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FIELD OF THE INVENTION
The present invention is in the field of recombinant DNA technology. More
specifically, the invention is directed to a ligase/polymerase-mediated
method for determining the identity of the nucleotide that is present at a
particular site, such as a single nucleotide polymorphic site, in the
genome of an animal. The invention further concerns the use of such
determinations to analyze entity, ancestry or genetic traits.
BACKGROUND OF THE INVENTION
I. The Determination of the Nucleotide Present at a Polymorphic Site
The genomes of viruses, bacteria, plants and animals naturally undergo
spontaneous mutation in the course of their continuing evolution (Gusella,
J. F., Ann. Rev. Biochem. 55:831-854 (1986)). Since such mutations are not
immediately transmitted throughout all of the members of a species, the
evolutionary process creates polymorphic alleles that co-exist in the
species populations. In some instances, such co-existence is in stable or
quasi-stable equilibrium. In other instances, the mutation confers a
survival or evolutionary advantage to the species, and accordingly, it may
eventually (i.e. over evolutionary time) be incorporated into the DNA of
every member of that species.
Several classes of polymorphisms have been identified. Variable nucleotide
type polymorphisms ("VNTRs"), for example arise from spontaneous tandem
duplications of di- or trinucleotide 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.,
European Patent Application 370,719; 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)). If such a variation alters the lengths of the
fragments that are generated by restriction endonuclease cleavage, the
variations are referred to as restriction fragment length polymorphisms
("RFLPs"). RFLPs have been widely used in human and animal genetic
analyses (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)).
Most polymorphisms arise from the replacement of only a single nucleotide
from the initially present gene sequence. In rare cases, such a
substitution can create or destroy a particular restriction site, and thus
may comprise an RFLP polymorphism. In many cases, however, the
substitution of a nucleotide in such single nucleotide polymorphisms
cannot be determined by restriction fragment analysis. In some cases, such
polymorphisms comprise mutations that are the determinative characteristic
in a genetic disease. Indeed, such mutations may affect a single
nucleotide in a protein-encoding gene in a manner sufficient to actually
cause the disease (i.e., hemophilia, sickle-cell anemia, etc.). Despite
the central importance of such polymorphisms in modern genetics, few
methods have been developed that could permit the comparison of the
alleles of two individuals at many such polymorphisms in parallel.
II. The Attributes of the Single Nucleotide Polymorphisms of the Present
Invention and the Advantages of Their Use in Genetic Analysis
A "polymorphism" is a variation in the DNA sequence of some members of a
species. A polymorphism is thus said to be "allelic," in that, due to the
existence of the polymorphism, some members of a species may have the
unmutated sequence (i.e. the original "allele") whereas other members may
have a mutated sequence (i.e. the variant or mutant "allele"). In the
simplest case, only one mutated sequence may exist, and the polymorphism
is said to be diallelic. In the case of diallelic diploid organisms, three
genotypes are possible. They can be homozygous for one allele, homozygous
for the other allele or heterozygous. In the case of diallelic haploid
organisms, they can have one allele or the other, thus only two genotypes
are possible. Diallelic polymorphisms are the preferred polymorphisms of
the present invention. The occurrence of alternative mutations can give
rise to trialleleic, etc. polymorphisms. An allele may be referred to by
the nucleotide(s) that comprise the mutation. The present invention is
directed to a particular class of allelic polymorphisms, and to their use
in genotyping a plant or animal. Such allelic polymorphisms are referred
to herein as "single nucleotide polymorphisms," or "SNPs." "Single
nucleotide polymorphism" are defined by their characteristic attributes. A
central attribute of such a polymorphism is that it contains a polymorphic
site, "X," most preferably occupied by a single nucleotide, which is the
site of the polymorphism's variation (Goelet, P. and Knapp, M., U.S.
patent application Ser. No. 08/145,145, herein incorporated by reference).
SNPs have several salient advantages over RFLPs and VNTRs. First, SNPs are
more stable than other classes of polymorphisms. Their spontaneous
mutation rate is approximately 10.sup.-9 (Kornberg, A., DNA Replication,
W. H. Freeman & Co., San Francisco, 1980), approximately 1,000 times less
frequent than VNTRs. Significantly, VNTR-type polymorphisms are
characterized by high mutation rates.
