Note: Descriptions are shown in the official language in which they were submitted.
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wo 98/00435 1 PCT/US97/11496
Q~,TGONUCLEOTIDE l~ KE~R AND TEC}~NIQUES INVO~VING~ IMMOB~,T7,1i,n
AND ~ Kl~D OLIGONUCLEO rIDES
Field of the Invention
The present invention relates generally to the production of single-stranded DNAmolecules, and more particularly to the production of single-stranded DNA using immobilized
metal affinity chromatography.
Rack~rDund Qf the Invention
lo Many of the most widely employed operations in molecular biology hinge upon the use
of single-stranded DNA as a probe or template. Whereas single strands of DNA cont~ining up to
approximately 100 residues can readily be produced by solid-phase synthesis, longer
oligodeoxynucleotides typically must be generated through enzymatic methods such as the
polymerase chain reaction (PCR) Because biochemical procedures for the synthesis of mixed-
sequence DNA yield double-skanded products, there exists a need for a method by which to
resolve a desired strand from its complement. Such resolution is rendered difficult by the
similarities in macroscopic physical properties such as size and charge of two complimentary
strands, and by the requirement that resolution be carried out in the presence of strong
denaturants such as urea or guanidinium hydrochloride.
Techniques exist for ~tt~rhing a single biotin onto an oligonucleotide~ then using a solid
- -matrix at which is immobilized avidin or streptavidin, to irnmobilize the oligonucleotide. A
problem with this approach is that the binding that is required for the technique to be effective
depends upon proteins being properly folded. Therefore, the technique cannot be used under
strong denaturing conditions. Also, if the technique is to be used to surface-immobilize a single
DNA strand for surface plasmon resonance studies, the avidin/biotin combination adds enough
mass to the surface that the technique, when used to detect binding of a single strand of DNA to
the immobilized strand, can lack precision.
It is, therefore, a object of the present invention to provide improved techniques for
linking an oligonucleotide such as DNA, or a single strand of DNA, to another moiety or a
surface, and to resolve a desired skand of DNA from its complement.
Summ~ry of the Invention
The present invention provides a nucleotide coupled to a linking moiety that can
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WO 98/00435 2 PCTrUS97/11496
coordinate a metal ion, namely, a species having a formula Ln-Ny~ where N represents the
- nucleotide and L represents the moiety which can coordinate a metal ion. n is at least 1, and y is
at least 1. According to one embodiment Ny is DNA, and L can be histaminylpurine where n is
approximately 6.
s According to another aspect, the invention provides a species having a formula R-Ch-M-
L"-Ny. According to this aspect, R represents a chemical moiety that can be linked to a surface,
or linked to a chemical or biological species. Ch represents a chelating agent that can coordinate
a metal ion, ~ represents a metal ion coordinated by the chelating agen~ represents a
nucleotide, L represents a moiety that can coordinate a metal ion, and n and y each is at least 1.
lo The chelating agent is, according to one embodiment, a quadradentate chelating agent, for
example nitrilotriacetic acid.
The invention also provides a single stranded oligonucleotide immobilized on a surface
and free of hybridization to any complim~nt~ry strand that is also immobilized on the surface to
the extent that the complimentary strand cannot be removed under conditions that cause-
denaturization of oligonucleotide strand from its complimentary strand. The single-stranded
immobilized oligonucleotide also will withstand conditions, while rem~ining immobilized,
harsher than 2 molar urea. The strand can be a strand of DNA.
According to another aspect the invention provides a method. The method involvesproviding first and second complementary strands of oligonucleotide, contacting a surface of a
solid phase with the first and second strands, and allowing the first strand to be immobilized at
the surface. The second strand is allowed to be carried away from the surface without
immobilization at the surface. According to one embodiment, this aspect involves providing first
and sccond complementary strands of DNA in a fluid medium, passing the fluid medium across a
surface of a solid phase, and allowing the first strand to be immobilized at the surface while the
second strand is allowed to be carried by the fluid medium away from the surface without
imrnobilization.
The various aspects of the invention can be combined in a variety of ways. For example,
the single stranded oligonucleotide irnmobilized on a surface and free of hybridization to any
complimentary strand that is also immobilized on the surface to the extent that the
complimentary strand cannot be removed under conditions that cause denaturization, can bc
immobilized via linkages such as Ln-Ny or R-Ch-M-Ln-Ny~ above. Additionally, the methods
can be practiced using any of the species that can facilitate immobilization of an oligonucleotide.
