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Description  |
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FIELD OF THE INVENTION
The invention relates generally to methods for sequencing polynucleotides,
and more particularly, to a method of sorting and sequencing many
polynucleotides simultaneously.
BACKGROUND
Presently there are two basic approaches to DNA sequence determination: the
chain termination method, e.g. Sanger et al, Proc. Natl. Acad. Sci.,
74:5463-5467 (1977); and the chemical degradation method, e.g. Maxam et
al, Proc. Natl. Acad. Sci., 74:560-564 (1977). The chain termination
method has been improved in many ways since its invention, and serves as
the basis for all currently available automated DNA sequencing machines,
e.g. Sanger et al, J. Mol. Biol., 143:161-178 (1980); Schreier et al, J.
Mol. Biol., 129:169-172 (1979); Smith et al, Nature, 321:674-679 (1987);
Prober et al, Science, 238:336-341 (1987); Hunkapiller et al, Science,
254:59-67 (1991); Bevan et al, PCR Methods and Applications, 1:222-228
(1992). Moreover, further improvements are easily envisioned that should
greatly enhance the throughput and efficiency of the approach, e.g. Huang
et al, Anal. Chem., 64:2149-2154 (1992)(capillary arrays); Best et al,
Anal. Chem., 66:4063-4067 (1994)(non-cross-linked polymeric separation
media for capillaries); better dye sets; and the like.
Nonetheless, even with such reasonably envisioned improvements, these
approaches still have several inherent technical problems that make them
both expensive and time consuming, particularly when applied to
large-scale sequencing projects. Such problems include i) the gel
electrophoretic separation step which is labor intensive, is difficult to
automate, and which introduces an extra degree of variability in the
analysis of data, e.g. band broadening due to temperature effects,
compressions due to secondary structure in the DNA sequencing fragments,
inhomogeneities in the separation gel, and the like; ii) nucleic acid
polymerases whose properties, such as processivity, fidelity, rate of
polymerization, rate of incorporation of chain terminators, and the like,
are often sequence dependent; iii) detection and analysis of DNA
sequencing fragments which are typically present in fmol quantities in
spacially overlapping bands in a gel; iv) lower signals because the
labelling moiety is distributed over the many hundred spacially separated
bands rather than being concentrated in a single homogeneous phase, v) in
the case of single-lane fluorescence detection, the availability of dyes
with suitable emission and absorption properties, quantum yield, and
spectral resolvability; and vi) the need for a separately prepared
sequencing template for each sequencing reaction to identify a maximum of
about 400-600 bases, e.g. Trainor, Anal. Biochem., 62:418-426 (1990);
Connell et al, Biotechniques, 5:342-348 (1987); Karger et al, Nucleic
Acids Research, 19:495-4962 (1991); Fung et al, U.S. Pat. No. 4,855,225;
Nishikawa et al, Electrophoresis, 12:623-631 (1991); and Hunkapiller et al
(cited above).
The need to prepare separate sequencing templates is especially onerous in
large-scale sequencing projects, e.g. Hunkapiller et al (cited above)(94.4
kilobase target--2399 templates); and Alderton et al, Anal. Biochem.,
201:166-169 (1992)(230 kilobase target--13,000 templates). Attempts to
automate template preparation have proved difficult, especially when
coupled with current sequencing methodolgies, e.g. Church et al, Science,
240:185-188 (1988); Beck et al, Anal. Biochem. 212:498-505 (1993); Wilson
et al, Biotechniques, 6:776-787 (1988); and the like.
In view of the above, a major advance in sequencing technology would take
place if there were means available for overcoming the
template-preparation bottleneck. In particular, the ability to prepare
many thousands of templates simulaneously without individual template
selection and handling would lead to significant increases in sequencing
throughput and significant lowering of sequencing costs.
SUMMARY OF THE INVENTION
An object of my invention is to provide a method for tagging and sorting
many thousands of fragments of a target polynucleotide for simultaneous
analysis and/or sequencing.
Another object of my invention is to provide a method, kits, and apparatus
for analyzing and/or sequencing many thousands of different
polynucleotides simultaneously.
A further object of my invention is to provide a method for greatly
reducing the number of separate template preparation steps required in
large scale sequencing projects.
Still another object of my invention is to provide a method for applying
single-base sequencing methodologies to many different target
polynucleotides simultaneously.
Another object of my invention is to provide a rapid and reliable method
for sequencing target polynucleotides having a length in the range of a
few hundred basepairs to several tens of thousands of basepairs.