Second, SNPs occur at greater frequency, and with greater uniformity than
RFLPs and VNTRs. The characterization of VNTRs and RFLPs is highly
dependent upon the method used to detect the polymorphism. In contrast,
because SNPs result from sequence variation, new polymorphisms can be
identified by sequencing random genomic or cDNA molecules. VNTRs and RFLPs
can also be considered a subset of SNPs because variation in the region of
a VNTR or RFLP can result in a single-base change in the region. SNPs can
also result from deletions, point mutations and insertions. Any single
base alteration, whatever the cause, can be a SNP. The greater frequency
of SNPs means that they can be more readily identified than the other
classes of polymorphisms. The greater uniformity of their distribution
permits the identification of SNPs "nearer" to a particular trait of
interest. The combined effect of these two attributes makes SNPs extremely
valuable. For example, if a particular trait (e.g. predisposition to
cancer) reflects a mutation at a particular locus, then any polymorphism
that is linked to the particular locus can be used to predict the
probability that an individual will be exhibit that trait.
SNPs can be characterized using any of a variety of methods. Such methods
include the direct or indirect sequencing of the site, the use of
restriction enzymes where the respective alleles of the site create or
destroy a restriction site, the use of allele-specific hybridization
probes, the use of antibodies that are specific for the proteins encoded
by the different alleles of the polymorphism, or by other biochemical
interpretation. However, no assay yet exists that is both highly accurate
and easy to perform.
III. Methods of Analyzing Polymorphic Sites
A. DNA Sequencing
The most obvious method of characterizing a polymorphism entails direct DNA
sequencing of the genetic locus that flanks and includes the polymorphism.
Such analysis can be accomplished using either the "dideoxy-mediated chain
termination method," also known as the "Sanger Method" (Sanger, F., et
al., J. Molec. Biol. 94:441 (1975)) or the "chemical degradation method,"
"also known as the "Maxam-Gilbert method" (Maxam, A. M., et al., Proc.
Natl. Acad. Sci. (U.S.A.) 74:560 (1977)). In combination with genomic
sequence-specific amplification technologies, such as the polymerase chain
reaction (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol.
51:263-273 (1986); Erlich H. et al., European Patent Appln. 50,424;
European Patent Appln. 84,796, European Patent Application 258,017,
European Patent Appln. 237,362; Mullis, K., European Patent Appln.
201,184; Mullis, K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat.
No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194)), may be
employed to facilitate the recovery of the desired polynucleotides, direct
sequencing methods are technically demanding, relatively expensive, and
have low throughput rates. As a result, there has been a demand for
techniques that simplify repeated and parallel analysis of SNPs.
B. Exonuclease Resistance
Mundy, C. R. (U.S. Pat. No. 4,656,127) discusses alternative methods for
determining the identity of the nucleotide present at a particular
polymorphic site. Mundy's methods employ 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 exonucleotide-resistant nucleotide
derivative present, then that derivative will be incorporated by a
polymerase 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 exonucleotide-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.
C. Microsequencing Methods
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); Syvanen, 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); Ugozzoll, L.
et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.
208:171-175 (1993)). These methods differ from Genetic Bit.TM. Analysis
("GBA.TM." discussed extensively below) 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 (Syvanen, 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.
D. Extension in Solution Using ddNTPs
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.
The method of Cohen has the significant disadvantage of being a
solution-based extension method that uses labeled dideoxynucleoside
triphosphates. The target DNA template is usually prepared by a DNA
amplification reaction, such as the PCR, that uses a high concentration of
deoxynucleoside triphosphates, the natural substrates of DNA polymerases.
These monomers will compete in the subsequent extension reaction with the
dideoxynucleoside triphosphates. Therefore, following the PCR, an
additional purification step is required to separate the DNA template from
the unincorporated dNTPs. Because it is a solution-based method, the
unincorporated dNTPs are difficult to remove and the method is not suited
for high volume testing.
E. Solid-Phase Extension Using ddNTPs
An alternative method, known as Genetic Bit Analysis.TM. or GBA.TM. is
described by Goelet, P. et al. (PCT Appln. No. 92/15712). In a preferred
embodiment, 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.
