U.S. patent application number 09/359147 was filed with the patent office on 2001-07-05 for microsphere attachment to dna.
Invention is credited to BALHORN, RODNEY L., BARRY, CHRISTOPHER H..
Application Number | 20010007024 09/359147 |
Document ID | / |
Family ID | 23412519 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010007024 |
Kind Code |
A1 |
BALHORN, RODNEY L. ; et
al. |
July 5, 2001 |
MICROSPHERE ATTACHMENT TO DNA
Abstract
Microscopic beads or other structures are attached to nucleic
acids (DNA) using a terminal transferase. The transferase adds
labeled dideoxy nucleotide bases to the ends of linear strands of
DNA. The labels, such as the antigens digoxigenin and biotin, bind
to the antibody compounds or other appropriate complementary
ligands, which are bound to the microscopic beads or other support
structures. The method does not require the synthesis of a
synthetic oligonucleotide probe. The method can be used to tag or
label DNA even when the DNA has an unknown sequence, has blunt
ends, or is a very large fragment (e.g., >500 kilobase
pairs).
Inventors: |
BALHORN, RODNEY L.;
(LIVERMORE, CA) ; BARRY, CHRISTOPHER H.; (FRESNO,
CA) |
Correspondence
Address: |
INTELLECTUAL PROPERTY LAW GROUP
LAWRENCE LIVERMORE NATIONAL LABORATORY
P.O. BOX 808 (L-703)
LIVERMORE
CA
94551
US
|
Family ID: |
23412519 |
Appl. No.: |
09/359147 |
Filed: |
July 21, 1999 |
Current U.S.
Class: |
536/23.1 ;
435/6.16; 435/6.19 |
Current CPC
Class: |
C12Q 1/6834 20130101;
Y10S 977/924 20130101; C12Q 2521/131 20130101; C12Q 2565/102
20130101; C12Q 2563/131 20130101; C07H 21/00 20130101; Y10T
436/143333 20150115; C12Q 1/6834 20130101 |
Class at
Publication: |
536/23.1 ;
435/6 |
International
Class: |
C07H 021/04; C12Q
001/68; C07H 021/02 |
Goverment Interests
[0001] The United States Government has rights in this invention
pursuant to Contract No. W-7405-ENG-48 between the United States
Department of Energy and the University of California for the
operation of Lawrence Livermore National Laboratory.
Claims
1. A method for attaching nucleic acids to a support structure,
comprising: providing a mixture of linear strands of nucleic acids
and excess labeled dideoxy nucleotide bases; adding terminal
transferase to the mixture, wherein the transferase binds a labeled
base to at least one end of a strand; removing the unbound bases
from the mixture; adding to the mixture a plurality of support
structures, wherein each structure comprises a first ligand that
binds to the labeled base at the end of the strand, whereby the end
of the strand is bound to a support structure.
2. The method as recited in claim 1, wherein the nucleotide bases
are labeled in equal molar amounts with a first label and a second
label.
3. The method as recited in claim 2, further comprising removing
strands from the mixture having both ends labeled with the same
label before adding the support structures.
4. The method as recited in claim 3, further comprising adding to
the mixture a plurality of support structures comprising a second
ligand that binds to the second labeled base at the end of the
strand, whereby the ends of the strand are bound to two different
support structures.
5. The method as recited in claim 2, wherein at least one of the
labels comprises an antigen.
6. The method as recited in claim 5, wherein the ligand comprises
an antibody capable of binding to the antigen.
7. The method as recited in claim 5, wherein the antigen is
selected from the group consisting of digoxigenin and biotin.
8. The method as recited in claim 6, wherein the antibody is
selected from the group consisting of anti-digoxigenin, avidin, and
streptavidin.
9. The method as recited in claim 1, wherein the linear strands
comprise blunt, double-stranded ends.
10. The method as recited in claim 9, wherein the linear strands
further comprise single-stranded ends.
11. The method as recited in claim 1, wherein the strands comprise
DNA having at least 500,000 base pairs.