My invention achieves these and other objects by providing a method and
materials for sorting polynucleotides with oligonucleotide tags.
Oligonucleotide tags of the invention are capable of hybridizing to
complementary oligomeric compounds consisting of subunits having enhanced
binding strength and specificity as compared to natural oligonucleotides.
Such complementary oligomeric compounds are referred to herein as "tag
complements." Subunits of tag complements may consist of monomers of
non-natural nucleotide analogs, referred to herein as "antisense monomers"
or they may comprise oligomers having lengths in the range of 3 to 6
nucleotides or analogs thereof, including antisense monomers, the
oligomers being selected from a minimally cross-hybridizing set. In such a
set, a duplex made up of an oligomer of the set and the complement of any
other oligomer of the set contains at least two mismatches. In other
words, an oligomer of a minimally cross-hybridizing set at best forms a
duplex having at least two mismatches with the complement of any other
oligomer of the same set. The number of oligonucleotide tags available in
a particular embodiment depends on the number of subunits per tag and on
the length of the subunit, when the subunit is an oligomer from a
minimally cross-hybridizing set. In the latter case, the number is
generally much less than the number of all possible sequences the length
of the tag, which for a tag n nucleotides long would be 4.sup.n. Preferred
antisense monomers include peptide nucleic acid monomers and nucleoside
phosphoramidates having a 3'-NHP(O)(O--)O-5 ' linkage with its adjacent
nucleoside. The latter compounds are referred to herein as N3.fwdarw.P5'
phosphoramidates.
In one aspect of my invention, tag complements attached to a solid phase
support are used to sort polynucleotides from a mixture of polynucleotides
each containing a tag. In this embodiment, tag complements are synthesized
on the surface of a solid phase support, such as a microscopic bead or a
specific location in an array of synthesis locations on a single support,
such that populations of identical sequences are produced in specific
regions. That is, the surface of each support, in the case of a bead, or
of each region, in the case of an array, is derivatized by only one type
of tag complement which has a particular sequence. The population of such
beads or regions contains a repertoire of tag complements with distinct
sequences, the size of the repertoire depending on the number of subunits
per oligonucleotide tag and the length of the subunits employed, where
oligomeric subunits are used. Similarly, the polynucleotides to be sorted
each comprises an oligonucleotide tag in the repertoire, such that
identical polynucleotides have the same tag and different polynucleotides
have different tags. Thus, when the populations of supports and
polynucleotides are mixed under conditions which permit specific
hybridization of the oligonucleotide tags with their respective
complements, subpopulations of identical polynucleotides are sorted onto
particular beads or regions. The subpopulations of polynucleotides can
then be manipulated on the solid phase support by micro-biochemical
techniques.
An important aspect of my invention is the use of the oligonucleotide tags
to sort polynucleotides for parallel sequence determination. Preferably,
this aspect of the invention comprises the following steps: (a) generating
from a target polynucleotide a plurality of fragments that covers the
target polynucleotide; (b) attaching an oligonucleotide tag from a
repertoire of tags to each fragment of the plurality (i) such that
substantially all the same fragments have the same oligonucleotide tag
attached and (ii) such that each oligonucleotide tag from the repertoire
comprises a plurality of subunits and each subunit of the plurality
consists of a complementary nucleotide of an antisense monomer or an
oligonucleotide having a length from three to six nucleotides, the
oligonucleotides being selected from a minimally cross-hybridizing set;
(c) sorting the fragments by specifically hybridizing the oligonucleotide
tags with their respective tag complements; (d) determining the nucleotide
sequence of a portion of each of the fragments of the plurality; and (e)
determining the nucleotide sequence of the target polynucleotide by
collating the sequences of the fragments.
Another important feature of my invention is a method of identifying or
fingerprinting, a population of mRNA molecules. Preferably, such a method
comprises the following steps: (a) forming a population of cDNA molecules
from the population of mRNA molecules, the cDNA molecules being
complementary to the mRNA molecules and each cDNA molecule having an
oligonucleotide tag attached, (i) such that substantially all of the same
cDNA molecules have the same oligonucleotide tag attached and (ii) such
that each oligonucleotide tag from the repertoire comprises a plurality of
subunits and each subunit of the plurality consists of a complementary
nucleotide of an antisense monomer or an oligonucleotide having a length
from three to six nucleotides, the oligonucleotides being selected from a
minimally cross-hybridizing set; (b) sorting the cDNA molecules by
specifically hybridizing the oligonucleotide tags with their respective
tag complements; (c) determining the nucleotide sequence of a portion of
each of the sorted cDNA molecules; and (d) identifying the population of
mRNA molecules by the frequency distribution of the portions of sequences
of the cDNA molecules.