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Other advantages, novel features, and objects of the invention will become apparent from
the following detailed description of the invention when considered in conjunction with the
accompanying drawings.
s ~rief Dc3~ ,1ion of the DI awi. ~.~
Fig. 1 is a schematic representation of double-stranded DNA immobilized, via an
attached linking moiety coordinated to a metal ion, to a solid phase surface;
Fig. 2 illustrates, schematically, primers and double-stranded PCR template used in one
set of examples; and
Fig. 3 is a photocopy of a ~len~tl-rin~ polyacrylamide gel electrophoresis analysis of DNA
strand resolution using a technique of the present invention.
Detailed Descri~>tion of the Invention
_ ~ Min, C., Cushing, T. Verdine, G., "Template-Directed Interference Footprinting of
s Protein-Adenine Contacts,", J. Am. Chem. Soc. 1996, 118, 6116-6120; and Min, C. Verdine, G.,
"Immobilized Metal Affinity Chromatography of DNA," Nucleic Acids Res. 1996, 24,3806-3810, both are incorporated herein by reference.
The present invention provides a linking functionality for attachment of an
oligonucleotide to another moiety or to a surface of a solid phase, where solid phase is defined as
any material insoluble in a medium used in conjunction with a procedure involving the
oligonucleotide. The techni~ue can be used, for example, to link an oligonucleotide to a solid
phase surface such as a chromatography solid phase, or to a surface of a biosensor element such
as a surface plasmon resonance (SPR) chip (for a discussion of SPR see, for example, Sternberg,
et al., "Quantitative Determination of Surface Concentration of Protein with Surface ~lasmon
Resonance Using Radiolabeled Proteins", Journal of Colloid and lnterface Science, 43 :2 513-
526, 1991, and references therein~. The technique also can be used, for example, to link an
oligonucleotide to a surface of a bead or plate used in a bioassay, to a label such as a fluorescent
label, or another molecule or solid entity.
The invention involves providing a species having the generalized formula Ln-Ny~ where
Ny represents a natural or synthetic, single or multiple-strand oligonucleotide typified in DNA or
a strand of DNA, and Ln represents a linking functionality where L represents a lig~tin~; moiety
that can coordinate a metal ion, and n is at least 1, preferably at least 2, and more preferably at
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W O 98/00435 4 PCT~US97/11496
Ieast 5._According to preferred embodiments, n is from 2 to 10, and most preferably is about 6.
L is a molecule that can be chemically attached to an oligonucleotide via, for example, the ester
bond of the oligonucleotide backbone, and Ln has a generalized formula I, below.
s Y O
I 11
(~~Z~~~P~O)n (I)
where Z is an organic moiety and Y is a ligand having ehe ability to coordinate a metal ion, Z and
Y together allowing incorporation of Ln into or on an oligonucleotide sequence. Those of
ordinary skill in the art can select app,ol.liately Y and Z to meet the requirement of compatibility
with an oligonucleotide sequence. Z and Y can be a single moiety in that a single organic moiety
15 can provide functionality compatible with an oligonucleotide sequence and also coordinate a
metal ion. Typically, Z is a hydrocarbon chain or a cyclic hydrocarbon group, optionally
interrupted by hetero groups, of a length of from about I to about 10 carbon atoms. As used
herein, "hydrocarbon" is meant to include alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl,
aralkyl, and the like. Hetero groups can include -O-, -CONH-, -CONHCO-, -NH-, -CSNH-,
20 -CO-, -CS-, -S-, -SO-, -(OCH2CH2)nR (where n=1-10), -(CF2)n- (where n--1-10)~ ole~ms, and the
like. ~ ---
Y (or Y and Z together) is a ligand that can coordinate free coordination sites on a metal
ion, such as a metal ion immobilized on a metal affinity chromatography solid phase without
complete coordination of the ion by the solid phase (see, for example, Hochulrt al., ~'New Metal
25 Chelate Adsorbent Selective for Proteins and Peptides ContS~ining Neighboring Histidine
~esidues" JournalofChronl~tography, 411(1987)177-1~4). YcanbeasdefinedaboveforZ,and preferably includes groups such as -N-, alcohols, thiols, carboxylates (including carboxylic
acid), and the like which can address a vacant coordination site on a metal ion. (~yclic and
heterocyclic organic compounds including -N- such as imidazoles, phenanthroline, adenine,
30 cytosine, and thymine are suitable. Additionally, amino acids satisfying these criteria, such as
histidine, lysine, cysteine, methionine, asparagine, tyrosine, glutamine, glut~m~t~, and aspartate
are suitable. Species such as hydroxamic acid also are suitable. In some cases, the above
moieties can serve as Z, with a different one of the above or another moiety serving as Y and
satisfying the criterion of coordination to a metal ion. For example, purine, thymine, ~ ?nine,
-
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W 098/00435 PCT~US97/11496
_
cytosine, or a combination could serve as Z, with another nucleic acid, amino acid, or nitrogen-
cont:~inin~ cyclic, multi-cyclic, or heterocyclic compound serving as Y. Where, for example,
purine, cytosine, or adenine are used as Z, the available -NH2- group can be used to couple Y
defined by, for example, hi.~t~mine. According to one preferred embodiment, 6-histaminyl
s purine defines Y, and Z is a cyclic ether (II, below).
