Nucleic acid arrays and methods of synthesis

Details Nucleic acid arrays and methods of synthesis Nucleic acid arrays and methods of synthesis

1999-04-08

2001-09-04

Whisenant, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091100, C436S094000, C536S023100, C536S024300, C536S024330

Reexamination Certificate

active

06284497

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to high density nucleic acid arrays and methods of synthesizing oligonucleotides on a solid surface. Specifically, the present invention contemplates the use of stabilized nucleic acid primer sequences immobilized on solid surfaces, and circular nucleic acid sequence templates combined with the use of isothermal rolling circle amplification to thereby increase oligonucleotide concentrations in a sample or on an array of oligonucleotides.
BACKGROUND OF THE INVENTION
It is estimated that the human genome encodes from 60,000 to 100,000 different genes, and that certain mutations in the genome lead to dysfunctional proteins, giving rise to a multitude of diseases. Assays capable of detecting the presence of particular mutations in a DNA sample are of substantial importance in forensics, medicine, epidemiology, public health, and in the prediction and diagnosis of disease. Such assays can be used, for example, to identify the causal agent of an infectious disease, to predict the likelihood that an individual will suffer from a genetic disease, to determine the purity of drinking water or milk, or to identify tissue samples.
Technologies are presently available that automate the processing and interpretation of such assays. For example, U.S. Pat. No. 5,874,219 to Rava, et al., teaches processing multiple chip assays by providing biological chips comprising molecular probe arrays. The biological chip is subjected to manipulation by fluid handling devices that automatically perform steps to carry out reactions between target molecules in the samples and probes. The chip is further subjecting to a reader that examines the probe arrays to detect any reactions between target molecules and probes. While this sophisticated technology is useful, the sensitivity of detection assays generally is often limited by the concentration at which a particular target nucleic acid molecule is present in a sample. Thus, methods that are capable of amplifying the concentration of nucleic acid molecules must be developed as important adjuncts to detection assays.
Methods of synthesizing desired single stranded DNA sequences are well known to those of skill in the art. In particular, methods of synthesizing oligonucleotides are found in, for example,
Oligonucleotide Synthesis: A Practical Approach,
Gait, ed., IRL Press, Oxford (1984). Methods of forming large arrays of oligonucleotides, peptides and other polymer sequences have been devised. Of particular note, Pirrung et al., U.S. Pat. No. 5,143,854, incorporated herein by reference, disclose methods of forming arrays of peptides, oligonucleotides and other polymer sequences using, for example, light-directed synthesis techniques. However, the above techniques produces only a relatively low concentrations of DNA; that is, the number of DNA on the array is limited to surface area.
One approach for overcoming the limitation of DNA concentration is to selectively amplify the nucleic acid molecule whose detection is desired prior to performing the assay. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments in vivo have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. An example of such methodologies are provided by, for example,
Molecular Cloning, A Laboratory, Manual,
2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989), incorporated herein by reference. However, these methods are limited because the concentration of a target molecule in a sample under evaluation is so low that it cannot be readily cloned.
In an effort to solve such limitations, other methods of in vitro nucleic acid amplification have been developed that employ template directed extension. In such methods, the nucleic acid molecule is used as a template for extension of a nucleic acid primer in a reaction catalyzed by polymerase. One such template extension method is the “polymerase chain reaction” (“PCR”); see Mullis, K. et al., Cold Spring Harbor,
Symp. Quant. Biol.,
51:263-273 (1986), incorporated herein by reference. PCR technology has several deficiencies. First, it requires the preparation of two different primers which hybridize to two oligonucleotide sequences of the target sequence flanking the region that is to be amplified. The concentration of the two primers can be rate limiting for the reaction. A disparity between the concentrations of the two primers can greatly reduce the overall yield of the reaction. The reaction conditions chosen must be such that both primers “prime” with similar efficiency. Since the two primers necessarily have different sequences, this requirement can constrain the choice of primers and require considerable experimentation. Finally, PCR requires the thermocycling of the molecules being amplified. The thermocycling requirement attenuates the overall rate of amplification because further extension of a primer ceases when the sample is heated to denature double-stranded nucleic acid molecules. Thus, to the extent that the extension of any primer molecule has not been completed prior to the next heating step of the cycle, the rate of amplification is impaired.
Other known nucleic acid amplification procedures include transcription-based amplification systems; for example, see Kwoh D. et al.,
Proc. Natl. Acad. Sci.
(
U.S.A.
), 86:1173 (1989). These methods are limited in that the amplification procedures depend on the time spent for all molecules to have finished a step in a cycling method. Particular molecules used to perform the method have different enzymatic rates. Molecules with slower enzymatic rates would slow down molecules with faster enzymatic rates in the cycle. This slowing down of the faster acting enzymes leads to a lower exponent of amplification, and hence, a lower concentration of DNA. Examples of others systems developed to amplify nucleotide sequences are described in U.S. Pat. No. 5,854,033 to Lizardi, incorporated herein by reference. Lizardi, however, does not describe solid surface immobilization of the primers used for extension, as the Lizardi method is performed in solution. This reference is therefore limited because it does not allow for the immobilization of the oligonucleotides, does not form an array, and hence suffers from the same deficiencies as the other methods described above.
Clearly, there is a great need for DNA arrays that allow for higher concentrations of DNA. Furthermore, approaches are needed to synthesize the arrays and particular target nucleic acid molecules at increased concentrations.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, for purposes of the present invention, the following terms are defined below.
The term “rolling circle amplification” (“RCA”) as used herein describes a method of DNA replication and amplification that results in a strand of nucleic acid comprising one or more copies of a sequence that is a complimentary to a sequence of the original circular DNA. This process for amplifying (generating complimentary copies) comprises hybridizing an oligonucleotide primer to the circular target DNA, followed by isothermal cycling (e.g., in the presence of a ligase and a DNA polymerase). A single round of amplification using RCA results in a large amplification of the sequences in the circular target to obtain a high concentration the desired oligonucleotide on a single strand of nucleic acid. Because the desired nucleic acid sequence becomes the predominant sequence (in terms of concentration) in the mixture, it is said to be “RCA amplified”. With RCA, it is possible to amplify a single

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