When used in combination with solid phase supports, such as microscopic
beads, my invention provides a readily automated system for manipulating
and sorting polynucleotides, particularly useful in large-scale parallel
operations, such as large-scale DNA sequencing, mRNA fingerprinting, and
the like, where many target polynucleotides or many segments of a single
target polynucleotide are sequenced and/or analyzed simultaneously.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a-1c illustrates structures of labeled probes employed in a
preferred method of "single base" sequencing which may be used with the
invention.
FIG. 2 illustrates the relative positions of the nuclease recognition site,
ligation site, and cleavage site in a ligated complex formed between a
target polynucleotide and a probe (SEQ ID. NO: 22) used in a preferred
"single base" sequencing method.
FIG. 3 is a flow chart illustrating a general algorithm for generating
minimally cross-hybridizing sets.
FIG. 4 illustrates a scheme for synthesizing oligonucleotide N3'.fwdarw.P5'
phosphoramidates.
FIG. 5 diagrammatically illustrates an apparatus for carrying out parallel
operations, such as polynucleotide sequencing, in accordance with the
invention.
DEFINITIONS
"Complement" or "tag complement" as used herein in reference to
oligonucleotide tags refers to an oligonucleotide to which a
oligonucleotide tag specifically hybridizes to form a perfectly matched
duplex or triplex. In embodiments where specific hybridization results in
a triplex, the oligonucleotide tag may be selected to be either double
stranded or single stranded. Thus, where triplexes are formed, the term
"complement" is meant to encompass either a double stranded complement of
a single stranded oligonucleotide tag or a single stranded complement of a
double stranded oligonucleotide tag.
The term "oligonucleotide" as used herein includes linear oligomers of
natural or modified monomers or linkages, including deoxyribonucleosides,
ribonucleosides, .alpha.-anomeric forms thereof, peptide nucleic acids
(PNAs), and the like, capable of specifically binding to a target
polynucleotide by way of a regular pattern of monomer-to-monomer
interactions, such as Watson-Crick type of base pairing, base stacking,
Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually
monomers are linked by phosphodiester bonds or analogs thereof to form
oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to
several tens of monomeric units. Whenever an oligonucleotide is
represented by a sequence of letters, such as "ATGCCTG," it will be
understood that the nucleotides are in 5'.fwdarw.3' order from left to
right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G"
denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoranilidate, phosphoramidate, and the like. It
is clear to those skilled in the art when oligonucleotides having natural
or non-natural nucleotides may be employed, e.g. where processing by
enzymes is called for, usually oligonucleotides consisting of natural
nucleotides are required.
"Perfectly matched" in reference to a duplex means that the poly- or
oligonucleotide strands making up the duplex form a double stranded
structure with one other such that every nucleotide in each strand
undergoes Watson-Crick basepairing with a nucleotide in the other strand.
The term also comprehends the pairing of nucleoside analogs, such as
deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may
be employed. In reference to a triplex, the term means that the triplex
consists of a perfectly matched duplex and a third strand in which every
nucleotide undergoes Hoogsteen or reverse Hoogsteen association with a
basepair of the perfectly matched duplex. Conversely, a "mismatch" in a
duplex between a tag and an oligonucleotide means that a pair or triplet
of nucleotides in the duplex or triplex fails to undergo Watson-Crick
and/or Hoogsteen and/or reverse Hoogsteen bonding.
As used herein, "nucleoside" includes the natural nucleosides, including
2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker,
DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in
reference to nucleosides includes synthetic nucleosides having modified
base moieties and/or modified sugar moieties, e.g. described by Scheit,
Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman,
Chemical Reviews, 90:543-584 (1990), or the like, with the only proviso
that they are capable of specific hybridization. Such analogs include
synthetic nucleosides designed to enhance binding properties, reduce
degeneracy, increase specificity, and the like.