F. Oligonucleotide Ligation Assay
Another solid phase method that uses different enzymology is the
"Oligonucleotide Ligation Assay" ("OLA") (Landegren, U. et al., Science
241:1077-1080 (1988). 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. OLA is capable of detecting point mutations. 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.
Assays, such as the OLA, require that each candidate dNTP of a
polymorphism be separately examined, using a separate set of
oligonucleotides for each dNTP. The major drawback of OLA is that ligation
is not a highly discriminating process and non-specific signals can be a
significant problem.
IV. Conclusions
As will be appreciated, most of the above-described methods require a
polymerase to incorporate a nucleotide derivative onto the 3'-terminus of
a primer molecule. It would be desirable to develop a more selective
process for discriminating single nucleotide polymorphisms. The present
invention satisfies this need by providing a ligase/polymerase-mediated
method of determining the identity of the nucleotide present at a
polymorphic site. The addition of a ligase to the process means that two
events are required to generate a signal, extension and ligation. This
grants the present invention a higher specificity and lower "noise" than
methods using either extension or ligation alone. Unlike the
oligonucleotide ligation assay, in the present invention, the
distinguishing step of extension is mediated by polymerase and polymerases
are more specific in their activity than ligases. Unlike the
polymerase-based assays, this method enhances the specificity of the
polymerase step by combining it with a second hybridization and a ligation
step for a signal to be attached to the solid phase.
SUMMARY OF THE INVENTION
The present invention is directed to a ligase/polymerase-mediated method
for determining the identity of the nucleotide present in a polymorphic
site of an organism (either a microorganism, plant, a non-human animal, or
a human). The invention is further directed to methods of using such
information in genetic analysis.
In detail, the invention provides a method for determining the identity of
a nucleotide present at a preselected single nucleotide site in a target
nucleic acid molecule, the method comprising the steps:
A) immobilizing a first oligonucleotide (either linker or primer) to a
solid support; the first oligonucleotide having a nucleotide sequence
complementary to that of the target molecule, and being capable of
hybridizing to the target molecule at a first region of the target
molecule such that one terminus of the hybridized first oligonucleotide is
immediately adjacent to the preselected site;
B) incubating the immobilized first oligonucleotide in the presence of the
target molecule, and in the further presence of a second oligonucleotide
(either linker or primer, the order of addition of the oligonucleotides
being immaterial; the second oligonucleotide having a sequence
complementary to that of the target molecule, and being capable of
hybridizing to the target molecule at a second region of the target
molecule, wherein the first and second regions are separated from one
another by the preselected site; the incubation being under conditions
sufficient to permit the first and second oligonucleotides to hybridize to
the target molecule to thereby form a hybridized product in which the
oligonucleotides are separated from one another by a space of a single
nucleotide, the space being opposite to the preselected site;
C) further incubating the hybridized product, in the presence of a
polymerase, a ligase, and a nucleoside triphosphate mixture containing at
least one deoxynucleoside triphosphate; the incubation being under
conditions sufficient to permit the template-dependent, polymerase
mediated, incorporation of the nucleoside triphosphate onto a 3'-terminus
of either of the immobilized first or second hybridized oligonucleotides,
and thereby fill the space between these hybridized oligonucleotides, and
cause the oligonucleotides to abut; the incorporation being dependent upon
whether the nucleoside triphosphate mixture contains a nucleoside
triphosphate that is complementary to the nucleotide present at the
preselected site;
D) permitting the ligase to ligate together any pair of abutting first and
second hybridized oligonucleotides;
E) further incubating the immobilized first oligonucleotide under
conditions sufficient to separate any non-covalently bonded target or
second oligonucleotide therefrom; and
F) determining the identity of the nucleotide of the preselected site by
determining whether the second oligonucleotide or one of the nucleoside
triphosphates has become immobilized to the solid support.
The invention further includes the embodiments of the above method wherein
the first and second oligonucleotides and the target molecule are DNA
molecules, RNA molecules, peptide nucleic acids and other modified DNA
molecules.
