a 25 mer oligonucleotide. This association of oligonucleotides
with detergents containing a hydrophobic tail will render the
oligonucleotide significantly hydrophobic and will lead to its
immobilization to the plate surface by hydrophobic interactions. In effect
these molecules appear to act as a linker between the hydrophobic areas of
the plate and the charged phosphate backbone of the oligonucleotide.
If the concentration of the detergent molecules is higher than their cmc,
micelles will be formed. Although oligonucleotides might still interact
with the detergent molecules, they will be included in the micelles, and
since the micelles have a hydrophobic core that is completely surrounded
by a polar surface, no hydrophobic interactions with the surface will
occur and therefore oligonucleotide immobilization will be diminished or
prevented. The different inhibitory concentrations observed for the
different reagents reflect the widely different concentrations at which
these reagents will form micelles. For CTAB, a very good detergent, the
cmc is very low, whereas EDC and octyldimethylamine hydrochloride, which
are very poor detergents, form micelles and inhibit the immobilization
only at relatively high concentrations.
An example of a suitable salt is sodium chloride (NaCl). When it is desired
to employ NaCl, concentrations of from about 50 mM to about 250 mM may be
used; a concentration of at least 50 mM is desirable in order to achieve
optimal immobilization (hydrophobic interactions are stronger at higher
salt concentrations). Examples of suitable cationic detergents are
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride, pH about 6.8
("EDC") and tertiary alkyl amines such as cetyl trimethyl ammonium bromide
and octyl dimethyl amine HCl. EDC may be employed at concentrations (in
water) of from about 30 mM to about 100 mM. Varga, J. M. et al. have shown
that various small biomolecules can be immobilized to polystyrene plates
by incubation with 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide
hydrochloride ("EDC") (Varga, J. M. et al., FASEB 4:2671-2677 (1990)).
However, no examples of oligonucleotide immobilization were provided, and
it is generally believed that short, single-stranded oligonucleotide
molecules are immobilized only very inefficiently to polystyrene supports.
Lacy et al. (J. Immunl. Methods 116:87-98 (1989)) have reported the
binding of calf thymus DNA to polystyrene in the presence of high salt and
high pH, but presented evidence that a synthetic oligonucleotide did not
bind.
A preferred tertiary alkyl amine is octyldimethylamine, which may be used
at concentrations of from about 50 mM to about 150 mM. Octyldimethylamine
has a structure that is very similar to that of EDC, however,
octyldimethylamine does not contain the reactive diimide functional group
of EDC. This demonstrates that EDC does not mediate covalent binding to
polystyrene, but indeed acts as a detergent (hydrophilic/hydrophobic
molecule). Tetramethylammonium chloride may also be used, preferably at a
concentration of from about 50 mM to about 250 mM.
The immobilization is achieved by incubation, preferably at room
temperature for 3 to 24 hours. After such incubation, the plates are
washed, preferably with a solution of 10 mM Tris HCl, pH 7.5, containing
150 mM NaCl and 0.05% vol. Tween 20 (TNTw). The latter ingredient serves
the important role of blocking all free oligonucleotide binding sites
still present on the polystyrene surface, so that no non-specific binding
of oligonucleotides can take place during any subsequent hybridization
step. The above procedure could immobilize at least 500 fmoles of
oligonucleotide per well (corresponding to a surface area of about 1
cm.sup.2). The oligonucleotides are immobilized to the surface of the
plate with sufficient stability and can only be removed by prolonged
incubations with 0.5M NaOH solutions at elevated temperatures. No
oligonucleotide is removed by washing the plate with water, TNTw (Tween
20), PBS, 1.5M NaCl, or other similar aqueous buffers.
Such procedures and reagents can effectively immobilize unmodified
oligonucleotides as well as modified (for example, biotinylated)
oligonucleotides. The immobilization mediated by these reagents is not
believed to reflect covalent bonding between the nucleic acid, and
reactive groups of the support. Without limitation to the scope of the
present invention, the immobilization is believed to be non-covalent, and
to reflect a combination of hydrophobic, ionic and hydrogen bonding
interactions to the polystyrene surface of the support.
Whatever the exact mechanism of immobilization, the reagents of the present
invention are capable of mediating an attachment of oligonucleotides to a
solid support that has sufficient stability to resist washing, and to
sustain a one hour treatment with 0.1N NaOH. Moreover, the immobilized
oligonucleotides can efficiently participate in hybridization reactions.
Although shorter oligonucleotides can also be immobilized, a length of at
least 12 bases was found to be required in order to be able to efficiently
hybridize to complementary DNA molecules. This observation suggests that
the process of immobilization of the oligonucleotide to the solid support
renders short portions of the immobilized molecules inaccessible to
hybridization.
In accordance with the present invention, the immobilization reagent is
incubated in the presence of the oligonucleotide that is to be immobilized
and the solid support. Suitable incubations may vary in duration, and
preferably will be maintained overnight. Incubation may be performed at
room temperature.
The nucleic acid molecules that are to be immobilized on the solid support
can be synthesized chemically, or can be recovered from a natural source.
Alternatively, such molecules can be produced via an in vitro
amplification protocol, such as PCR. Short oligonucleotides are more
preferably obtained via chemical synthesis.
II. The Use of Immobilized Oligonucleotides in Genetic Analysis
The methods of the present invention are particularly useful in producing
immobilized oligonucleotides for solid phase hybridization, for solid
phase dideoxy sequencing studies, and for analysis of DNA polymorphisms.
A. Hybridization Detection Of PCR Products
Thus, for example, they may be used to detect specific PCR products by
hybridization where the capture probe is immobilized on the solid phase
(Ranki et al., Gene 21: 77-85, 1983; Keller et al., J. Clin. Microbiol.