"Stable" in reference to the formation of a covalent linkage and/or
non-covalent complex between binding moieties means that melting
temperature of the oligonucleotide clamp incorporating the given pair(s)
of binding moieties and its target polynucleotide is increased by at least
twenty-five percent over the melting temperature of oligonucleotide
moieties of the clamp alone, wherein melting temperature is measured by
standard techniques, e.g. half maximum of 260 nm absorbance v. temperature
as described more fully below. Preferably, stable means that melting
temperature of the oligonucleotide clamp incorporating the given pair(s)
of binding moieties and its target polynucleotide is increased by at least
fifty percent over the melting temperature of oligonucleotide moieties of
the clamp alone.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a method of labeling and sorting molecules,
particularly polynucleotides, by the use of oligonucleotide tags. In one
aspect, the oligonucleotide tags of the invention comprise a plurality of
"words" or subunits selected from minimally cross-hybridizing sets of
subunits. Subunits of such sets cannot form a duplex or triplex with the
complement of another subunit of the same set with less than two
mismatched nucleotides. Thus, the sequences of any two oligonucleotide
tags of a repertoire that form duplexes will never be "closer" than
differing by two nucleotides. In particular embodiments, sequences of any
two oligonucleotide tags of a repertoire can be even "further" apart, e.g.
by designing a minimally cross-hybridizing set such that subunits cannot
form a duplex with the complement of another subunit of the same set with
less than three mismatched nucleotides, and so on. Usually,
oligonucleotide tags of the invention and their complements are oligomers
of the natural nucleotides so that they may be conveniently processed by
enzymes, such as ligases, polymerase, terminal transferases, and the like.
In another aspect of the invention, tag complements consist of monomers
referred to herein as "antisense monomers." This term is meant to
encompass a range of compounds typically developed for antisense
therapeutics that have enhance binding strength and enhance specificity
for polynucleotide targets. As mentioned above under the definition of
"oligonucleotide," the compounds may include a variety of different
modifications of the natural nucleotides, e.g. modification of base
moieties, sugar moieties, and/or monomer-to-monomer linkages. Such
compounds also include oligonucleotide loops, oligonucleotide "clamps,"
and like structures, described more fully below, that promote enhanced
binding and specificity.
By the use of such compounds, the invention finds particular utility in
labeling and sorting polynucleotides for parallel operations, such as
sequencing, fingerprinting or other types of analysis.
Constructing Oligonucleotide Tags from Minimally Cross-Hybridizing Sets of
Subunits
The nucleotide sequences of the subunits for any minimally
cross-hybridizing set are conveniently enumerated by simple computer
programs following the general algorithm illustrated in FIG. 3, and as
exemplified by program minhx whose source code is listed in Appendix I.
Minhx computes all minimally cross-hybridizing sets having subunits
composed of three kinds of nucleotides and having length of four.
The algorithm of FIG. 3 is implemented by first defining the characteristic
of the subunits of the minimally cross-hybridizing set, i.e. length,
number of base differences between members, and composition, e.g. do they
consist of two, three, or four kinds of bases. A table M.sub.n, n=1, is
generated (100) that consists of all possible sequences of a given length
and composition. An initial subunit S.sub.1 is selected and compared (120)
with successive subunits S.sub.i for i=n+1 to the end of the table.
Whenever a successive subunit has the required number of mismatches to be
a member of the minimally cross-hybridizing set, it is saved in a new
table M.sub.n+1 (125), that also contains subunits previously selected in
prior passes through step 120. For example, in the first set of
comparisons, M.sub.2 will contain S.sub.1 ; in the second set of
comparisons, M.sub.3 will contain S.sub.1 and S.sub.2 ; in the third set
of comparisons, M.sub.4 will contain S.sub.1, S.sub.2, and S.sub.3 ; and
so on. Similarly, comparisons in table M.sub.j will be between S.sub.j and
all successive subunits in M.sub.j. Note that each successive table
M.sub.n+1 is smaller than its predecessors as subunits are eliminated in
successive passes through step 130. After every subunit of table M.sub.n
has been compared (140) the old table is replaced by the new table
M.sub.n+1, and the next round of comparisons are begun. The process stops
(160) when a table M.sub.n is reached that contains no successive subunits
to compare to the selected subunit S.sub.i, i.e. M.sub.n =M.sub.n+1.
As mentioned above, preferred minimally cross-hybridizing sets comprise
subunits that make approximately equivalent contributions to duplex
stability as every other subunit in the set. Guidance for selecting such
sets is provided by published techniques for selecting optimal PCR primers
and calculating duplex stabilities, e.g. Rychlik et al, Nucleic Acids
Research, 17:8543-8551 (1989) and 18:6409-6412 (1990); Breslauer et al,
Proc. Natl. Acad. Sci., 83:3746-3750 (1986); Wetmur, Crit. Rev. Biochem.
Mol. Biol., 26:227-259 (1991); and the like. For shorter tags, e.g. about
30 nucleotides or less, the algorithm described by Rychlik and Wetmur is
preferred, and for longer tags, e.g. about 30-35 nucleotides or greater,
an algorithm disclosed by Suggs et al, pages 683-693 in Brown, editor,
ICN-UCLA Symp. Dev. Biol., Vol. 23 (Academic Press, New York, 1981) may be
conveniently employed.