NH~
N-H~
~o ~
Species Ln-Ny finds use in conjunction with a metal ion partially coordinated by a
- chelating agent that is linlced, or can be linked, to a moiety or surface of interest. That is, the
20 species finds usc in a combination of the formula:
R-Ch-Ln-Ny (III)
where R represents a chemical or physical m-oiety, Ch represents a chelating agent that can
2s coordinate a metal ion, and M represents a metal ion coordinated by the chelating agent. R can
be a particulate species such as gold sol, a label such as a fluorescent label, a biological molecule
of intercst such as a binding partner for interaction with another species, a chromatography solid
phase, or a linking or linkable moiety that can effect coupling of species III to any desired
surface or species. The particular species or surface to which species III is coupled is not
30 important, but the invention lies in the ability to coordinate Ny to R via Ln.
Co-pending, commonly-owned U.S. Patent Application Serial No. 081312,388, filed
September 26, 1994, now U.S. PatentNo. 5,620,850, and incorporated herein by reference,
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describes a variety of combinations of Ch and M suitable for use in the invention. In particular,
the metal ion is preferably selected from those that have at least four coordination sites,
preferably six coordination sites. A non-limiting list of metal ions suitable includes Co3+, Cr3t,
H 2i pd2-' Pt~ Pd4~ Pt4+ Rh3+ Ir3+ Ru3~ Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Pb2+, Mn2~ Fe3~, Fe~,
s Au3+, Aui, Ag+, Cu+, MO22+, Tl3+, Tl+, Bi3~, CH3Hg+, Al3+, Ga3+, Ce3+, UO2~, and La3+.
The chelating agent is preferably selected from bidentate, tridentate. and quadradentate,
chelating agents, and is selected in conjunction with the metal ion so that when the chelating
agent coordinates the metal ion, at least two free coordination sites of the metal remain. The
chelating agent and metal are selected so that the chelating agent can coordinate the metal ion
10 with a degree of stability great enough that the metal ion will remain immobilized at the surface
by the chelating agent.
Additionally, the chelating agent is selected as one that has a chelating moiety and a
non-chelating linker moiety, such that it can be covalently linked via its linker moicty to the
moiety R while leaving the chelating moiety undisturbed by the covalent linkage and free to
1S coordinate a metal ion. Alternatively, the chelating agent can bc selected as one that can be
modified via routine organic synthesis to include a non-chelating linker moiety, if such synthesis
leaves undisturbed the chelating moiety. One of ordinary skill in the art will appreciate that the
non-chelating linker moiety should provide functionality suitable for chemical linkage such as,
for example, an amine, alcohol, carbamate, carboxylic acid, thiol, aldehyde~ olefin. etc., for
20 formation of an ester linkage, formation of an amide linkage, thiol displacement and thiol ether
formation, and the like.