12. The method as recited in claim 1, wherein the strands comprise
DNA having ends of unknown sequence.
13. The method as recited in claim 1, wherein the strands are only
labeled at the ends.
14. The method as recited in claim 1, wherein the support
structures are selected from the group consisting of beads, flat
substrates, and optical handles.
15. The method as recited in claim 1, wherein the support
structures comprise materials selected from the group consisting of
latex, polystyrene, magnetic materials, paramagnetic materials,
glass, mica, and silicon.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to a method for attaching nucleic
acids, such as DNA, to microscopic beads or other support
structures using a terminal transferase.
[0004] 2. Description of Related Art
[0005] Recent technical advancements in nanomanipulation have
allowed the mechanical behavior of single DNA molecules to be
studied. These techniques include the use of microspheres, magnetic
beads, microfibers, microneedles, optical traps, and hydrodynamic
flow. The attachment of microspheres to DNA has proven useful to
manipulate DNA for placement or immobilization on a selected
substrate or mechanical support, where the DNA strand can be
confined in an extended conformation. Once the DNA is affixed to a
substrate, a variety of processes (e.g., laser tweezers, scanning
probe microscopy) can be used to sequence or map gene locations of
the DNA. In addition, tethering microspheres to DNA may be useful
in purification or separation methods that selectively isolate
labeled or tagged DNA fragments.
[0006] Conventional techniques of tethering or attaching DNA to
microspheres rely on hybridization and ligation of manufactured,
labeled single-stranded DNA probes to known DNA sequences. U.S.
Pat. No. 5,674,743 to Ulmer discusses methods in the art for
attaching the DNA to a microscopic bead and is incorporated herein
by reference. One method is to first couple specific
oligonucleotide linkers to the bead using known techniques, and
then to use DNA ligase to link the DNA to the linker on the bead.
Oligonucleotide linkers can be employed which specifically
hybridize to unique sequences at the end of the DNA fragment, such
as the overlapping end from a restriction enzyme site or the
"sticky ends" of bacteriophage lambda based cloning vectors.
[0007] Another method for coupling DNA to beads uses specific
ligands attached to the end of the DNA to link to ligand-binding
molecules attached to the bead. Possible ligand-binding partner
pairs include biotin-avidin/streptavidin, avidin/streptavidin, or
various antibody/antigen pairs such as digoxygenin-antidigoxygenin
antidigoxygenin antibody (Smith et al., "Direct Mechanical
Measurements of the Elasticity of Single DNA Molecules by Using
Magnetic Beads," Science 258:11122-1126 (1992)). Smith et al.
(1992) describe the attachment of the ends of Lambda DNA fragments
to magnetic beads and glass plates using ligated 97-kbp dimers of
methylated phage DNA. The left sticky end of the dimer is
hybridized and ligated to a 12-base oligo, 3' end-labeled
previously with digoxigenin. The right sticky end is similarly
attached to a 12-base oligo constructed with a 3' biotin end-label.
The glass plates and beads were then labeled with antidigoxygenin
and avidin/streptavidin, respectively. In this procedure, the
sticky ends of the dimer are known, and the fragment of DNA is
relatively small.
[0008] In Baumann et al., "Ionic effects on the elasticity of
single DNA molecules", Proc. Natl. Acad. Sci. U.S.A. 94:6185-6190
(1997), lambda phage DNA molecules are tethered between two
streptavidin-coated latex beads (d=3.5 .mu.m). One bead is held by
a micropipette while the other is optically trapped by
force-measuring laser tweezers. The 5'-overhangs of .lambda. DNA
were biotinylated with the Klenow fragment of DNA polymerase using
biotin-11-dCTP (Sigma), DATP, dGTP, and dUTP. The Klenow fragment
is the E. coli DNA polymerase I fragment. The polymerase
catalytically synthesizes new strands of DNA in vitro by moving
along the preexisting single DNA strand and creating a new
complementary strand by incorporating single nucleotides one at a
time into the new strand.
