Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-10-22
2002-05-14
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S091500, C536S022100, C536S023100
Reexamination Certificate
active
06387636
ABSTRACT:
TECHNICAL FIELD
This invention relates to arrays of biopolymers used in diagnostics, screening, gene expression analysis, and other applications. In particular, the invention relates to a method of fabricating biopolymer arrays, such as DNA arrays.
BACKGROUND ART
Polynucleotide arrays, such as DNA or RNA arrays, are known and are used, for example, as diagnostic or screening tools. Such arrays comprise a plurality of different polynucleotide probes arranged in a predetermined configuration on a substrate. The polynucleotides of the plurality differ by having a different nucleotide sequence. Different polynucleotide probes are located at different regions (also known as features or spots) on the substrate, wherein in each region, multiple copies of the same polynucleotide are usually present.
The array is exposed to a sample of biological material to be evaluated, also known as the “target”. Upon exposure to the target sample, the array will exhibit a binding pattern, wherein complementary target polynucleotides will hybridize or bind to the array polynucleotide probes during an assay. This binding pattern can be observed, for example, by labeling all polynucleotide targets (for example, DNA) in the sample with a suitable label (such as a fluorescent compound), and accurately observing the fluorescence pattern on the array. Assuming that the different sequence polynucleotide probes were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the target sample.
Biopolymer arrays can be fabricated using either methods of deposition of intact biopolymer species or using in situ synthesis methods. The deposition methods basically involve depositing intact biopolymers at predetermined locations on a substrate that are suitably activated such that the intact biopolymers can link thereto. The intact species of biopolymers, each having different monomer sequences, may be deposited at different regions of the substrate to yield the completed array having a predetermined configuration. Typical procedures known in the art for deposition of intact polynucleotide species, particularly DNA such as whole oligomers or cDNA, are to load a small volume of DNA in solution in one or more drop dispensers such as the tip of a pin or in an open capillary and, touch the pin or capillary to the surface of the substrate. Such a procedure is described in U.S. Pat. No. 5,807,522. When the fluid touches the substrate surface, some of the fluid is transferred from the pin or capillary to the substrate location. The pin or capillary must be washed prior to picking up the next type of DNA for spotting onto the array. This process is repeated for the plurality of different polynucleotides and, eventually, the desired array having a predetermined configuration is formed. Alternatively, the DNA can be loaded into a drop dispenser in the form of an inkjet head and fired onto the substrate. Such a technique has been described, for example, in PCT publications WO 95/25116 and WO 98/41531, and elsewhere.
The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA) using phosphoramidite or other chemistry. Such in situ synthesis methods can be basically regarded as iterative steps of depositing droplets of. (a) a protected monomer onto predetermined locations on a substrate to link with either a suitably activated substrate surface (or with a previously deposited deprotected monomer); (b) deprotecting the deposited monomer so that it can now react with a subsequently deposited protected monomer; and (c) depositing another protected monomer for linking. Different monomers may be deposited at different regions on the substrate during any one cycle so that the different regions of the completed array will carry the plurality of different biopolymer sequences as desired in the completed array. In situ synthesis methods may require one or more intermediate further steps in each iteration, such as oxidation and washing steps, as are well known in the art.
In order for an assay to yield accurate results, it is important that the different biopolymer features actually be present on the array, that they are put down accurately in the desired or predetermined pattern, that the biopolymers are of the correct size, and that each different feature be uniformly populated with the respective biopolymer.
In polynucleotide arrays, the conventional in situ synthesis methods use phosphoramidite nucleoside monomers. In order for the phosphoramidite group to link to a hydroxyl of a previously deposited deprotected polynucleotide monomer, it must first be activated usually by using a weak acid, such as tetrazole. However, an activated phosphoramidite is highly reactive with moisture in the air. Therefore, unless some precaution is taken, the activated phosphoramidite can be used up before the desired reaction is complete. As a result there is a reduction in the deposited phosphoramidite monomer available for forming the complete polynucleotide. This problem is present even when the synthesis is performed in a nitrogen chamber.
Further, the size (volume) of the synthesis droplet on the substrate surface could be very small, such as a few pico- or nano-liters, such that the ratio of surface to volume is very high. A high surface to volume ratio favors the diffusion of moisture into the droplets. Initially, the moisture from the air tends to be adsorbed at the surface of the synthesis droplet. Therefore, the phosphoramidite concentration at the surface of the droplet will tend to be lowest. Consequently, the concentration of a completed probe polynucleotide at a feature on the array tends to decrease from the center of a feature toward its perimeter. Variations in completed probe concentration within a feature result in a decrease in the concentration of target sample that consequently hybridizes to the respective polynucleotide probe. Therefore, the total signal that should be available from the hybridized target is diminished at the particular feature location during optical evaluation of the array. Further, it should be noted that the water vapor concentration in the ambient atmosphere might vary. Therefore, the signal from the hybridized target may also vary from array to array, leading to inconsistency in absolute signal generated from different arrays of a batch when the same concentration of a target is encountered.
The foregoing problems exist particularly where the phosphoramidite is mixed with the activator and the mixture is deposited as a droplet on the substrate, and even where the activator is deposited onto a previously deposited droplet containing the phosphoramidite, both as such are described in PCT publication WO 98-41531. In either case, ambient moisture presents a problem. Furthermore, when one droplet is deposited on the other, there is no guarantee of efficient mixing such that the activated phosphoramidite will be evenly present at the substrate surface.
Thus, it would be advantageous to have a means of fabricating biopolymer arrays that lessens the likelihood of deleterious environmental influences on the accuracy of the fabrication. In particular, it would be desirable, in the fabrication of arrays of biopolymers using biomonomers with a linking group that must be activated (such as a phosphoramidite), to provide a means by which the potential reactivity of the activated biomonomer with an ambient atmosphere component (such as water vapor in air) can be kept low.
SUMMARY OF THE INVENTION
The present invention provides a method of fabricating an array of biopolymers on a substrate. The method is useful for shielding biosynthesis reactions and the bio-reactants from the ambient environment. Moreover, the method is useful for shielding pre-synthesized biopolymers during their attachment to an array substrate. In
Lefkowitz Steven M.
Perbost Michel G. M.
Agilent Technologie,s Inc.
Celsa Bennett
Prasthofer Thomas
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