With the above considerations in mind, suitable chelating agents and corresponding metal
ions can be selected by those of ordinary skill in the art. In accordance with such selection
reference can be made to "Chelating Agents and Metal Chelates", Dwyer, F. P.; Mellor, D. P.,
2s Academic Press, and "Critical Stability Constants", Martell, A. E.; Smith, R. M., Plenum Press,
New York. These works describe a variety of chelating agents, and discuss the stability of
coordination between chelating agents and metal ions. Preferably, a chelating agent and metal
ion is selected such that the dissociation constant of the combination in aqueous solution is better
than 10 nM at physiological pH, that is, such that at least one half of the metal ions are
30 coordinated by chelating agent at a concentration of 10 nM.
A non-limiting exemplary list of suitable chelating agents includes nitrilotriacctic acid,
- 2,2'-bis(salicylideneamino3-6 ,6' -demethyldiphenyl, and 1 ,8-bis(a-pyridyl)-3 ,6-dithiaoctane.
.
_
-
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As an example of one, ~lefelled arrangement using species III, reference is made to Fig.
1, in which a species 25 linked to a solid phase surface includes an oligonucleotide, in particular
DNA 10 including a first strand 12 and a second, complementary strand 14 including a linking
functionality (Ln) 16 defined by 6 histaminyl purines, two of which (18 and 20, respectively) are
s coordinated to a metal ion 22. ~etal ion 22 also is coordinated by chelating agent 24 (a
nitrilotriacetic acid derivative, as illustrated) which coordinates four of the six coordination sites
of the metal ion, leaving two free coordination sites addressable by linking functionality 16.
Chelating agent 24 is attached, via linker 26 (a hydrocarbon chain, as illustrated), to a surface 28
of a solid phase 30. Linkage of chelating agent 24 to surface 28 via linker 26 can be
lo accomplished by any means such as covalent zltt~çhment, or the like.
A single-stranded oligonucleotide also is usefully immobilized as shown in FIG. 1 in
accordance with the invention. According to one embodiment, linker 26 is selected as one that
promotes formation of a self-assembled monolayer of a plurality of linkers 26 at surface 28, and
linker 26 termin~tes at its end opposite the chelating agent in a functionality that is attracted to
15 surface 28 and thereby promotes formation of a self-assembled monolayer. Such an arrangement
of a metal-coor-lin~ting chelating agent immobilized at a surface as part of a self-assembled
monolayer is described in commonly-owned co-pending U.S. patent application serial number
08/312,388, now U.S. Patent No. 5,620,850, referenced above. Self-assembled monolayer-
forming species also are described in U.S. Patent No. 5,512,131, issued ~pril 30, 1996 to Kumar
20 and Whitesides, incorporated herein by reference. Where a self-assembled monolayer cont~ining
a chelating agent 24 is provided for use with species Ln-Ny such as DNA 10 including linking
functionality 16, it can be advantageous to form a self-assembled mixed monolayer (as defined in
application serial number 08/312~388~ and U.S. Patent No. 5~620~850 referenced above), that is, a
heterogeneous self-assembled monolayer including an ordered assembly of at least another self-
25 assembled monolayer-forrning species other than species 25. A heterogeneous monolayer
de~1ned by less than about 50% of species 25 and greater than about 50% of another species such
as one termin~ting in a moiety that does not interact with Ln or Ny can be formulated, as well as
- one with a ratio of less than about 30% species 25, or less than about 20% species 25, etc.
- It is to be understood that coordination of linking functionality 16 to M may involve
30 linkage via two neighboring ligating moieties L (H, for histaminyl purine, as illustrated in FIG.
1), or another arrangement. Additionally, linking functionality 16 may include neighboring
ligating moieties L and/or ligating moieties L separated by one or more intermediate moieties
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W O 98/00435 8 PCTrUS97111496
that do not have li~ting capacity, SO long as linking functionality 16 possesses the ability to
coordinate free coordination sites at M.
Referring again to formula I, Y (and Y in combination with Z if Z has coort1in~in~
capacity) and n should be selected to provide the oligonucleotide with coor-lin~tin~ capacity
s sufficient to link the oligonucleotide to a metal ion to the extent that the oligonucleotide does not
become detached from the metal ion during the course of a procedure involving the
oligonuclcotide coordinated to the metal ion so as, for example, to immobilize the
oligonucleotide at a solid phase. Coordination should not, however, be so strong that Ln will
compete with coordination of the metal ion by the chelating agent to the extent that the metal ion
o is stripped from the chelating agent (thus, e.g., stripped from a solid phase, label, or other
moicty). For example, in most cases ligands such as ethylenediarnine tetra acetic acid would be
unsuitable. Those of ordinary skill in the art, with reference to "Chelating Agents and Metal
Chelates," Dwyer, F.P.; Mellor, D.P., Academic Press, and "Critical Stability Constants",
Martell, A.E.; Smith, R.M., Plenum Press, New York, can select a suitable group Y (and
15 optionally Z) and the number of repeating units n for a particular metal ion including a particular
number of free coordination sites.