[0009] In Smith et al., "Overstretching B-DNA: The elastic response
of individual double-stranded and single-stranded DNA molecules",
Science 271:795-799 (1996), the DNA was labeled at both ends. Two
oligonucleotides were constructed: a 20-nucleotide strand
complementary to the right overhand of .lambda., and a
5'-biotinylated 8-nucleotide complementary to the remaining 8 base
pairs of the 20-nucleotide fragment. These oligos were hybridized
to each other and to the right end of .lambda., and then ligated
with T4 ligase. The left end of .lambda. was then biotinylated with
Klenow enzyme and bio-11-dCTP.
[0010] In Strick et al., "Behavior of Supercoiled DNA", Biophysical
Jour. 74:2016-2028 (1998), linear DNA molecules (60-kb) were bound
to strepavidin-coated superparamagnetic beads. A segment of
photochemically labeled DNA was affixed to each end of a 48.5-kb
phage .lambda. DNA. A 5-kb fragment tagged roughly every 200-400-bp
with a biotin label was annealed and ligated to the cohesive left
end of the .lambda.DNA with T4 DNA ligase. A 6-kb fragment was
similarly tagged with digoxigenin molecules, and then annealed and
ligated to the cohesive right end of the .lambda.-DNA.
[0011] Covalent chemical attachment of the DNA to the bead can be
accomplished by using standard coupling agents to link the
5'-phosphate on the DNA to coated microspheres through a
phosphoamidate bond. In a particular embodiment in which the DNA
contains the appropriate single-stranded telomeric recognition
site, telomere terminal transferase (Greider et al., Cell
51:887-898 (1987)) can be used to incorporate a biotinylated
nucleotide at the 3' end of the DNA which can then be bound to
avidin immobilized on the bead. In another embodiment, calf thymus
terminal transferase (Kato et al., J. Biol. Chem. 242:2780 (1967))
can be used to incorporate a ligand-linked nucleotide onto the 3'
end of any DNA molecule with a free 3' hydroxyl group. In still
another approach, a DNA-binding protein can be coupled to the bead
by chemistries well known in the art and in such a fashion that the
DNA-binding site is unperturbed. DNA containing the recognition
sequence for the DNA-binding protein can thereby be coupled to the
bead.
[0012] As an alternative to microscopic beads, bead-like structures
referred to as "optical handles" can be chemically synthesized at
the end of a DNA molecule to provide a particle with dimensions and
refractive properties appropriate for manipulation by an optical
trap. The first step in the synthesis of such particle-like
structures at the end of a DNA molecule can be accomplished by
methods similar to those described above, with the sequential
addition of branched oligonucleotides or by modification of the
techniques for the synthesis of starburst dendrimers.
[0013] In general, the first step in the synthesis of an "optical
handle" at a specific target sequence requires the binding of a
bifunctional binding agent to the target sequence. The first
functionality of the binding agent provides a means for uniquely
recognizing and binding to the target DNA sequence. The second
functionality provides a means for binding or linkage to initiate
the first cycle of growth of the optical handle. Such
functionality, for example, can be provided by a biotin group
attached to the DNA recognition and binding functionality so as to
be capable of binding to streptavidin. Adding streptavidin to the
bifunctional binding agent already complexed with its target DNA
sequence then results in a complex composed of target DNA,
bifunctional linker-biotin, and streptavidin. Streptavidin contains
four binding sites for biotin, with two each on opposite sides of
the streptavidin protein molecule. Only one of these sites will be
occupied by binding to the biotin group of the bifunctional linker,
leaving the three other sites available for binding.
[0014] A second linker is introduced which is made of two biotin
molecules joined by a spacer. The spacer couples the two biotin
groups in such a way that each biotin is fully capable of binding
to streptavidin, but the length and rigidity of the spacer is
selected so that both biotin groups cannot bind to the same
streptavidin molecule. Addition of biotin-spacer-biotin to the
complex will result in the binding of one, two or three such
molecules to the previously unoccupied sites in the single
streptavidin molecule, providing one, two or three exposed biotin
groups for subsequent binding to additional streptavidin molecules.
