Testing multiple fluid samples with multiple biopolymer arrays

Details Testing multiple fluid samples with multiple biopolymer arrays Testing multiple fluid samples with multiple biopolymer arrays

1999-06-30

2002-06-04

Warden, Jill (Department: 1743)

Chemistry: analytical and immunological testing

Including sample preparation

Volumetric liquid transfer

C436S063000, C436S172000, C435S305100, C435S305200, C435S305300, C422S091000, C422S105000, C422S105000

Reexamination Certificate

active

06399394

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to arrays, particularly biopolymer 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 spots or features) 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 fluorescent signal 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 in situ synthesis methods or deposition of the previously obtained biopolymers. 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). 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. Washing or other additional steps may also be used. Procedures known in the art for deposition of polynucleotides, particularly DNA such as whole oligomers or cDNA, are described, for example, in U.S. Pat. No. 5,807,522 (touching drop dispensers to a substrate), and in PCT publications WO 95/25116 and WO 98/41531, and elsewhere (use of an ink jet type head to fire drops onto the substrate).
In array fabrication, the quantities of DNA available for the array are usually very small and expensive. Sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require use of arrays with large numbers of very small, closely spaced spots. During use of an array, such as for gene expression monitoring or for patient testing, it will often be desirable to test very large numbers of such small samples against the many of the same or different array patterns. Thus, it is desirable to provide a convenient means by which many samples can be exposed to many arrays in a highly parallel process.
U.S. Pat. Nos. 5,874,219 and 5,545,531 provide a DNA chip wafer to which a plate carrying multiple channels can be mounted, to provide many test wells. Grace Bio-Labs, Inc., of Bend, Oreg., manufactures “Perfusion Chambers” which include covers with openings and which can be placed on specimen slides. However, the present invention appreciates that sample fluid loss can occur in chambers with openings, particularly as a result of evaporation under the elevated temperatures used over a number of hours during hybridizations of nucleic acid arrays. Such losses can potentially result in inaccurate results. Sample contamination may also occur through uncontrolled openings. Furthermore, it may be difficult to provide positive or negative pressure to the chambers to load or empty them while avoiding sample loss. The present invention recognizes that when chambers become very thin to accommodate small sample volumes, capillary forces become significant and some positive means of loading and/or emptying the chamber should preferably be provided which at the same time will avoid sample loss. As well, the present invention recognizes that any closed chamber system for arrays which uses assembled components should be provided with some way of avoiding pushing apart of chamber components as a result of internal pressure increases during heating.
As already mentioned, the testing of multiple samples on multiple arrays on a single substrate has potential to expedite and simplify multiple sample handling. However, such a technique also has the potential to propagate multiple errors. For example, in the case of hybridizing multiple samples to a contiguous substrate carrying multiple polynucleotide arrays, elevated temperatures over a lengthy predetermined time may be required. If for any reason inadequate conditions were provided (for example, by failure of a heating system to reach and maintain the required temperature for the required time), poor results may be obtained. It has been previously disclosed to use control oligonucleotide probes and reference nucleic acid sequences with single arrays. The reference sequences are mixed with sample and the mixture exposed to the array. Hybridization of reference sequences to corresponding reference features, is used as an indication of overall assay performance. However, since sample is present together with reference sequences, there is a potential of interference from similar sequences in a sample. In a conventional situation, where a single sample is tested on a single array, and the inadequate hybridization conditions are not detected, this might lead to a single error. However, with a single substrate carrying multiple arrays, this might suggest system failure and lead to invalidating multiple test results, when the error may in fact be due to interference of the test sample on the hybridization of the reference target to the reference features.
The present invention realizes that it would be desirable then, to provide apparatus and methods for testing multiple samples with multiple arrays, particularly biopolymer arrays such as DNA or RNA arrays, which retain the samples in readily accessible chambers and yet which will not likely suffer sample loss or contamination. The present invention further realizes that it would be desirable that an apparatus and/or method for testing multiple samples with multiple biopolymer arrays, should preferably be able to provide features which include one or more of the following: the ability to allow samples to be positively loaded into or withdrawn from the chamber while avoiding sample leakage; tolerance for increased temperatures without adverse sample loss; of relatively simple constructions; be easy to clean and preferably with any components subject to wear being readily replaceable; and the ability to avoid multiple undetected errors.
SUMMARY OF THE INVENTION
The present invention then, provides in one aspect a method of testing multiple fluid samples with multiple biopolymer arrays. This, or any other aspects of the method, may use any suitable apparatus as described herein. Any of the fluid samples may be of the same or different compositions. The method includes assembling a cover to a contiguous substrate which carries on a first side, multiple arrays each with multiple regions of biopolymers linked to the substrate. As a result, the cover and the substrate together form a plurality of chambers each containing a biopolymer array and each being accessible through its own port. The method further optionally includes introducing multiple fluid samples into respective chambers through a port of each such that the fluid samples contact respective arrays, and observing the binding pattern of the arrays. The binding pattern may be observed in any suitable manner, whether directly or indirectly.
The method may particularly use an apparatus in which each chamber is accessible through a first and a second port. In this case, fluid samples may be introduced into respective chambers through respective first ports while venting through respective second ports. This introduction of multiple fluid samples may optionally be performed simultaneously. The ports may

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