A preferred embodiment of minimally cross-hybridizing sets are those whose
subunits are made up of three of the four natural nucleotides. As will be
discussed more fully below, the absence of one type of nucleotide in the
oligonucleotide tags permits target polynucleotides to be loaded onto
solid phase supports by use of the 5'.fwdarw.3' exonuclease activity of a
DNA polymerase. The following is an exemplary minimally cross-hybridizing
set of subunits each comprising four nucleotides selected from the group
consisting of A, G, and T:
TABLE I
______________________________________
Word: w.sub.1 w.sub.2 w.sub.3
w.sub.4
Sequence: GATT TGAT TAGA TTTG
Word: w.sub.5 w.sub.6 w.sub.7
w.sub.8
Sequence: GTAA AGTA ATGT AAAG
______________________________________
In this set, each member would form a duplex having three mismatched bases
with the complement of every other member.
Further exemplary minimally cross-hybridizing sets are listed below in
Table I. Clearly, additional sets can be generated by substituting
different groups of nucleotides, or by using subsets of known minimally
cross-hybridizing sets.
TABLE II
______________________________________
Exemplary Minimally Cross-Hybridizing Sets of 4-mer Subunits
______________________________________
CATT ACCC AAAC AAAG AACA AACG
CTAA AGGG ACCA ACCA ACAC ACAA
TCAT CACG AGGG AGGC AGGG AGGC
ACTA CCGA CACG CACC CAAG CAAC
TACA CGAC CCGC CCGG CCGC CCGG
TTTC GAGC CGAA CGAA CGCA CGCA
ATCT GCAG GAGA GAGA GAGA GAGA
AAAC GGCA GCAG GCAC GCCG GCCC
AAAA GGCC GGCG GGAC GGAG
AAGA AAGC AAGG ACAG ACCG ACGA
ACAC ACAA ACAA AACA AAAA AAAC
AGCG AGCG AGCC AGGC AGGC AGCG
CAAG CAAG CAAC CAAC CACC CACA
CCCA CCCC CCCG CCGA CCGA CCAG
CGGC CGGA CGGA CGCG CGAG CGGC
GACC GACA GACA GAGG GAGG GAGG
GCGG GCGG GCGC GCCC GCAC GCCC
GGAA GGAC GGAG GGAA GGCA GGAA
______________________________________
The oligonucleotide tags of the invention and their complements are
conveniently synthesized on an automated DNA synthesizer, e.g. an Applied
Biosystems, Inc. (Foster City, Calif.) model 392 or 394 DNA/RNA
Synthesizer, using standard chemistries, such as phosphoramidite
chemistry, e.g. disclosed in the following references: Beaucage and Iyer,
Tetrahedron, 48:2223-2311 (1992); Molko et al, U.S. Pat. No. 4,980,460;
Koster et al, U.S. Pat. No. 4,725,677; Caruthers et al, U.S. Pat. Nos.
4,415,732; 4,458,066; and 4,973,679; and the like. Alternative
chemistries, e.g. resulting in non-natural backbone groups, such as
phosphorothioate, phosphoramidate, and the like, may also be employed
provided that the resulting oligonucleotides are capable of specific
hybridization. In some embodiments, tags may comprise naturally occuring
nucleotides that permit processing or manipulation by enzymes, while the
corresponding tag complements may comprise non-natural nucleotide analogs,
such as peptide nucleic acids, or like compounds, that promote the
formation of more stable duplexes during sorting.
When microparticles are used as supports, repertoires of oligonucleotide
tags and tag complements are preferably generated by subunit-wise
synthesis via "split and mix" techniques, e.g. as disclosed in Shortle et
al, International patent application PCT/US93/03418. Briefly, the basic
unit of the synthesis is a subunit of the oligonucleotide tag. Preferably,
phosphoramidite chemistry is used and 3' phosphoramidite oligonucleotides
are prepared for each subunit in a minimally cross-hybridizing set, e.g.
for the set first listed above, there would be eight 4-mer
3'-phosphoramidites. Synthesis proceeds as disclosed by Shortle et al or
in direct analogy with the techniques employed to generate diverse
oligonucleotide libraries using nucleosidic monomers, e.g. as disclosed in
Telenius et al, Genomics, 13:718-725 (1992); Welsh et al, Nucleic Acids
Research, 19:5275-5279 (1991); Grothues et al, Nucleic Acids Research,
1:1321-1322 (1993); Hartley, European patent application 90304496.4; Lam
et al, Nature, 354:82-84 (1991); Zuckerman et al, Int. J. Pept. Protein
Research, 40:498-507 (1992); and the like. Generally, these techniques
simply call for the application of mixtures of the activated monomers to
the growing oligonucleotide during the coupling steps.