By alternating the addition of streptavidin and
biotin-spacer-biotin, the optical handle is synthesized as an
exponentially growing complex of cross-linked streptavidin
molecules. A sufficient number of cycles are carried out to provide
an optical handle of sufficient size and optical properties for
manipulation by optical tweezers.
[0015] One of the disadvantages of the above techniques for
attaching DNA to microscopic beads or other structures is that the
sequence at the end of the DNA fragment must be known in order to
synthesize an oligonucleotide linker or probe. Thus, completely
unknown DNA fragments cannot be attached to microspheres or
supports using these methods. In addition, if small (<18 bp)
synthesized oligonucleotides are used, these oligonucleotides can
attach to the targeted sequence within large DNA fragments, e.g.,
>500-kb, not just at the ends. Finally, the hybridization
attachment methods are limited to DNA fragments with cohesive,
single-strand ends.
[0016] The present invention addresses the above-mentioned problems
and provides a simple method for attaching microscopic beads and
other particle-like (e.g., optical handles) or support structures
to fragments of DNA of unknown sequences, of any length, and having
either single-stranded or blunt (double-stranded) ends. The present
method is capable of labeling both ends of the double-stranded DNA,
differentiating one end from the other, and labeling the ends
independently of sequence and regardless of 3' or 5' recessive
ends. The present invention also provides for a covalent tethering
of both DNA ends with an appropriate cross-linker and does not
require the synthesis of a synthetic oligonucleotide probe.
SUMMARY OF THE INVENTION
[0017] The object of the invention is to attach nucleic acids,
specifically DNA, to microscopic beads or other support structures
using a terminal transferase. The present invention accomplishes
this attachment without the synthesis of oligonucleotide probes or
known target oligonucleotide or amino acid sequences.
[0018] In the present method, DNA is placed in solution with
labeled dideoxy nucleotide bases. Nucleotides with antigen labels
such as digoxigenin and biotin may be used. When linear
double-stranded DNA is present, terminal transferase is added,
which attaches a single labeled base to each of the 3' ends of the
DNA strands. When two different labeled bases are used (e.g.,
digoxigenin and biotin), the yield of DNA strands with different
labels on the ends is predictably 50%. The differently labeled ends
prevent both ends of a single DNA molecule from attaching to a
single support structure. The labeled DNA is attached to the
microscopic beads or other support structures, which are labeled
with the appropriate antibody (e.g., anti-digoxigenin or
avidin/streptavidin) or other complementary ligand.
[0019] Another object of the present invention is to provide a
method that can be used with DNA strands having blunt
(double-stranded) or single-stranded (cohesive, sticky) ends.
Furthermore, the invention can attach microscopic beads, bead-like
structures such as optical handles, or other support structures to
DNA of any size, including fragments greater than 500 kilobase
pairs (kb).
[0020] Other objects, features, and advantages of the present
invention will become apparent from the following description and
accompanying drawings.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention is a method for attaching microscopic
beads or other structures to nucleic acids using a terminal
transferase. The present method is particularly useful for
attaching microscopic structures to DNA. The microscopic structures
include microspheres and microscopic beads made from latex,
polystyrene, and magnetic or paramagnetic materials. Other
structures include substrates made of glass, mica, or silicon
materials. The present method can be used to tag or label DNA even
when the DNA has an unknown sequence, or is very large (e.g.,
>500 kilobase pairs). The present invention does not require the
synthesis of a synthetic oligonucleotide probe.
[0022] In the present method, DNA is added to a buffered solution
containing labeled dideoxy nucleotide bases, an enzyme, and the
salts and metals needed for the enzyme to be chemically active. The
bases are typically labeled in equal molar amounts with two
different labels, such as the antigens digoxigenin and biotin. The
labels can bind to the antibody compound (e.g., anti-digoxigenin or
avidin/streptavidin) or other appropriate complementary ligand to
form a tight or strong bond. The antibodies or ligands are bound to
the microscopic beads or support structures by methods known in the
art. Two different labels are typically used so that both ends of
the DNA strand do not attach to the microscopic bead or other
support structure. Many types of labels can be used: fluorescent
labels, bioreactive labels, chemiluminescent labels, photolabile
labels, photoreactive labels, radioactive labels, and chemically
reactive labels.
