Organic compounds -- part of the class 532-570 series – Organic compounds – Silicon containing
Utility Patent
1997-10-16
2001-01-02
Ceperley, Mary E. (Department: 1641)
Organic compounds -- part of the class 532-570 series
Organic compounds
Silicon containing
C204S600000, C435S006120, C436S524000, C436S525000, C436S527000, C436S532000, C562S083000
Utility Patent
active
06169194
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the preparation of covalently immobilized nucleic acids onto solid supports using a novel trichlorosilane adhesion agent which forms a monolayer film and provides a reactive thiol functionality to which oligonucleotides can be attached in very high density. The oligonucleotides can be modified using novel linking agents which allow nucleic acids to be attached to the silanized surface either permanently or reversibly using a chemoselective method in buffered aqueous solution at room temperature. A protocol to create highly reproducible silane monolayers by moisture and humidity control is described. Included in the invention are processes for the synthesis of the silane, synthesis of novel bifunctional linkers for nucleic acid modification, and immobilization protocols.
BACKGROUND OF THE INVENTION
General Scope
Medical diagnosis and treatment of diseases at the genetic level is quickly becoming a reality. Drug design strategies will increasingly depend on developing new methods for regulating gene expression. Early detection of infectious viral diseases and genetic mutations using fast, reliable diagnostic techniques combined with gene-therapy strategies create the possibility of effecting a cure before symptoms of the disease appear. New technologies based on gene isolation and purification, synthesis, amplification, and detection are required to meet these challenges. These emerging technologies require improved methods for oligonucleotide immobilization in several key fields including: nucleic acid separation/purification; nucleic acid amplification (solid-phase PCR); oligonucleotide synthesis; isolation of nucleic acid binding proteins and drugs; and detection of oligonucleotides through hybridization; and sequencing.
Amplification of Oligonucleotides by Solid Phase PCR
The polymerase chain reaction (PCR) is a rapid procedure for producing many copies of a specific segment of DNA in vitro. This technique has now made possible many applications such as molecular genetic research, gene sequencing, forensic/criminal and clinical investigations and many others in which only a minuscule quantity of DNA is available. PCR was originally developed for the solution phase and requires four essential ingredients; Taq DNA polymerase which is the enzyme responsible for building the new DNA copies, the original DNA strand to be amplified, the four triphosphate bases, and finally, the priming sequences from which the new DNA copies will grow. A much improved method is to attach the priming sequences to a solid support which allows the amplified DNA to be chemoselectively removed after the reaction is completed.
Oligonucleotide Synthesis Via Solid Phase Methods
Short fragments of single-stranded DNA or RNA of any desired sequence can be rapidly synthesized using an automated DNA synthesizer which is now commonplace in many laboratories. Oligonucleotides are made by the sequential addition of activated monomers to a growing chain that is linked to an insoluble solid support. The solid-phase synthetic method has the following advantages: reaction yields can be near-quantitative by using excess reagents which can then be easily removed by filtration processes; the repetitive synthesis is readily automated; handling is minimized thereby decreasing the risk of contamination; and wastage of expensive reagents is also minimized. Functionalized polystyrene beads or carboxyl-derivatized controlled-pore-glass are commonly used as supports, with the powdered material being sealed into a tube which has porous frits at both ends. Usually, the first nucleotide is attached to the solid support via a carboxylic ester link to the 3′ hydroxyl, and synthesis is carried out in the 3′-5′ direction. A high ratio of oligonucleotide to surface area is required to optimally perform the synthesis to prevent wasteage of reagents. It is advantageous to attach special tethering groups to one of the chain ends to act as a chemical “handle” so the single-stranded nucleic acid can be attached to other solid surfaces such as affinity columns or biosensor devices. It should be mentioned that solid phase synthesis and the immobilization technology to which it is dependant, can be applied to the synthesis of other types of polymers as well including peptides, proteins, and combinatorial synthesis of diverse arrays of any class of molecule.
Separation, Isolation and Purification of Oligonucleic Acids, Oligonucleotide—Other Molecule Complexes and Other Biomolecules
Biomolecules in general can be purified by electrophoretic, chromatographic, filtration or by affinity techniques. Electrophoresis is widely used to separate nucleic acid fragments in a gel matrix. The fragments are usually transfered or “blotted” onto a membrane which has an affinity for the nucleic acids so that further processing can be accomplished. Reverse-Phase Liquid Chromatography (RPLC) has been used to separate mixtures of nucleic acids, proteins and other biomolecules on coated solid supports. Microfiltration is used to remove impurities from biomolecule preparations.
Improved separations can be achieved by immobilizing various sequences of nucleic acids onto the stationary media to produce an “affinity” hybrid technique. Single-stranded nucleic acids can be immobilized onto a solid support, to which the complementary strand can specifically hybridize; such a technique is referred to as hybridization. Impurities are washed away, while the complementary strand remains affixed, and elution selectively occurs when variables such as buffer strength are changed. In such a manner, improved electrophoresis membranes for “Northern blots”, nucleic acid chromatographic supports, and nucleic acid binding filtration media can be made.
Molecules other than nucleic acids can specifically recognize immobilized nucleic acids. Research to discover new treatments for genetic diseases requires the development of novel methods for investigating the interactions of genes with regulatory proteins. Gene transcription, replication and repair are mediated by many DNA or RNA binding proteins. Drugs such as cis-diaminedichloroplatinum (II) known as “cisplatin” which has antitumor activity for the treatment of ovarian, bladder and testicular cancer, anthracycline antibiotics and polycyclic aromatic compounds can intercalate into DNA structures. Antisense drug therapy innovations are directed at strongly binding a complementary segment of nucleic acid material to the target gene in a highly selective fashion. Similar to nucleic acid purification through immobilized hybridization as mentioned earlier, proteins, drugs and any type of nucleic acid binding molecule can be purified through selective interactions with immobilized oligonucleotides. In all cases, a high density of immobilized oligonucleotides will result in increased efficiency, together with minimization of waste production.
Detection of Oligonucleotides, Antisense Compounds and Small Molecules
Detection of oligonucleotides for diagnostic assays through hybridization and sequencing is also dependant on high density surface immobilization of oligonucleotides. Determining genetic sequences is a well established field. Standard techniques such as electrophoretic separation of partially digested nucleic acid fragments are generally too slow for clinical work. A different approach is sequencing by hybridization or SBH in which a library of short oligonucleotide probes, labelled in some way, and of known sequence, are presented to unknown DNA. When complementary sequences are found, a process known as hybridization occurs which allows for signalling the presence of a particular sequence in the gene. New techniques such as micromachined capillary electrophoresis arrays require high density immobilization techniques, as each channel possesses a very minute surface area.
Immobilized nucleic acid probes on sensor surfaces can provide much faster analyses at a fraction of the cost. Such is the basis for a “gene chip” in which vast arrays of different genetic probes,
McGovern Mark E.
Thompson Michael
Ceperley Mary E.
Ridout & Maybee
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