Replica amplification of nucleic acid arrays

Details Replica amplification of nucleic acid arrays Replica amplification of nucleic acid arrays

1998-08-28

2002-08-13

Marschel, Ardin H. (Department: 1631)

Chemical apparatus and process disinfecting, deodorizing, preser

Analyzer, structured indicator, or manipulative laboratory...

Means for analyzing liquid or solid sample

C422S050000, C435S006120, C536S022100, C536S023100

Reexamination Certificate

active

06432360

ABSTRACT:

FIELD OF THE INVENTION
The invention relates in general to the reproducible, mass-production of nucleic acid arrays.
BACKGROUND OF THE INVENTION
Arrays of nucleic acid molecules are of enormous utility in facilitating methods aimed at genomic characterization (such as polymorphism analysis and high-throughput sequencing techniques), screening of clinical patients or entire pedigrees for the risk of genetic disease, elucidation of protein/DNA- or protein/protein interactions or the assay of candidate pharmaceutical compounds for efficacy; however, such arrays are both labor-intensive and costly to produce by conventional methods. Highly ordered arrays of nucleic acid fragments are known in the art (Fodor et al., U.S. Pat. No. 5,510,270; Lockhart et al., U.S. Pat. No. 5,556,752). Chetverin and Kramer (WO 93/17126) are said to disclose a highly ordered array which may be amplified.
U.S. Pat. No. 5,616,478 of Chetverin and Chetverina reportedly claims methods of nucleic acid amplification, in which pools of nucleic acid molecules are positioned on a support matrix to which they are not covalently linked. Utermohlen (U.S. Pat. No. 5,437,976) is said to disclose nucleic acid molecules randomly immobilized on a reusable matrix.
There is need in the art for improved methods of nucleic acid array design and production.
SUMMARY OF THE INVENTION
The invention provides a method of producing a plurality of a nucleic acid array, comprising, in order, the steps of amplifying in situ nucleic acid molecules of a first randomly-patterned, immobilized nucleic acid array comprising a heterogeneous pool of nucleic acid molecules affixed to a support, transferring at least a subset of the nucleic acid molecules produced by such amplifying to a second support, and affixing the subset so transferred to the second support to form a second randomly-patterned, immobilized nucleic acid array, wherein the nucleic acid molecules of the second array occupy positions that correspond to those of the nucleic acid molecules from which they were amplified on the first array, so that the first array serves as a template to produce a plurality.
As used herein in reference to nucleic acid arrays, the term “plurality” is defined as designating two or more such arrays, wherein a first (or “template”) array plus a second array made from it comprise a plurality. When such a plurality comprises more than two arrays, arrays beyond the second array may be produced using either the first array or any copy of it as a template.
As used herein, the terms “randomly-patterned” or “random” refer to a non-ordered, non-Cartesian distribution (in other words, not arranged at pre-determined points along the x- and y axes of a grid or at defined ‘clock positions’, degrees or radii from the center of a radial pattern) of nucleic acid molecules over a support, that is not achieved through an intentional design (or program by which such a design may be achieved) or by placement of individual nucleic acid features. Such a “randomly-patterned” or “random” array of nucleic acids may be achieved by dropping, spraying, plating or spreading a solution, emulsion, aerosol, vapor or dry preparation comprising a pool of nucleic acid molecules onto a support and allowing the nucleic acid molecules to settle onto the support without intervention in any manner to direct them to specific sites thereon.
As used herein, the terms “immobilized” or “affixed” refer to covalent linkage between a nucleic acid molecule and a support matrix.
As used herein, the term “array” refers to a heterogeneous pool of nucleic acid molecules that is distributed over a support matrix; preferably, these molecules differing in sequence are spaced at a distance from one another sufficient to permit the identification of discrete features of the array.
As used herein, the term “heterogeneous” is defined to refer to a population or collection of nucleic acid molecules that comprises a plurality of different sequences; it is contemplated that a heterogeneous pool of nucleic acid molecules results from a preparation of RNA or DNA from a cell which may be unfractionated or partially-fractionated.
An “unfractionated” nucleic acid preparation is defined as that which has not undergone the selective removal of any sequences present in the complement of RNA or DNA, as the case may be, of the biological sample from which it was prepared. A nucleic acid preparation in which the average molecular weight has been lowered by cleaving the component nucleic acid molecules, but which still retains all sequences, is still “unfractionated” according to this definition, as it retains the diversity of sequences present in the biological sample from which it was prepared.
A “partially-fractionated” nucleic acid preparation may have undergone qualitative size-selection. In this case, uncleaved sequences, such as whole chromosomes or RNA molecules, are Us selectively retained or removed based upon size. In addition, a “partially-fractionated” preparation may comprise molecules that have undergone selection through hybridization to a sequence of interest; alternatively, a “partially-fractionated” preparation may have had undesirable sequences removed through hybridization. It is contemplated that a “partially-fractionated” pool of nucleic acid molecules will not comprise a single sequence that has been enriched after extraction from the biological sample to the point at which it is pure, or substantially pure.
In this context, “substantially pure” refers to a single nucleic acid sequence that is represented by a majority of nucleic acid molecules of the pool. Again, this refers to enrichment of a sequence in vitro; obviously, if a given sequence is heavily represented in the biological sample, a preparation containing it is not excluded from use according to the invention.
As used herein, the term “biological sample” refers to a whole organism or a subset of its tissues, cells or component parts (e.g. fluids). “biological sample” further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. Lastly, “biological sample” refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as nucleic acid molecules.
As used herein, the term “organism” refers to all cellular life-forms, such as prokaryotes and eukaryotes, as well as non-cellular, nucleic acid-containing entities, such as bacteriophage and viruses.
As used herein, the term “feature” refers to each nucleic acid sequence occupying a discrete physical location on the array; if a given sequence is represented at more than one such site, each site is classified as a feature. In this context, the term “nucleic acid sequence” may refer either to a single nucleic acid molecule, whether double or single-stranded, to a “clone” of amplified copies of a nucleic acid molecule present at the same physical location on the array or to a replica, on a separate support, of such a clone.
As used herein, the term “amplifying” refers to production of copies of a nucleic acid molecule of the array via repeated rounds of primed enzymatic synthesis; “in situ amplification” indicates that such amplifying takes place with the template nucleic acid molecule positioned on a support according to the invention, rather than in solution.
As used herein, the term “support” refers to a matrix upon which nucleic acid molecules of a nucleic acid array are immobilized; preferably, a support is semi-solid.
As used herein, the term “semi-solid” refers to a compressible matrix with both a solid and a liquid component, wherein the liquid occupies pores, spaces or other interstices between the solid matrix elements.
As used herein in reference to the physical placement of nucleic acid molecules or features and/or their orientation relative to one another on an array of the invention, the terms “correspond” or “corresponding” refer to a molecule occupying a position on a second array that is either identical t

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