29: 638-641, 1991; Urdea et al., Gene 61: 253-264, 1987). A preferred
method would be to prepare a single-stranded PCR product before
hybridization. A sample, suspected to contain the target molecule, or an
amplification product thereof, would then be added to the well and
permitted to hybridize to the bound oligonucleotide.
The methods of the present invention do not require that the target nucleic
acid contain only one of its natural two strands. Thus, the methods of the
present invention may be practiced on either double-stranded DNA, or on
single-stranded DNA obtained by, for example, alkali treatment of native
DNA. The presence of the unused (non-template) strand does not affect the
reaction.
Where desired, however, any of a variety of methods can be used to
eliminate one of the two natural stands of the target DNA molecule from
the reaction. Single-stranded DNA molecules may be produced using the
single-stranded DNA bacteriophage M13 (Messing, J. et al., Meth. Enzymol.
101:20 (1983); see also, Sambrook, J., et al. (In: Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989)).
Several alternative methods can be used to generate single-stranded DNA
molecules. Gyllensten, U. et al., (Proc. Natl. Acad. Sci. (U.S.A.)
85:7652-7656 (1988) and Mihovilovic, M. et al., (BioTechniques 7:14
(1989)) describe a method, termed "asymmetric PCR," in which the standard
"PCR" method is conducted using primers that are present in different
molar concentrations. Higuchi, R. G. et al. (Nucleic Acids Res. 17:5865
(1985)) exemplifies an additional method for generating single-stranded
amplification products. The method entails phosphorylating the 5'-terminus
of one strand of a double-stranded amplification product, and then
permitting a 5' 3' exonuclease (such as exonuclease) to preferentially
degrade the phosphorylated strand.
Other methods have also exploited the nuclease resistant properties of
phosphorothioate derivatives in order to generate single-stranded DNA
molecules (Benkovic et al., U.S. Pat. No. 4,521,509; Jun. 4, 1985);
Sayers, J. R. et al. (Nucl. Acids Res. 16:791-802 (1988); Eckstein, F. et
al., Biochemistry 15:1685-1691 (1976); Ott, J. et al., Biochemistry
26:8237-8241 (1987)).
Most preferably, such single-stranded molecules will be produced using the
methods described by Nikiforov, T. (commonly assigned U.S. patent
application Ser. No. 08/155,746, herein incorporated by reference). In
brief, these methods employ nuclease resistant nucleotide derivatives, and
incorporate such derivatives, by chemical synthesis or enzymatic means,
into primer molecules, or their extension products, in place of naturally
occurring nucleotides.
Suitable nucleotide derivatives include derivatives in which one or two of
the non-bridging oxygens of the phosphate moiety of a nucleotide has been
replaced with a sulfur-containing group (especially a phosphorothioate),
an alkyl group (especially a methyl or ethyl alkyl group), a
nitrogen-containing group (especially an amine), and/or a
selenium-containing group, etc. Phosphorothioate deoxyribonucleotide or
ribonucleotide derivatives are the most preferred nucleotide derivatives.
Methods of producing and using such phosphorothioate derivatives are
disclosed by Nikiforov, T. (U.S. patent application Ser. No. 08/155,746).
B. Solid Phase DNA Sequencing
The methods of the present invention may also be used in the practice of
solid-phase sequencing as described by Khrapko, K. R. et al. (DNA Seq.: 1,
375-388, 1991) and Drmanac, R. and Crkvenjakov, R.,Int. J. Genome RES.: 1,
1-1, 1992), both herein incorporated by reference.
C. GBA.TM. Genetic Bit Analysis
The methods of the present invention may also be used to immobilize the
oligonucleotides that are used in the GBA.TM. Genetic Bit Analysis
(Goelet, P. et al., PCT Appln. No. 92/15712). Oligonucleotides having a
defined sequence complementary to a region that lies immediately proximal
or distal to the variable nucleotide of a polymorphism would thus be
provided to a polystyrene microtiter well, and incubated with a salt, in
accordance with the above-described methods.
The immobilized primer is then incubated in the presence of a DNA molecule
(preferably a genomic DNA molecule) having a single nucleotide
polymorphism whose immediately 3'-distal sequence is complementary to that
of the immobilized primer. Preferably, such incubation occurs in the
complete absence of any dNTP (i.e. dATP, dCTP, dGTP, or dTTP), but only in
the presence of one or more chain terminating nucleotide triphosphate
derivatives (such as a dideoxy derivative), and under conditions
sufficient to permit the incorporation of such a derivative on to the
3'-terminus of the primer. As will be appreciated, where the polymorphic
site is such that only two or three alleles exist (such that only two or
three species of ddNTPs, respectively, could be incorporated into the
primer extension product), the presence of unusable nucleotide
triphosphate(s) in the reaction is immaterial. In consequence of the
incubation, and the use of only chain terminating nucleotide derivatives,
a single dideoxynucleotide is added to the 3'-terminus of the primer. The
identity of that added nucleotide is determined by, and is complementary
to, the nucleotide of the polymorphic site of the polymorphism.
In this embodiment, the nucleotide of the polymorphic site is thus
determined by assaying which of the set of labeled nucleotides has been
incorporated onto the 3'-terminus of the bound oligonucleotide by a
primer-dependent polymerase. Most preferably, where multiple
dideoxynucleotide derivatives are simultaneously employed, different
labels will be used to permit the differential determination of the
identity of the incorporated dideoxynucleotide derivative.
D. Ligase-Mediated GBA.TM.
The methods and reagents of the present invention can also be used in
concert with a polymerase/ligase mediated polymorphic interrogation assay.
This assay, termed ligase-mediated GBA.TM. genetic bit analysis, is a more
specific version of the GBA.TM. genetic bit ana | | |