Double stranded forms of tags are made by separately synthesizing the
complementary strands followed by mixing under conditions that permit
duplex formation. Such duplex tags may then be inserted into cloning
vectors along with target polynucleotides for sorting and manipulation of
the target polynucleotide in accordance with the invention.
In embodiments where specific hybridization occurs via triplex formation,
coding of tag sequences follows the same principles as for duplex-forming
tags; however, there are further constraints on the selection of subunit
sequences. Generally, third strand association via Hoogsteen type of
binding is most stable along homopyrimidine-homopurine tracks in a double
stranded target. Usually, base triplets form in T-A*T or C-G*C motifs
(where "-" indicates Watson-Crick pairing and "*" indicates Hoogsteen type
of binding); however, other motifs are also possible. For example,
Hoogsteen base pairing permits parallel and antiparallel orientations
between the third strand (the Hoogsteen strand) and the purine-rich strand
of the duplex to which the third strand binds, depending on conditions and
the composition of the strands. There is extensive guidance in the
literature for selecting appropriate sequences, orientation, conditions,
nucleoside type (e.g. whether ribose or deoxyribose nucleosides are
employed), base modifications (e.g. methylated cytosine, and the like) in
order to maximize, or otherwise regulate, triplex stability as desired in
particular embodiments, e.g. Roberts et al, Proc. Natl. Acad. Sci.,
88:9397-9401 (1991); Roberts et al, Science, 258:1463-1466 (1992);
Distefano et al, Proc. Natl. Acad. Sci., 90:1179-1183 (1993); Mergny et
al, Biochemistry, 30:9791-9798 (1991); Cheng et al, J. Am. Chem. Soc.,
114:4465-4474 (1992); Beal and Dervan, Nucleic Acids Research,
20:2773-2776 (1992); Beal and Dervan, J. Am. Chem. Soc., 114:4976-4982
(1992); Giovannangeli et al, Proc. Natl. Acad. Sci., 89:8631-8635 (1992);
Moser and Dervan, Science, 238:645-650 (1987); McShan et al, J. Biol.
Chem., 267:5712-5721 (1992); Yoon et al, Proc. Natl. Acad. Sci.,
89:3840-3844 (1992); Blume et al, Nucleic Acids Research, 20:1777-1784
(1992); Thuong and Helene, Angew. Chem. Int. Ed. Engl. 32:666-690 (1993);
and the like. Conditions for annealing single-stranded or duplex tags to
their single-stranded or duplex complements are well known, e.g. Ji et al,
Anal. Chem. 65:1323-1328 (1993).
When oligomeric subunits are employed, oligonucleotide tags of the
invention and their complements may range in length from 12 to 60
nucleotides or basepairs; more preferably, they range in length from 18 to
40 nucleotides or basepairs; and most preferably, they range in length
from 25 to 40 nucleotides or basepairs. When constructed from antisense
monomers, oligonucleotide tags and their complements preferably range in
length from 10 to 40 monomers; and more preferably, they range in length
from 12 to 30 monomers.
Solid Phase Supports
Solid phase supports for use with the invention may have a wide variety of
forms, including microparticles, beads, and membranes, slides, plates,
micromachined chips, and the like. Likewise, solid phase supports of the
invention may comprise a wide variety of compositions, including glass,
plastic, silicon, alkanethiolate-derivatized gold, cellulose, low
cross-linked and high cross-linked polystyrene, silica gel, polyamide, and
the like. Preferably, either a population of discrete particles are
employed such that each has a uniform coating, or population, of
complementary sequences of the same tag (and no other), or a single or a
few supports are employed with spacially discrete regions each containing
a uniform coating or population, of complementary sequences to the same
tag (and no other). In the latter embodiment, the area of the regions may
vary according to particular applications; usually, the regions range in
area from several .mu.m.sup.2, e.g. 3-5, to several hundred .mu.m.sup.2,
e.g. 100-500. Preferably, such regions are spacially discrete so that
signals generated by events, e.g. fluorescent emissions, at adjacent
regions can be resolved by the detection system being employed. In some
applications, it may be desirable to have regions with uniform coatings of
more than one tag complement, e.g. for simultaneous sequence analysis, or
for bringing separately tagged molecules into close proximity.
