Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-21
2002-07-16
Sisson, B. L. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S183000, C435S007100, C435S287200, C436S501000, C436S094000, C530S387100, C536S023100, C536S025320
Reexamination Certificate
active
06420112
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for attaching nucleic acids, such as DNA, to microscopic beads or other support structures using a terminal transferase.
2. Description of Related Art
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.
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.
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, or various antibody/antigen pairs such as digoxygenin-antidigoxygenin antibody (Smith et al., “Direct Mechanical Measurements of the Elasticity of Single DNA Molecules by Using Magnetic Beads,”
Science
258:1122-1126 (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. Individual multimer of &lgr; DNA (48.5 kbp) were chemically attached by one of their ends to a glass slide and by their other end to a magnetic bead. Ligated 97-kbp dimers of methylated phage &lgr; DNA, strain c1857ind1Sam7(NEB) were used. The left sticky end of the dimer is hybridized and ligated to a 12-base oligo. 3′ end-labeled previously with digoxigenin (Boehringer Genius-5). The right sticky end is similarly attached to a 12-base oligo constructed with a 3′ biotin end-label (Glenn Research). The glass microscope slide is successively coated with &ggr;-aminopropyltriethoxysilane (Pierce), protein A, polyclonal antidigoxigenin (Boehringer), and finally cross-linked with dimethyl pimelimidate (Pierce). The molecule ends were labeled differently to prevent attachment of both ends of the DNA to either the glass of the bead. The DNA is free to swivel about either point of attachment.
In Baumann et al., “Ionic effects on the elasticity of single DNA molecules”,
Proc. Natl. Acad. Sci. USA
94:6185-6190 (1997), lambda phage DNA molecules are tethered between two streptavidin-coated latex beads (d=3.5 &mgr;m). One bead is held by a micropipette while the other is optically trapped by force-measuring laser tweezers. The 5′-overhangs of &lgr; 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. &lgr;-DNA molecules were tethered between two streptavidin-coated latex beads (diameter b
53
.
54
mm). One bead was held by a micropipette while the other was optically trapped by force-measuring laser tweezers.
The 5′-overhangs of 1 DNA (methylated c1857ind1Sam7; New England Biolabs) were biotinylated with the Klenow fragment of DNA polymerase using biotin-11-dCTP (Sigma), dATP, dGTP, and dUTP . . . Single-strand nicks were repaired with DNA ligase. After biotinyla-tion and nick ligation, DNA stocks were stored in an EDTA-containing buffer.
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 &lgr;, 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 &lgr;, and then ligated with T4 ligase. The left end of &lgr; was then biotinylated with Klenow enzyme and bio-11-dCTP. Each end of a single &lgr;-phage dsDNA molecule (48.5 kbp) was attached to a separate microscopic latex bead. Carboxylate-polystyrene beads (3.54 &mgr;m in diameter, CV=2.7%, Spherotech) were covalently coated with streptavidin using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). Each molecule was pulled both right and left from the pipette to determine the point of attachment of the molecule on the pipette bead. DNA molecules are compact random coils in solution except when one attaches by an end to the bead on the pipette tip. Extra beads are carried by buffer and one is caught by the laser trap.
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 &lgr; 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 &lgr;-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 &lgr;-DNA. Fifty micrograms of phage 1-DNA (Boehringer-Mannheim, Meylan, France) are recipitated and resuspended in distilled water to eliminate any organic buffers. Two batches of this DNA are aliquoted; the first is subjected to two successive rounds of photolabeling in the presence of 25 mg of photoactivatable biotin (Pierce, Montlucon, France). Photolabeling reactions are carried out according to the Pierce protocol: DNA at a concentration of 1 mg/ml is mixed with an equal volume of photobiotin (also at a concentration of 1 mg/ml). The reaction tube is left open and placed in an ice bath, 10 cm away from a 40-W, 360-nm sunlamp for 10 min. The process is repeated, with the addition of another 25 ml of photobiotin. A second aliquot of purified DNA is subjected to the same sequence of labeling reactions, except that photoactivatable digoxigenin (Boehringer-Mannheim) is used as the labeling molecule. Both batches of labeled DNA are then precipitated in cold ethanol and resuspended in Tris-HCl (10 mM) and EDTA (1 mM) (T10 E1). They are then digested by 10 units of restriction enzyme Nru1 (New England Biolabs, Montignyle-Bretonneux, France) at 37° C. in the enzyme's buffer. The cohesive left (4600 bp) and cohesive right (6700
Balhorn Rodney L.
Barry Christopher H.
Lee Ann M.
Scott Eddie E.
Sisson B. L.
The Regents of the University of California
Thompson Alan H.
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