Biopolymer arrays and their fabrication

Details Biopolymer arrays and their fabrication Biopolymer arrays and their fabrication

1999-07-16

2001-10-23

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S022100, C536S025340, C536S027400

Reexamination Certificate

active

06306599

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to arrays, particularly polynucleotide arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
BACKGROUND OF THE INVENTION
Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions (sometimes referenced as features or spots) of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. The arrays, when exposed to a sample, will exhibit a binding pattern. 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 polynucleotides 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 sample.
Biopolymer arrays can be fabricated using either deposition of the previously obtained biopolymers or in situ synthesis methods. The deposition methods basically involve depositing biopolymers at predetermined locations on a substrate which are suitably activated such that the biopolymers can link thereto. Biopolymers of different sequence may be deposited at different regions of the substrate to yield the completed array. Typical procedures known in the art for deposition of previously obtained polynucleotides, 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 surface, some of the fluid is transferred. 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 many different sequences and, eventually, the desired array 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 iterating the sequence 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 different biopolymer sequences as desired in the completed array. One or more intermediate further steps may be required in each iteration, such as oxidation and washing steps.
It is important in such arrays that features actually be present, that they are put down accurately in the desired pattern, are of the correct size, and that the DNA is uniformly coated within the feature. However, in the conventional in situ methods for polynucleotide arrays, phosphoramidite nucleoside monomers are used. 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. This leads to a reduction in deposited monomer available for reaction. Furthermore, since water tends to be adsorbed initially at the surface of a droplet which is being used in one cycle of forming a polynucleotide at a feature, 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 of the array, tends to decrease from the center of a feature toward its perimeter. This leads to a decrease in the total signal that should be available when a target to which that polynucleotide hybridizes, is detected. Furthermore, since water vapor concentration the ambient atmosphere may vary, such signal 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 particularly exist where the phosphoramidite is mixed with activator and the mixture deposited as a droplet on the substrate, such as described in PCT publication WO98-41531. The foregoing reference also states that the activator can be deposited onto a previously deposited droplet containing the phosphoramidite. However, the potential for the activated phorphoramidite to react with moisture in the ambient atmosphere still exists. 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.
It would be desirable then, in the fabrication of arrays of biopolymers using biomonomers with a linking group which must be activated (such as a phosphoramidite), to provide a means by which 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 then, provides a method of fabricating an addressable array of biopolymers on a substrate using a biomonomer with a first linking group which must be activated for linking to a substrate bound moiety. The method includes forming on a region of the substrate carrying the substrate bound moiety, a solid activator composition. A biomonomer containing fluid composition is deposited on the region so that the solid activator activates the first linking group and the biomonomer links to the substrate bound moiety. Typically, the foregoing steps are repeated, with a biomonomer deposited and linked to the substrate bound moiety in one cycle acting as the substrate bound moiety for the next cycle, so as to form the biopolymer. However, it will be appreciated that one or more such cycles can be performed.
In the fabrication of a typical array with multiple features, all of the foregoing steps are repeated at each of multiple different regions on the same substrate, where it is desired to form the features. Generally, the biomonomer containing fluid will be deposited after forming the solid activator composition on the region or regions. As to of forming the solid activator composition at the region, one way of accomplishing this is to deposit a composition of solid activator as a fluid composition, and allowing fluid to evaporate. In this case, the fluid composition may have less than 20% by weight of solid activator content, for example 3% to 20% by weight (or even less than 10% by weight). In one aspect of the method, a sufficient amount of the biomonomer fluid composition may be deposited at a region so as to cover an area greater than that covered by the solid activator composition at the same region (or greater than the area covered by the activator fluid composition at the same region, when the solid activator is deposited as a fluid composition).
The biopolymers may in particular be polynucleotides (for example, DNA), in which case the biomonomer is a nucleoside monomeric unit. The activated biomonomer may particularly react with a component in ambient atmosphere. For exampl

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