Tag complements may be used with the solid phase support that they are
synthesized on, or they may be separately synthesized and attached to a
solid phase support for use, e.g. as disclosed by Lund et al, Nucleic
Acids Research, 16:10861-10880 (1988); Albretsen et al, Anal. Biochem.,
189:40-50 (1990); Wolf et al, Nucleic Acids Research, 15:2911-2926 (1987);
or Ghosh et al, Nucleic Acids Research, 15:5353-5372 (1987). Preferably,
tag complements are synthesized on and used with the same solid phase
support, which may comprise a variety of forms and include a variety of
linking moieties. Such supports may comprise microparticles or arrays, or
matrices, of regions where uniform populations of tag complements are
synthesized. A wide variety of microparticle supports may be used with the
invention, including microparticles made of controlled pore glass (CPG),
highly cross-linked polystyrene, acrylic copolymers, cellulose, nylon,
dextran, latex, polyacrolein, and the like, disclosed in the following
exemplary references: Meth. Enzymol., Section A, pages 11-147, vol. 44
(Academic Press, New York, 1976); U.S. Pat. Nos. 4,678,814; 4,413,070; and
4,046,720; and Pon, Chapter 19, in Agrawal, editor, Methods in Molecular
Biology, Vol. 20, (Humana Press, Totowa, N.J., 1993). Microparticle
supports further include commercially available nucleoside-derivatized CPG
and polystyrene beads (e.g. available from Applied Biosystems, Foster
City, Calif.); derivatized magnetic beads; polystyrene grafted with
polyethylene glycol (e.g., TentaGel.TM., Rapp Polymere, Tubingen Germany);
and the like. Selection of the support characteristics, such as material,
porosity, size, shape, and the like, and the type of linking moiety
employed depends on the conditions under which the tags are used. For
example, in applications involving successive processing with enzymes,
supports and linkers that minimize steric hinderance of the enzymes and
that facilitate access to substrate are preferred. Exemplary linking
moieties are disclosed in Pon et al, Biotechniques, 6:768-775 (1988);
Webb, U.S. Pat. No. 4,659,774; Barany et al, International patent
application PCT/US91/06103; Brown et al, J. Chem. Soc. Commun.,
1989:891-893; Damha et al, Nucleic Acids Research, 18:3813-3821 (1990);
Beattie et al, Clinical Chemistry, 39:719-722 (1993); Maskos and Southern,
Nucleic Acids Research, 20:1679-1684 (1992); and the like.
As mentioned above, tag complements may also be synthesized on a single (or
a few) solid phase support to form an array of regions uniformly coated
with tag complements. That is, within each region in such an array the
same tag complement is synthesized. Techniques for synthesizing such
arrays are disclosed in McGall et al, International application
PCT/US93/03767; Pease et al, Proc. Natl. Acad. Sci., 91:5022-5026 (1994);
Southern and Maskos, International application PCT/GB89/01114; Maskos and
Southern (cited above); Southern et al, Genomics, 13:1008-1017 (1992); and
Maskos and Southern, Nucleic Acids Research, 21:4663-4669 (1993).
Preferably, the invention is implemented with microparticles or beads
uniformly coated with complements of the same tag sequence. Microparticle
supports and methods of covalently or noncovalently linking
oligonucleotides to their surfaces are well known, as exemplified by the
following references: Beaucage and Iyer (cited above); Gait, editor,
Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984);
and the references cited above. Generally, the size and shape of a
microparticle is not critical; however, microparticles in the size range
of a few, e.g. 1-2, to several hundred, e.g. 200-1000 .mu.m diameter are
preferable, as they facilitate the construction and manipulation of large
repertoires of oligonucleotide tags with minimal reagent and sample usage.
Preferably, commercially available controlled-pore glass (CPG) or
polystyrene supports are employed as solid phase supports in the
invention. Such supports come available with base-labile linkers and
initial nucleosides attached, e.g. Applied Biosystems (Foster City,
Calif.). Preferably, microparticles having pore size between 500 and 1000
angstroms are employed.
Synthesis of Oligonucleotide N3'.fwdarw.P5' Phosphoramidates
Tag complements comprising oligonucleotide N3'.fwdarw.P5' phosphoramidates
may be synthesized on a solid support using the step-by-step elongation
procedure outlined in FIG. 4. The synthetic cycle for addition of a single
aminonucleoside consists essentially of the following operations:
detritylation (step a); phosphitylation of the 5' hydroxyl group to
generate a 5'-hydrogen phosphonate diester (steps b and c); and
Atherton-Todd type coupling of a 5'-DMT-3'-aminonucleoside (e.g. as
disclosed by Glinski et al, Chem. Comm., pp. 915-916 (1970)) with the 5'
hydrogen phosphonate in the presence of carbon tetrachloride (step d).