[0023] When linear double-stranded DNA is formed, such as by
heating, terminal transferase is added to the DNA solution.
Terminal transferase is a calf thymus enzyme that adds one labeled
nucleotide to each 3' end of the DNA strands and provides a method
for creating cohesive ends on blunt-ended DNA fragments. The
labeled nucleotide is added independent of sequence or whether the
strands have overhangs (single-stranded) or blunt (double-stranded)
ends. Since the nucleotides add randomly to the ends, the two
labels have equal probability of attaching to each end. Thus, fifty
percent of the labeled DNA strands have different labels at the
ends.
[0024] Since the transferase adds the bases at the ends of the DNA
strands, there is labeling along the strand unless there is a break
in the DNA. The present invention can easily be used with large DNA
sequences, for example, greater than 500 kb. This lack of
"intra-strand" labeling can be a distinct advantage of the present
invention over conventional techniques. Conventional techniques
that depend on hybridizing short oligonucleotide sequences (5-20
bases) that can occur (be repeated) elsewhere along the strand (as
well as at the end) may result in undesirable intra-strand
labeling. Indeed, the longer the DNA sequence and the shorter the
synthesized oligonucleotide (e.g., <18 bp), the more likely the
target sequence will be present within the molecule. Thus,
conventional techniques may produce unsatisfactory results with
large DNA sequences.
[0025] After the reaction is completed, the excess nucleotides are
removed and the DNA is purified of the label. The 50% yield of DNA
strands that have the same label on both ends are removed by
binding the strands first to beads or resin with avidin and washing
off with biotin. The beads (or resin) are then bound with antibody
to digoxigenin. Only those strands having both (different) labels
will bind the second time. The dual-labeled DNA is then mixed with
the microscopic beads or other support structures that are coated
or otherwise bound to the complementary ligand. The end of each DNA
strand with the complementary ligand forms a covalent bond with
bead or support structure. The non-attached end of the DNA, having
a different label, can then be attached to microscopic beads or
support structures that are coated or otherwise bound to a second
complementary ligand.
EXAMPLE
[0026] Terminal Transferase Labeling of Lambda DNA
[0027] The Lambda DNA molecule (Gibco) is added to a buffer
solution containing components required for enzyme activity (e.g.,
enzyme and salts/metals) and dideoxy nucleotide bases labeled in
equal molar amounts with two antigens, such as digoxigenin or
biotin. The solution is heated to denature the cos site of Lambda,
which creates linear double-stranded DNA. Terminal transferase is
added and the solution is heated for one or more hours. The
transferase adds one nucleotide at the 3' end of each of the DNA
strands before the reaction is arrested. The nucleotide is added
independent of sequence or whether the strands have overhangs or
blunt ends. The nucleotides will add randomly to the 3' ends, so
either ddNTP-DIG or ddNTP-BIO has an equal opportunity of binding
to either end of the DNA. Thus, there is an acceptable yield of 50%
labeled DNA fragments containing the digoxigenin label at one end
and a biotin on the other.
[0028] After the excess nucleotides are removed and the DNA is
purified of contaminated label, the DNA is attached to spheres
coated with the complementary antibody. This provides a covalent
tethering of both DNA ends with an appropriate cross-linker. The
antibodies are attached to the spheres by methods known in the
art.
[0029] The foregoing description of preferred embodiments of the
invention is presented for purposes of illustration and description
and is not intended to be exhaustive or to limit the invention to
the precise form disclosed. Many modifications and variations are
possible in light of the above teaching. The embodiments were
chosen and described to best explain the principles of the
invention and its practical application to thereby enable others
skilled in the art to best use the invention in various embodiments
and with various modifications suited to the particular use
contemplated.
* * * * *