In some cases it can be advantageous to test a particular Ln/M-Ch system to determine
whether coordination will be sufficiently stable for use in the present invention, but will not be so
stable that the metal ion is stripped from the chelating agent. For example, it is within the realm
20 of routine exI~erimentation to one of ordinary skill in the art to prepare a very simple
polynucleotide containing as few as two nucleotides or up to approximately 6 or 8 with an
attached linking functionality Ln~ i.e., 3'-NNNNN-L6, where N is any nucleotide or a variety of
nucleotides. The test oligonuclcotide then can be labeled radioactively at the 5' end by mixing
with T4 polynucleotide kinase, then with ~32P-ATP. Then the oligonucleotide can be mixed with
2~ beads of resin containing immobilized, coordinated metal ion, the beads rinsed, and
determination can be made as to whether radioactivity is present at the beads. If radioactivity is
present, then the selection of L (i.e. Y or ~ and R together) and n is a~plul)l iate to provide
coordination capacity sufficient to immobilize the oligonucleotide, but not to the extent that the
metal ion is stripped from the chelating agent.
If radioactivity is not present at the beads, then determination of whether the L~ n
combination results in insufficient coordination or coordination to an excessive extent such that
metal ion is stripped can be made by subjecting the beads, and/or the solution used to flush the
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W O 98/00435 9 PCTrUS97/11496
beads, to ultraviolet spectroscopy or ICP to determine the location of the metal ion. These
simple screening techniques can be performed by one of ordinary skill in the art without the
necessity of synthe~i~ing an entire oligonucleotide such as a DNA fragment useful in a particular
procedure.
~ 5 In some cases one would want to be able to separate the linker-nucleotide from th~metal
ion, as in elution from a column, and in some cases one would want essentially irreversible
binding. Accordingly, as a second test, beads can be flushed with an eluting fluid such as
200mM imidazole and detprmin~tion made as to whether the radioactivity is still present at the
beads.
o The present invention also represents an improvement over immobilization of single-
stranded oligonucleotide at a surface via biotin/avidin or biotin/streptavidin linlcage. That
linkage will withstand conditions only up to 2 molar urea, or less. The present invention
involves immobilization that withstands conditions harsher than 2 molar urea.
The function and advantage of these and other embodiments of the present invention will
be more fully understood *om the examples below. The following examples are intended to
illustrate the benefits of the present invention, but do not exemplify the full scope of the
invention.
F~mllle 1 Synthesis Of A Nucleotide Including A Moiety That Can Coordinate A Metal Ion
A nucleotide that can coordinate a metal ion due to incorporation of a moiety for that
purpose (having a formula Ln-Ny)~ specifically, DNA sequencing primers con~ining six
successive 6-histaminylpurine residues added on to the S'-end of the DNA was synthesized. The
6-histaminylpurine moiety was introduced through the convertible nucleotide approach by
analogy to reported proccdures (~erentz, A.E.; Verdine, G.L., J. Am. Chem. Soc. 1991. 113,
~s 4000-4002: ~erentz, A.E.; Verdine, G.L. In Nucleic ~cids and Molecular Biology Eckstein, F.,
-- Lilley, D.M.J. F,ds.; Springer-Verlag: Berlin, 1994; Vol. 8, pp. 14-40. Briefly, resin-bound
oligonucleotides cont~ining 06-phenyl-2'-deoxyinosine (~dl) in place of the dH residue were
dcprotected from the resin by mild âmmonia treatment (conc NH40H, nt, 4b) Iyophilized to ~~~
dryness, then treated with 5 M aqueous histamine (55~C, 14 h) to convert the ~dl residues to dH.
The crude oligonucleotides were purified by clP~ polyacrylamide gel electrophoresis. An
additional dG residue was added at the 5'-end to ensure e~ficient end-labeling using
polynucleotide kinase.
