Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-06
2001-06-12
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024330
Reexamination Certificate
active
06245518
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to spatially-addressable arrays of molecules, particularly biological molecules such as peptides and oligonucleotide probes, and methods of making and using the same.
2. BACKGROUND OF THE INVENTION
Recent advances in the ability to construct arrays of biological molecules has greatly facilitated the ease and speed with which certain biological assays can be performed. For example, in the areas of nucleic acid sequencing and analysis, the advent of new technologies for constructing arrays of immobilized target nucleic acids or oligonucleotide probes has enabled the rapid screening and sequencing of nucleic acids. Arrays of peptides and small biomolecules have also proven useful in binding assays used in pharmaceutical development. The usefulness of these arrays depends on the ability to generate arrays with spatially addressable regions of defined composition or sequence.
Several technologies have been developed for producing these arrays of biological molecules. Several researchers have devised methods for in situ synthesis of arrays of biological polymers, such as nucleic acids, peptides, and carbohydrates. These methods use, for example, physical barriers to separate regions, devices (such as inkjet printers) for precise delivery of reagents to regions, or masking techniques that allow the use of light to determine the course of synthesis. See, e.g., WO 90/03382; Fodor et al., 1991, Science 251:767-73; Pease et aL., 1994, Proc. Natl. Acad. Sci. 91:5022-26; U.S. Pat. No. 5,424,186, to Fodor et al. Alternatively, presynthesized biomolecules or biological polymers may be attached directly to the substrate at precise positions using a variety of techniques, ranging from simple spotting to robotic delivery systems. A variety of different substrates and techniques for attaching the biomolecules to the substrates are also available.
As noted above, arrays of nucleic acids have proven particularly valuable. The ability to perform many previously available techniques has been greatly enhanced by availability of arrays, which permit many assays to be performed simultaneously on a single array rather than having to do each assay individually. Other techniques that would have been virtually impossible are now possible using polynucleotide arrays.
One technique that has been particularly enhanced by the availability of arrays of nucleic acids is sequencing by hybridization (SBH). SBH is a technique for rapidly sequencing nucleic acids without using gels. In SBH, polynucleotides having overlapping sequences are hybridized to a target nucleic acid. The sequences of the polynucleotides that hybridize are then determined and the common sequences overlapped to generate the sequence of the nucleic acid. The use of arrays has allowed the generation of sufficient hybridization information to make SBH feasible on a large scale.
SBH is divided into three formats, depending on the nature of the array and the way in which it is interrogated. In Format I, the target nucleic acid is immobilized and the labeled polynucleotides are in solution. In Format II, the polynucleotides are immobilized and the labeled target nucleic acid is in solution. In Format III, immobilized polynucleotides are hybridized with an unlabeled target nucleic acid and labeled oligonucleotide probes. Hybridization is assayed by ligating the labeled oligonucleotide probes to the immobilized polynucleotides. All three formats require the ability to distinguish perfectly matched hybrids from hybrids that contain a single mismatch at any position. For a more detailed discussion of SBH and the three formats, see WO 98/31836, particularly at pages 1-3.
While the demand for biological arrays, and in particular polynucleotide arrays, is high, current methodologies for constructing such arrays still suffer from certain difficulties. The most common difficulty is assaying the quality and integrity of an array once it has been fabricated. While the chemistries involved in producing the arrays are relatively well understood, methods for synthesizing arrays still suffer from lack of reliability and reproducibility, and even failure. However, identifying regions of attachment failures is very difficult, particularly with the small spots found in miniaturized arrays. Thus, quality control of produced arrays is very difficult to maintain. Furthermore, even minor variations in attachment efficiencies can make interpretation of results generated from such arrays very difficult, as the researcher may not be able to tell whether a difference in signal is real or merely an artifact of the attachment process. This problem is particularly acute in applications such as sequencing by hybridization, which require extremely accurate differentiation of even minor differences in hybridization.
3. SUMMARY OF THE INVENTION
These and other shortcomings in the art are overcome by the present invention, which in one aspect provides spatially addressable arrays of immobilized molecules in which each spot in the array contains an amount of a detectable label which is proportional to the amount of molecule immobilized at that spot. The label can be any molecule which is capable of producing a detectable, quantifiable signal, such as a radioisotope, fluorophore, chromophore, chemiluminescent moiety, etc. The labels at each spot may be the same or different, but are preferably the same.
In another aspect, the invention provides methods of making arrays of immobilized molecules in which each spot in the array contains an amount of a detectable label which is proportional to the amount of molecule immobilized at that spot. In the method, a molecule to be immobilized at a particular spot on the array is “spiked” with a detectable label capable of immobilizing to the substrate with the same efficiency as the molecule. The molecules to be immobilized at different spots are each “spiked” with the same proportion of label. Thus, following immobilization, each spot in the array contains an amount of label which is proportional to the efficiency of the immobilization technique. Following synthesis, the array can be scanned or otherwise analyzed for detectable signal to monitor the fidelity of the array synthesis.
In a preferred embodiment, the label is attached to, incorporated within, or otherwise associated with the same type of molecule as that to be immobilized. Accordingly, in this preferred embodiment of the methods, the molecule to be immobilized is spiked with a small amount of labeled molecule of the same type. Again, the molecules to be immobilized at different locations are each spiked with the same proportion of labeled molecule.
In another aspect, the invention provides methods of increasing the accuracy of array-based assays. In the method, background signals produced from an array of spatially addressable immobilized molecules according to the invention are quantified and recorded.
The array is contacted with a target molecule capable of interacting with at least one of the immobilized molecules. The target molecule is labeled in some manner to produce an assay signal, or the interaction between the target and immobilized molecule is such that only those spots on the array where interaction has taken place produce a detectable assay signal.
Following contact and optional washing, the array is scanned or otherwise analyzed for detectable assay signal, and the signal from each labeled spot quantified. The intensities of the signals from the respective spots are then normalized, typically by obtaining the ratio I
a
/I
b
(where I
a
is the assay signal intensity and I
b
is the background signal intensity), to account for signal differences caused by deviations in the quantities of immobilized molecules. This normalization process permits signal intensities from different spots on the array to be directly compared, regardless of the fidelity of the array synthesis.
The labels giving rise to the background signals and assay signals, i.e., the moieties used to label the array spots and target molecules, respect
Brusca John S.
Hyseq Inc.
Kim Young
Pennie & Edmonds LLP
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