The invention also encompasses the embodiments of the above methods wherein
in step A, the 3'-terminus of the first oligonucleotide (the "linker") is
immobilized to the solid support, and wherein in step C, the conditions
permit the incorporation of the nucleoside triphosphate onto the
3'-terminus of the second hybridized oligonucleotide (the "primer") or
wherein in step A, the 5'-terminus of the first oligonucleotide is
immobilized to the solid support, and wherein in step C, the conditions
permit the incorporation of the nucleoside triphosphate onto the
3'-terminus of the first hybridized oligonucleotide (primer). Following
incorporation, the primer and linker oligonucleotides are ligated together
and the identity of the polymorphic nucleotide is determined from the
signal associated with the solid phase.
The invention additionally concerns the embodiment of the above methods
wherein one of the nucleoside triphosphates is detectably labeled (as with
a hapten, an enzyme label, a fluorescent label, a radioisotopic label, or
a chemiluminescent label).
The invention particularly concerns the embodiments of the above methods
wherein in step C, the nucleoside triphosphate mixture contains one or
more detectably labeled nucleoside triphosphate(s), the other unlabeled
nucleoside triphosphates being either deoxynucleoside triphosphates or
dideoxynucleoside triphosphates, and wherein in step F, the identity of
the nucleotide of the preselected site is determined by detecting the
label of the immobilized labeled deoxy- or dideoxynucleoside triphosphate.
The invention also concerns the embodiment of the above methods wherein the
second oligonucleotide is detectably labeled. Wherein in step C, the
nucleoside triphosphate mixture contains only one nucleoside triphosphate,
the nucleoside triphosphate being a deoxynucleoside triphosphate with or
without the other three dideoxynucleotide triphosphates, and wherein in
step F, the identity of the nucleotide of the preselected site is
determined by detecting the label of the immobilized labeled second
oligonucleotide.
In another embodiment, steps A-D may be performed in solution and the
ligated oligonucleotides captured onto a solid phase for detection.
In yet another embodiment, steps A-D may be performed in solution and
detection of the ligated oligonucleotides performed in solution.
The invention includes the use of the above-described methods to analyze a
polymorphism of any diploid organism including an animal selected from the
group consisting of a horse, a sheep, a bovine, a canine, a feline, a
plant and a human, as well as haploid organisms including bacteria, fungi
and viruses.
DESCRIPTION OF THE FIGURES
FIG. 1 is a diagram of a Ligase-Mediated GBA.TM. procedure using a labeled
dNTP. In (1), a 5' phosphorylated linker oligonucleotide is bound to the
surface of a microwell. In (2), template DNA is allowed to hybridize to
the linker. In (3), a primer oligonucleotide hybridizes to the immobilized
template. In (4), in the presence of DNA polymerase, ligase, a labeled
dNTP and unlabeled dNTP(s), a labeled dNTP is incorporated and the linker
and primer are ligated. In (5) The well is washed with alkali to remove
all unligated DNA. In (6), The labeled base is detected using an enzyme
conjugated antibody and substrate.
FIG. 2 is a diagram of a Ligase-Mediated GBA.TM. procedure using a labeled
primer. In (1), a 5' phosphorylated linker oligonucleotide is bound to the
surface of a microwell by its 3' end. In (2), template DNA is allowed to
hybridize to the linker. In (3), a biotinylated primer oligonucleotide is
allowed to hybridize to the immobilized linker. In (4), in the presence of
DNA polymerase, ligase, a labeled dNTP and three unlabeled ddNTPs, the
dNTP is incorporated and the linker and primer are ligated. In (5) the
well is washed with alkali to remove all unligated DNA. In (6), the
labeled base is detected using an enzyme conjugated antibody and
substrate.
FIG. 3 is a diagram of a Ligase-Mediated GBA.TM. procedure using a labeled
linker. In (1), a primer oligonucleotide is bound to the surface of a
microwell by its 5' end. In (2), template DNA is allowed to hybridize to
the linker. In (3), a 5' phosphorylated 3' biotinylated linker
oligonucleotide hybridizes to the immobilized template. In (4), in the
presence of DNA polymerase, ligase, a labeled dNTP and three ddNTPs, the
dNTP is incorporated and the linker and primer are ligated. In (5) the
well is washed with alkali to remove all unligated DNA. In (6), the
labeled base is detected using an enzyme conjugated antibody and
substrate.