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- 10-
For the present IMAC-based strategy to be of significant practical value, it must be
capable of resolving PCR-amplified duplex DNA into its two component strands. To test this
application directly, we used one H6-tagged primer plus one unmodified primer to amplify a 183
base-pair segment of the plasmid pUCl 8-rnAE~RE2, thereby to furnish a duplex product with an
5 H6-tag on only one strand. The DNA template for PCE~ amplification, pUC 1 8-mARRE2, was
derived from the commercial cloning vector pUCI 8 by inserting a segment of the murine
interleukin-2 enhancer into the BamHI site (Chen, L.; Jain, J.; Oakley, M.G.; glover, J.N.M.:
Dervan, P.B.: Hogan, P.G.: Rao, A.; Verdine, G.L. Curr. Biol. 1995, 5, 882-889). pUC18 and
close relatives are more widely used in bacterial cloning than each of the unbound and bound
10 fractions was added 900 ,ul absolute ethanol (stored at 20~C), then the tubes were vortexed
briclly and chilled for 3û min on dry powdered CO2. The tubes were microcentrifuged for 30
min at 16,000 x g. The supernatant was removed and the pellet washed with 200 1ll 80%
aqueous EtOH (-20 ~C). Following removal of the ethanol solution, the tubes were dried by
- centrifugal Iyophilization (SpeedVac, Savant). To each dry tube was added 50 ,ul TE buffer.
15 The DNA concentration was determined by UV spectrophotometry. The designation of "top"
and "bottom" strands refers to the sequence as mapped in Figure 2. The "top" strand also
corresponds to the coding strand of the lacZ'o~ peptide, part of which was encoded within the
polylinker region of pUC18. That is, the PCR product generated using primers l a ~ 2b will bear
the H6-tag on the "top" strand only, whereas that generated using 2a + I b will bear the H6-tag on
20 thc "bottom" strand only. The yield of I'CR product formed in these reactions was no less than
that observed in parallel reactions with only unmodified primers (data not shown), thus
indicating that the E~6-tag does not affect PCR amplification adversely.
Primers la/lb contain a sequence that is identical to a stretch (shaded) of thc C'top'' strand
of the plasmic pUCl 8-mAE~R~e2; they are extended in the rightward dircction (5'-3') during PCR.
2s Primers 2a/2b contain a sequence that is identical to a stretch (shaded) of the ~'bottom" strand of
pUC18-mAE~RE2; they are extended in the leftward direction. The shaded sequence in primers
1 a and 2a denotes the E~6-tag. E and H denote the EcoR I and Hnd III sites, respectively, of the
pUCl 8 polylinker; a stretch of the murine IL-2 enhancer (mARRE2, batched) was inserted to the
BamII I site to generate pucl8--mAE?RE2. (c) Sch~mzltic illustration ofthc procedure for PCR
30 amplification to produce duplex DNA cont~ining a H6-tag on one strand, and resolution o~the
two constituent strands using IMAC.
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Exam~le 2: Immobilization Of Ln-Ny At A Surface
The nucleotide including a moiety that can coordinate a metal ion was immobilized at a
surface. Specifically, to assess the ability of the H6-tag to endow DNA with selective retentivity
on a Ni2~--charged chelating resin, the H6-tagged oligonucleotides (2a/2b) and their unrnodified
5 counterparts (lb/2b) were 5'-end-labeled with 32p, then passed in parallel through a Ni+2-
nitrilotriactic acid (NTA)-agarose resin. In particular, the PCR products were denatured in 6- M
guanidinum HCI, then incubated batch wise with Ni7+-NTA-agarose.
Fxslmple 3: Immobilization of a First Strand of First and Second Coml ~enlent~ry Strands of
10 nNA at a Surface with Selective Removal of the Second Strand~ and Ouantitative Determin~tisn
- of ~ound Versus Unbound Strands
Following the procedure of Example 2~ it was demons~rated that the H6-tag confers, upon
an oligonucleotide, the ability to be selectively and reversibly retained on a surface. specifically
Ni2~-NTA-agarose. In particular, it was demonstrated that the technique results in single-
stranded oligonucleotide immobilized on a surface and free of hybridization to any
complimentary strand that is also immobilized on the surface to the extent that the
complimentary strand cannot be removed under conditions that cause der~t-lri7~tion of
oligonucleotide strand from its complimentary strand.