Coupling yields range between 94-96% per cycle. The resulting
oligonucleotide phosphoramidate is cleaved and deprotected with ammonia
and thereafter purified by ion exchange high performance liquid
chromatography. The following references provide guidance for carrying out
the above synthesis: Atherton et al, J. Chem. Soc., pp. 660-663 (1945);
Gryaznov et al, Nucleic Acids Research, 20:3403-3409 (1992); Gryaznov et
al, Vest. Mosk. Univ. Ser. 2: Khim 27:421-424 (1986); and Gryaznov et al,
Tetrahedron Lett., 31:3205-3208 (1990). Fung and Gryaznov, International
application PCT/US94/03087, also show that 3'-amino-oligonucleotides may
be enzymatically ligated to 5'-phosphorylated oligonucleotides in a
standard template-driven ligation reaction.
Oligonucleotide Clamps
Tag complements of the invention may comprise an oligonucleotide clamp,
which is a compound capable of forming a covalently closed macrocycle or a
stable circular complex after specifically binding to a target
polynucleotide, which in the case of the present invention is its
corresponding oligonucleotide tag. Generally, oligonucleotide clamps
comprise one or more oligonucleotide moieties capable of specifically
binding to a tag and one or more pairs of binding moieties covalently
linked to the oligonucleotide moieties. Upon annealing of the
oligonucleotide moieties to the target polynucleotide, the binding
moieties of a pair are brought into juxtaposition so that they form a
stable covalent or non-covalent linkage or complex. The interaction of the
binding moieties of the one or more pairs effectively clamps the
specifically annealed oligonucleotide moieties to the target
polynucleotide.
In one preferred form oligonucleotide clamps comprise a first binding
moiety, a first oligonucleotide moiety, a hinge region, a second
oligonucleotide moiety, and a second binding moiety, for example, as
represented by the particular embodiment of the following formula:
X-O.sub.1 -G-O.sub.2 -Y
wherein O.sub.1 and O.sub.2 are the first and second oligonucleotide
moieties, G is the hinge region, X is the first binding moiety and Y is
the second binding moiety such that X and Y form a stable covalent or
non-covalent linkage or complex whenever they are brought into
juxtapositon by the annealing of the oligonucleotide moieties to a target
polynucleotide. Preferably, in this embodiment, one of O.sub.1 and O.sub.2
undergoes Watson-Crick binding with the target polynucleotide while the
other of O.sub.1 and O.sub.2 undergoes Hoogsteen binding.
Preferably, stability of oligonucleotide clamp/target polynucleotide
complexes are determined by way of melting, or strand dissociation,
curves. The temperature of fifty percent strand dissociation is taken as
the melting temperature, T.sub.m, which, in turn, provides a convenient
measure of stability. T.sub.m measurements are typically carried out in a
saline solution at neutral pH with target and clamp concentrations at
between about 1.0-2.0 .mu.M. Typical conditions are as follows: 150 mM
NaCl and 10 mM MgCl.sub.2 in a 10 mM sodium phosphate buffer (pH 7.0) or
in a 10 mM Tris-HCl buffer (pH 7.0); or like conditions. Data for melting
curves are accumulated by heating a sample of the oligonucleotide
clamp/target polynucleotide complex from room temperature to about
85.degree.-90.degree. C. As the temperature of the sample increases,
absorbance of 260 nm light is monitored at 1.degree. C. intervals, e.g.
using a Cary (Australia) model 1E or a Hewlett-Packard (Palo Alto, Calif.)
model HP 8459 UV/VIS spectrophotometer and model HP 89100A temperature
controller, or like instruments.
Hinge regions consist of nucleosidic or non-nucleosidic polymers which
preferably facilitate the specific binding of the monomers of the
oligonucleotide moieties with their complementary nucleotides of the
target polynucleotide. Hinge regions may also include linkages to solid
phase supports, e.g. via a derivatized base of a nucleotide, or the like.
Generally, the oligonucleotide moieties may be connected to hinge regions
and/or binding moieties in either 5'.fwdarw.3' or 3'.fwdarw.5'
orientations. For example, in the embodiment described above comprising a
first binding moiety, a first oligonucleotide moiety, a hinge region, a
second oligonucleotide moiety, and a second binding moiety, the
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