FIG. 4 is a diagram of a Ligase-Mediated GBA.TM. procedure in solution. In
(1), a 5' phosphorylated, 3' fluoresceinated linker oligonucleotide is
incubated with template DNA and a primer oligonucleotide. In (2), the
three DNA molecules are allowed to hybridize in solution. In (3), in the
presence of DNA polymerase, ligase, a labeled dNTP and unlabelled dNTP(s),
a labeled dNTP is incorporated and the linker and primer are ligated. In
(4) the ligated oligonucleotides are captured onto a solid phase and the
well is washed to remove unligated DNA. In (5), the labeled base is
detected using an enzyme conjugated antibody and substrate.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. The Ligase/Polymerase-Mediated Assay of the Present Invention
A. Sample Preparation
Nucleic acid specimens may be obtained from an individual of the species
that is to be analyzed using either "invasive" or "non-invasive" sampling
means. A sampling means is said to be "invasive" if it involves the
collection of nucleic acids from within the skin or organs of an animal
(including, especially, a murine, a human, an ovine, an equine, a bovine,
a porcine, a canine, or a feline animal). Examples of invasive methods
include blood collection, semen collection, needle biopsy, pleural
aspiration, etc. Examples of such methods are discussed by Kim, C. H. et
al. (J. Virol. 66:3879-3882 (1992)); Biswas, B. et al. (Annals NY Acad.
Sci. 590:582-583 (1990)); Biswas, B. et al. (J. Clin. Microbiol.
29:2228-2233 (1991)).
In contrast, a "non-invasive" sampling means is one in which the nucleic
acid molecules are recovered from an internal or external surface of the
animal. Examples of such "non-invasive" sampling means include "swabbing,"
collection of tears, saliva, urine, fecal material, sweat or perspiration,
etc. As used herein, "swabbing" denotes contacting an applicator/collector
("swab") containing or comprising an adsorbent material to a surface in a
manner sufficient to collect surface debris and/or dead or sloughed off
cells or cellular debris. Such collection may be accomplished by swabbing
nasal, oral, rectal, vaginal or aural orifices, by contacting the skin or
tear ducts, by collecting hair follicles, etc.
B. Amplification of Target Sequences
The detection of polymorphic sites in a sample of DNA may be facilitated
through the use of DNA amplification methods. Such methods specifically
increase the concentration of sequences that span the polymorphic site, or
include that site and sequences located either distal or proximal to it.
Such amplified molecules can be readily detected by gel electrophoresis or
other means.
The most preferred method of achieving such amplification employs PCR,
using primer pairs that are capable of hybridizing to the proximal
sequences that define a polymorphism in its double-stranded form.
C. Preparation of Single-Stranded DNA
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 single-stranded DNA obtained
by, for example, alkali treatment or native DNA. The presence of the
unused (non-template) strand does not affect the reaction.
Where desired, 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(1):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'.fwdarw.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. (U.S. patent application Ser. No.
08/005,061, 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, for example. Phosphorothioate
deoxyribonucleotide or ribonucleotide derivatives (e.g. a nucleoside
5'-O-1-thiotriphosphate) are the most preferred nucleotide derivatives.
Any of a variety of chemical methods may be used to produce such
phosphorothioate derivatives (see, for example, Zon, G. et al., Anti-Canc.
Drug Des. 6:539-568 (1991); Kim, S. G. et al., Biochem. Biophys. Res.
Commun. 179:1614-1619 (1991); Vu, H. et al., Tetrahedron Lett.
32:3005-3008 (1991); Taylor, J. W. et al., Nucl. Acids Res. 13:8749-8764
(1985); Eckstein, F. et al., Biochemistry 15:1685-1691 (1976); Ott, J. et
al., Biochemistry 26:8237-8241 (1987); Ludwig, J. et al., J. Org. Chem.
54:631-635 (1989), all herein incorporated by reference).
Importantly, the selected nucleotide derivative must be suitable for in
vitro primer-mediated extension and provide nuclease resistance to the
region of the nucleic acid molecule in which it is incorporated. In the
most preferred embodiment, it must confer resistance to exonucleases that
attack double-stranded DNA from the 5'-end (5'.fwdarw.3' exonucleases).
Examples of such exonucleases include bacteriophage T7 gene 6 exonuclease
("T7 exonuclease") and the bacteriophage lambda exonuclease
("exonuclease"). Both T7 exonuclease and exonuclease are inhibited to a
significant degree by the presence of phosphorothioate bonds so as to
allow the selective degradation of one of the strands. However, any
double-strand specific, 5'.fwdarw.3' exonuclease can be used for this
process, provided that its activity is affected by the presence of the
bonds of the nuclease resistant nucleotide derivatives. The preferred
enzyme when using phosphorothioate derivatives is the T7 gene 6
exonuclease, which shows maximal enzymatic activity in the same buffer
used for many DNA dependent polymerase buffers including Taq polymerase.
The 5'.fwdarw.3' exonuclease resistant properties of phosphorothioate
derivative-containing DNA molecules are discussed, for example, in Kunkel,
T. A. (In: Nucleic Acids and Molecular Biology, Vol. 2, 124-135 (Eckstein,
F. et al., eds.), Springer-Verlag, Berlin, (1988)). The
3'.fwdarw.5'-exonuclease resistant properties of phosphorothioate
nucleotide containing nucleic acid molecules are disclosed in Putney, S.
D., et al. (Proc. Natl. Acad. Sci. (U.S.A.) 78:7350-7354 (1981)) and
Gupta, A. P., et al. (Nucl. Acids. Res., 12:5897-5911 (1984)).
D. Methods of Immobilization
Any of a variety of methods can be used to immobilize the linker or primer
oligonucleotide to the solid support. One of the most widely used methods
to achieve such an immobilization of oligonucleotide primers for
subsequent use in hybridization-based assays consists of the non-covalent
coating of these solid phases with streptavidin or avidin and the
subsequent immobilization of biotinylated oligonucleotides (Holmstrom, K.
et al., Anal. Biochem. 209:278-283 (1993)). Another recent method
(Running, J. A. et al., BioTechniques 8:276-277 (1990); Newton, C. R. et
al. Nucl. Acids Res. 21:1155-1162 (1993)) 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.
In another published method (Kawai, S. et al., Anal. Biochem. 209:63-69
(1993)), short oligonucleotide probes were ligated together to form
multimers and these were ligated into a phagemid vector. Following in
vitro amplification and isolation of the single-stranded form of these
phagemids, they were immobilized onto polystyrene plates and fixed by UV
irradiation at 254 nm. The probes immobilized in this way were then used
to capture and detect a biotinylated PCR product.
A method for the direct covalent attachment of short, 5'-phosphorylated
primers to chemically modified polystyrene plates ("Covalink" plates,
Nunc) has also been published (Rasmussen, S. R. et al., Anal. Biochem.
198:138-142 (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.
Most preferably, the immobilization of the oligonucleotides of the present
invention is accomplished using a method that can be used directly,
without the need for any pretreatment of commercially available
polystyrene microwell plates (ELISA plates) or microscope glass slides
(Nikiforov, T. and Knapp, M., U.S. patent application Ser. No. 08/162,397,
herein incorporated by reference). Since 96 well polystyrene plates are
widely used in ELISA tests, there has been significant interest in the
development of methods for the immobilization of short oligonucleotide
primers to the wells of these plates for subsequent hybridization assays.
Also of interest is a method for the immobilization to microscope glass
slides, since the latter are used in the so-called Slide Immunoenzymatic
Assay (SIA) (de Macario, E. C. et al., BioTechniques 3:138-145 (1985)).
The solid support can be glass, plastic, paper, etc. The support can be
fashioned as a bead, dipstick, test tube, or a variety of other shapes. In
a preferred embodiment, the support will be a microtiter dish, having a
multiplicity of wells. The conventional 96-well microtiter dishes used in
diagnostic laboratories and in tissue culture are a preferred support. The
use of such a support allows the simultaneous determination of a large
number of samples and controls, and thus facilitates the analysis.
Automated delivery systems can be used to provide reagents to such
microtiter dishes. S | | |
