Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-16
2003-05-06
Siew, Jeffrey (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091200, C435S287200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06558907
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods and compositions for fabrication of high-density nucleic acid arrays for use in biological assays.
2. Background
Hybridization is a hydrogen-bonding interaction between two nucleic acid strands that obey the Watson-Crick complementary rules. All other base pairs are mismatches that destabilize hybrids. Since a single mismatch decreases the melting temperature of a hybrid by up to 10° C., conditions can be found in which only perfect hybrids can survive. Many hybridization experiments can be simultaneously carried out on a single solid support on which multiple nucleic acids “probes” have been immobilized by either covalent or non-covalent methods. The tethered “probe” is hybridized with target nucleic acids that usually bear a radioactive, fluorescent label, or haptens that could be visualized by chemiluminescent or other detection methods. The resulting hybrids are separated from the unreacted labeled strands by washing the support. Hybrids are recognized by detecting the label bound to the surface of the support.
Oligonucleotide hybridization is widely used to determine the presence in a nucleic acid of a sequence that is complimentary to the oligonucleotide probe. In many cases, this provides a simple, fast, and inexpensive alternative to conventional sequencing methods. Hybridization does not require nucleic acid cloning and purification, carrying out base-specific reactions, or tedious electrophoretic separations. Hybridization of oligonucleotide probes has been successfully used for various purposes, such as analysis of genetic polymorphisms, diagnosis of genetic diseases, cancer diagnostics, detection of viral and microbial pathogens, screening of clones, genome mapping and ordering of fragment libraries.
An oligonucleotide array is comprised of a number of individual oligonucleotide species tethered to the surface of a solid support in a regular pattern, each species in a different area, so that the location of each oligonucleotide is known. An array can contain a chosen collection of oligonucleotides, e.g., probes specific for all known clinically important pathogens or specific for all known clinically important pathogens or specific for all known sequence markers of genetic diseases. Such an array can satisfy the needs of a diagnostic laboratory. Alternatively, an array can contain all possible oligonucleotides of a given length n. Hybridization of a nucleic acid with such a comprehensive array results in a list of all its constituent n-mers, which can be used for unambiguous gene identification (e.g., in forensic studies), for determination of unknown gene variants and mutations (including the sequencing of related genomes once the sequence of one of them is known), for overlapping clones, and for checking sequences determined by conventional methods. Finally, surveying the n-mers by hybridization to a comprehensive array can provide sufficient information to determine the sequence of a totally unknown nucleic acid.
Oligonucleotide arrays can be prepared by synthesizing all the oligonucleotides, in parallel, directly on the support, employing the methods of solid-phase chemical synthesis in combination with site-directing masks as described in U.S. Pat. No. 5,510,270. Four masks with non-overlapping windows and four coupling reactions are required to increase the length of tethered oligonucleotides by one. In each subsequent round of synthesis, a different set of four masks is used, and this determines the unique sequence of the oligonucleotides synthesized in each particular area. Using an efficient photolithographic technique, miniature arrays containing as many as 10
5
individual oligonucleotides per cm
2
of area have been demonstrated.
Another technique for creating oligonucleotide arrays involves precise drop deposition using a piezoelectric pump as described in U.S. Pat. No. 5,474,796. The piezoelectric pump delivers minute volumes of liquid to a substrate surface. The pump design is very similar to the pumps used in ink jet printing. This picopump is capable of delivering 50 micron diameter (65 picoliter) droplets at up to 3000 Hz and can accurately hit a 250 micron target. The pump unit is assembled with five nozzles array heads, one for each of the four nucleotides and a fifth for delivering, activating agent for coupling. The pump unit remains stationary while droplets are fired downward at a moving array plate. When energized, a microdroplet is ejected from the pump and deposited on the array plate at a functionalized binding site. Different oligonucleotides are synthesized at each individual binding site based on the microdrop deposition sequence.
A popular method for creating high-density arrays utilizes pins that are dipped into solutions of the sample fluids and then touched to a surface. The nucleic acid, e.g., DNA, is typically solubilized in an aqueous medium (also sometimes referred to as an “ink”) that contains salts, which are used as components of buffers that are compatible with biological macromolecules. 3×SSC (450 mM sodium chloride and 45 mM sodium citrate) is a standard printing ink. See, e.g., U.S. Pat. No. 5,807,522 (Example 1).
Printing with 3×SSC is considered useful since the salt particles that are deposited on the arrays after printing enable the verification of the printing process. This verification can be achieved by an imaging method that uses the principle of compound microscopy to photograph printed grids of an HDA, and then electronically determine the presence or absence of the salt spots printed with DNA. By using an oblique white light source, principally the salt deposits are detected by the imaging method (also referred to herein as the 100% Dot Inspection System) and not DNA. Therefore, if one wishes to use such a verification process it is imperative that the ink contains salts.
However, the use of SSC inks can be problematic. The first problem encountered in manufacturing DNA arrays using a 3×SSC ink is that the rate of evaporation of the aqueous medium is very high compared to the time required to print multiple slides. This is a major obstacle to scaling up the manufacturing process. Additionally, it has been observed by the present inventors that not only is the 3×SSC ink incapable of printing the required number of slides but variable arrays result due to a rapidly changing concentration of DNA because of the evaporation of aqueous medium.
Therefore, there is a need for an ink composition for printing HDA of nucleic acids that overcome the disadvantages seen in the art.
SUMMARY OF THE INVENTION
In an attempt to solve the problem related to evaporation discussed above, we tested an ink composition consisting of 50% DMSO (dimethylsulfoxide) by volume and SSC at a final concentration of 1× (150 mM NaCl+15 mM sodium citrate). This composition was considered optimal since DMSO is an organic solvent with high vapor pressure and it is highly miscible with water. Moreover, DMSO is a hygroscopic solvent and is thereby able to compete with the net losses of the solvent due to evaporation of the aqueous component by absorbing moisture from the air. The 1:1 ratio of DMSO and SSC was found to give minimal evaporation of the ink over several print runs and was successfully used to print various HDAs.
To further test the ink, genetic material was suspended in the 1:1 DMSO:SSC (1×) composition and used to print human and yeast arrays using the contact printing method. Many arrays were printed on CMT-GAPS™ glass slides (Corning) with this ink for over a period of 2 months and the hybridization performance obtained on these slides was acceptable. However, after ~4-5 weeks, these genetic materials failed to give satisfactory hybridization performance. After repeated failed attempts at printing this DNA, the genetic materials were analyzed for the presence of contaminants that could potentially degrade the DNA (for example the DNase enzyme is a common cause of DNA degradation). Gel electrophorese
Koroulis Melanie C.
Pal Santona
Beall Thomas R.
Corning Incorporated
Kung Vincent T.
Siew Jeffrey
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