Following removal of the supernatant, which contains unbound DNA, the resin was
washed and the bound DNA eluted with 200 mM imidazole. Aliquots of the crude PCR product
and the two DNA-containing fractions from the IMAC step were 5'-end labeled with 3~P and
analyzed by denaturing polyacrylamide gel electrophoresis (PAGE). The use of PAGE as an
assay for strand resolution was made possible by the slightly reduced mobility of H6-tagged
DNA strands relative to their complimentary strands generated by PCT. The retarded mobility of
the H6-tagged strand relative to the unmodified strand may arise from the partial positive charge
of its 6 pendant imidazole moieties at pH 8 and a greater length, due to incomplete
polymerization of the T6-stretch opposite H6 during PCR. The identities of the strands were
independently verified by PAGE analysis of PCR reactions that employed one ~2P-labeled primer
and one nonradioactive primer. Although this slight difference in mobility provides a convenient
- 30 means of assaying the strand resolution by IMAC, it is not sufficiently large to permit
preparative strand resolution by gel electrophoresis and extraction.
Fig. 3 is a photocopy of the results of denaturing PAGE of DNA strand resolution using
IMAC. Each panel of three lanes represents the results obtained using a pair of PCR primers,
.
.
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- 12 -
one of which (1 a or 2a) contains an H6-tag and the other of which (2b or 1 b, respectively) is
unmodified. C (lanes 1 and 4): controls showing the mixture of strands obtained directly by
PCR, prior to IMAC resolution; U (1anes 2 and 5): unbound fractions from IMAC; B (lanes 3
and 6~: bound fraction from IMAC, after elution with 200 mM imidazole.
The unbound fractions from IMAC in Fig. 3 contained primarily the faster-migrati~g
species, which corresponds to the respective unmodified strands, whereas the imidazole-eluted
bound fractions contained primarily the respective H6-tagged strands. Quantitative
phosporoim~gin~ analysis from repeated runs of the strand resolution procedure revealed that the
unrnodified strand typically comprise >90% of the unbound DNA and the H6-tagged DNA
comprise 95% of the bound fractions. Thus we conclude that I~fAC cleanly resolves a uniquely
~6-Laggcd PCR product into its two component strands. All but a trace amount of the bound
radioactivity could bc eluted from thc resin upon washing with a 200mM aqueous imidazole.
Fx~n~ple 4: Template-Directed Polymeri~tion Usin~ Surface-Immobilized Nn-Ny
To assay the biological activity of single-stranded DNA generated through the PCR-
IMAC sequence, we employed the resolved strands as templates in Sanger dideoxy sequencing
runs. The experiment was carried out using oligonucleotide strands that had been synthesized so
as to be attachable to a surface, in accordance with the invention, but werc not attached to the
surface during the experiment. Thus, the experiment proves thc concept that the single-stranded
oligonucleotide, immobilized at a surface in accordance with the invention, retains its biological
activity.
Specifically, single-stranded DNA was immobilized at a surface of a column, as
dcscribcd above, and then thc surface-immobilized DNA was flushed from the column, and a
chelatin~ agent was used to complex any rem~ining or free nickel ion in solution. Initially, the
=
unmodi~lcd strands were found to be cxcellent sequencing templates, but the H6-tagged strands
failed to support template-directed polymerization. Eventually, it was dlscovered that the H6-
taggcd DNA solutions contain adventitious Ni2~ in amounts sufficient to cause profound
inhibition of the DNA polymerase enzyme. This problem was overcome simply by adding 1,10
phenanthroline to the imidazole-conl~inin~ eluate prior to precipitation with ethanol. Single-
stranded templates prepared in this manner give uniformly high-quality DNA sequence data.
Those skilled in the art would readily appreciate that all parameters listed herein are
meant to be exemplary and actual parameters will depend upon the specific application for which
_
. CA 02259493 1998-12-30
W 0 98/00435 -13- PCTrUS97/11496
the methods and arrangements of the present invention are being used. It is, therefore, to be
understood that the foregoing embodiments are presented by way of example only and at that,
within the scope of the appended claims and equivalence thereto, the invention may be practiced
otherwise and as specifically described.
s
-- What is claimed is:
