Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-12
2001-07-31
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007500, C435S180000, C435S181000, C436S501000, C436S518000, C422S050000, C422S068100, C422S105000, C530S333000, C530S334000, C530S402000, C530S810000, C530S811000, C530S815000
Reexamination Certificate
active
06268141
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Area of the Art
The invention relates generally to solid supports with immobilized biopolymers and specifically to solid supports with immobilized unmodified biopolymers and methods of immobilizing unmodified biopolymers to solid supports.
2. Description of the Prior Art
Biopolymer synthesis and biopolymer analysis often require the attachment of biopolymers to solid supports. For example, organic and inorganic materials have been utilized for the solid phase synthesis of peptides, oligonucleotides and small organic molecules. The synthesis involves the stepwise addition of activated monomers such as amino acid derivatives or nucleotide derivatives to a growing oligomeric chain attached at one end to a solid support. At the completion of the synthesis, the newly synthesized biopolymers may be cleaved from the solid support and subsequently utilized in biochemical research or diagnostic applications or, alternatively, be utilized without cleaving the biopolymers from the solid support.
For biopolymer analysis, biopolymers may be attached to a solid support in several ways. In blotting techniques, native biopolymers are first captured onto a membrane and subsequently immobilized on the membrane by heat, radiation or chemical techniques. The immobilized biopolymers are then available for subsequent analyses, such as those associated with southern blotting applications and reverse hybridization analytical techniques.
Additionally, presynthesized or natural oligonucleotides have been immobilized by covalently attaching activated oligonucleotides to the solid support. Current methodology for the covvalent attachment of nucleic acids to solid supports (substrates) involves modification of the DNA (or RNA). For example, oligonucleotides are usually derivatized to a 5′-amino terminus, making the DNA more reactive for covalent attachment to an activated surface. Other methods of attachment have employed reactions with terminal phosphate groups or Sulfhydral groups with surface carbodiimide or other activation chemistries (see Lund et al,
Nucleic Acid Res
. 16:10861-80, 1988, Bischoffet al,
Analyt. Biochem
. 164: 336-344, 1987).
It is generally understood that reactive groups present within native polynucleotides are weak and therefore make for inefficient attachment. In addition, when native polynucleotides are exposed to highly reactive surface groups, excessive crosslinking may occur. This crosslink may prevent the attached nucleic acid from fully participating in hybridization. These conditions are most noticeable for short fragments of double-stranded DNA or oligonucleotides. Thus, oligonucleotides often have to be modified, for example, derivatized to a 5′-amino terminus, for an effective attachment. The 5′ amino-linker allows selective binding of the amino-containing DNA to silylated slides through a Schiff's base reaction with aldehyde groups on the chip surface. The selectivity of amino-modified versus natural, unmodified DNA is about 10:1 for cDNAs and about 10,100:1 for single-stranded 15-mers. DNA molecules of intermediate lengths exhibit intermediate discrimination ratios. In addition, the 5′ end attachment of the DNA to the chip via the amino group permits steric accessibility of the bound molecules during the hybridization reaction. Therefore, post-modification has been perceived as obligatory for attachment of, e.g., oligonucleotide probes for creation of arrays. Such post-modification processes require additional time-consuming steps at substantial costs.
Therefore, it is desirable to develop a more effective method for attaching biopolymers, particularly unmodified biopolymers to a solid support. It is particularly desirable to develop a method to directly attach unmodified biopolymers, such as polynucleotides, to a solid support.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that both short and long fragments of single-stranded or double-stranded DNA may be efficiently attached to acyl fluoride activated supports directly without spacer arms and without modifying the polynucleotide. It is also based on the discovery that other biopolymers, such as protein A, antibodies, streptavidin, ect., may also be attached to a solid support without modifications to the biopolymers.
Accordingly, one aspect of the present invention provides a method of attaching unmodified biopolymers to a solid support. The method comprises the steps of:
(a) providing unmodified biopolymers;
(b) providing a solid support having at least one surface comprising pendant acyl fluoride lunctionalities, and
(c) contacting the unmodified biopolymers with the solid support under a condition sufficient for allowing the attachment of the biopolymers to the solid support.
According to embodiments of the present invention, the biopolymers may be nucleic acids, polypeptides, proteins, carbohydrates, lipids and analogues thereof In one embodiment of the present invention, the biopolymer is a polynucleotide, and the polynucleotide may be synthesized oligonucleotide, amplified DNA, cDNA, single stranded DNA, double stranded DNA, PNA, RNA or mRNA. Also according to embodiments of the present invention, the solid support may be polymeric materials, glasses, ceramics, natural fibers, silicons, metals and composites thereof
Another aspect of the present invention provides a method of analyzing a biopolymer target in a sample. The method comprises the steps of:
(a) providing a solid support fabricated of a material having pendent acyl fluoride groups on at least one surface;
(b) providing an agent that can form a complex with the biopolymer target, wherein the agent comprises a second biopolymer;
(c) contacting the solid support with either the agent or the biopolymer target under a condition that allows the attachment of either the unmodified agent or the unmodified biopolymer to the solid support, wherein the agent and the biopolymer target are unmodified;
(d) contacting the solid support attached with the unmodified agent with the biopolymer target, or contacting the solid support with the attached, unmodifed biopolymer target with the agent under a condition that allows the formation of a complex comprising the agent and the biopolymer target;
(e) detecting and determining the presence of the complex as a measurement for presence or the amount of the biopolymer target contained in the sample.
A further aspect of the present invention provides a device comprising a plurality of unmodified biopolymer and a solid support. The solid support has at least one surface comprising pendant acyl fluoride functionalities, and the biopolymer is attached to the solid support by reaction with the pendant acyl fluoride functionalities.
The present invention is well-suited for use in creating polynucleotide arrays, such as sensor arrays and other array-based systems such as differential gene expression micro-arrays. The polynucleotide arrays may be used for the evaluation or identification of biological activity. The present invention may also be used in creating polynucleotide arrays for the purpose of polynucleotide sequencing. Further, the present invention may be used in hybridization assays and immunoassays.
The present invention provides many advantages. It allows for the attachment of unmodified biopolymers directly to a solid support. It thus simplifies and further increases the versatility of a process for creating biopolymer arrays.
There is both an economic advantage as well as a technical advantage to this invention. First, costly production of modified biopolymers, such as amino-modified DNA, may be avoided. Post-modification processing of oligonucleotides is time-consuming and can substantially increase costs by as much as two-fold. Second, the task of making arrays is greatly simplified, since post-modification processes are no longer required.
REFERENCES:
patent: 5011861 (1991-04-01), Coull et al.
patent: 5554501 (1996-09-01), Coassin et al.
patent: 5602207 (1997-02-01), Boyd et al.
patent: 5717075 (1998-02-01), Boyd et al.
p
Matson Robert S.
Milton Raymond C.
Beckman Coulter Inc.
Gabel Gailene R.
Grant Arnold
Le Long V.
May William H.
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