Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-05
2004-06-22
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S252800, C435S174000, C435S183000, C382S129000, C382S133000, C382S153000, C382S173000, C382S286000, C382S291000, C702S019000, C702S022000, C536S022100
Reexamination Certificate
active
06753145
ABSTRACT:
TECHNICAL FIELD
This invention relates to biological assays. In particular, the invention is directed to materials and methods of hybridizing microarrays of nucleic acid molecules for analytical, therapeutic and diagnostic purposes.
BACKGROUND ART
Microarrays of DNA or RNA polynucleotides or oligonucleotides are state-of-the-art biological tools used in the investigation and evaluation of genes for analytical, diagnostic, and therapeutic purposes. Microarrays typically comprise a plurality of oligomers, synthesized or deposited on a glass support or substrate in an array pattern. The support-bound oligomers are called “probes” and function to bind or hybridize with a sample of DNA or RNA material under test, called a “target” in hybridization experiments. However, some investigators bind the target sample under test to the microarray substrate and put the oligomer probes in solution for hybridization. Moreover, some investigators use the reverse definition, referring to the surface-bound oligonucleotides as targets and the solution sample of nucleic acids as probes. Either of the “targets” or “probes” may be the one that is to be evaluated by the other (thus, either one could be an unknown mixture of polynucleotides to be evaluated by binding with the other). All of these iterations are within the scope of this discussion herein. In use, the microarray surface is contacted with one or more targets under conditions that promote specific, high-affinity binding of the target to one or more of the probes. The targets are typically labeled with an optically detectable label, such as a fluorescent tag, so that the hybridized targets and probes are detectable with scanning equipment. DNA array technology offers the potential of using a multitude (hundreds of thousands) of different oligonucleotides to analyze changing mRNA populations.
There are numerous types of substrates used in hybridization assays. Common substrates or supports used for microarray assays are siliceous substrates, such as glass. The surface of the substrates are typically treated or derivatized to facilitate binding of the probes to the substrate. For in situ synthesis of probes, the first monomers of the oligomer probe sequences are attached to the substrate surface that is derivatized with a silane or other compounds known in the art to facilitate the bonding of the first monomers. Subsequent monomers are added directly to the monomers of the growing oligomer chain. For deposition of presynthesized or whole probes, such as cDNA probes, the probe is attached to a polymer adsorbed or coated on the surface of the substrate to facilitate bonding. The adsorbed polymer is coated and dried on the substrate surface. The substrate surface derivatizations enable and facilitate the attachment of nucleic acids to the surface of microarray substrates for the manufacture of the microarrays. Surface treatments or derivatization techniques, including those mentioned above, are well known in the art.
Microarrays of oligomer probes, such as oligonucleotides or polynucleotides, are hybridized using conventional methods and hybridization solutions. J. Sambrook, E. F. Fritsch, T. Maniatis,
Molecular Cloning: A Laboratory Manual
, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ed. 2
nd
, 1989, vol. 1-3, incorporated herein by reference, describe the considerations and conditions for hybridization of oligonucleotide probes. Probe length, hybridization temperature, as well as other factors that are well known in the art affect hybridization conditions. Typically, hybridizations using synthetic oligomers are usually carried out under conditions that are 5-10° C. below the calculated melting temperature T
m
of a perfect hybrid to minimize mismatched or non-Watson/Crick base pairing between the probe and target, and maximize the rate at which Watson/Crick base pairs form. Other factors influencing the rate of hybrid formation include the salt concentration, the presence of surfactants, solvents or co-solvents, the concentration of nucleic acid in solution, the length of hybridization, and the degree and method of agitation.
The hybridization solution typically comprises a salt (monovalent cation), either SSPE or SSC buffer that provides buffering capacity between pH 6.8-8.5 (more typically between pH 7.0-7.5), a divalent cation chelating agent (e.g. ethylenediaminetetraacetic acid, EDTA), and agents for blocking non-specific binding of targets to the array surface (surfactants, proteins and/or carrier DNA from an organism unrelated to the experiment at hand). More specifically, a typical hybridization solution contains 6×SSPE (0.9 M NaCl, 60 mM sodium phosphate (pH 7.4); 6 mM EDTA); or 6×SSC (0.9 M NaCl, 90 mM sodium citrate (pH 7.0)), 0.5% w/v sodium dodecyl sulfate (SDS); 100 &mgr;g/ml denatured, fragmented salmon sperm DNA; and 0.1% nonfat dried milk.
The microarray is hybridized for a period of time ranging from about 2 hours to about 2 days, depending on the make-up of the probes (i.e., probe length and diversity of probe composition) and the complexity of the target, for example, at a controlled temperature, which typically ranges from 20° C. to 70° C., depending on the melting temperature T
m
, as discussed above. Low temperature hybridizations are performed at about 20° C. to about 50° C. (typically about 37-45° C.). High temperature hybridizations are performed at or around 55° C. to about 70° C. (typically 60° C. to 65° C.). However, for most nucleic acid microarrays, high temperature hybridizations are preferred in the art since the higher temperature maximizes the rate of Watson/Crick base pairing of nucleotides, while low temperature hybridizations typically maximize Watson/Crick base pairings by use of a co-solvent to lower the T
m
. The typical time period for hybridization of a microarray is overnight or longer (i.e., anywhere from 8 hours to 24 hours) so as to hybridize the target. The array is then washed and dried and optically scanned to measure the degree of hybridization using conventional methods and equipment that are well known in the art.
A problem in the DNA microarray hybridization art is sporadic poor hybridization assay performance characterized by low-intensity or missing features on the microarray substrate, high backgrounds, and visually “blotchy” substrates. For microarrays containing DNA on adsorbed polymer substrate surfaces, this problem has been observed using conventional hybridization conditions, such as using a solution comprising 20×SSC (3.0 M NaCl, 300 mM Sodium Citrate (pH 7.0), 10% SDS) at high hybridization temperature of about 65° C. and within conventional hybridization times of about 6 hrs. to about 24 hours.
Thus, it would be advantageous to have materials, conditions and methods of hybridizing arrays of oligomers on siliceous substrates that have been treated or coated with a surface adsorbed polymer in biological assays at the preferred higher hybridization temperature range and longer hybridization times without affecting the hybridization assay performance.
SUMMARY OF THE INVENTION
The present invention provides a buffer composition and method for hybridizing nucleic acid microarrays with other nucleic acid materials used in high throughput analytical, therapeutic, and diagnostic applications. The method uses an envelope of hybridization conditions for performing assays at high hybridization temperatures for long periods of time. The hybridization conditions of the method advantageously are compatible with siliceous substrates having adsorbed polymer surfaces. The buffer composition and method of the present invention work particularly well on adsorbed polycationic polymer coated siliceous surfaces. The envelope of conditions addresses solution pH and buffer type, salt composition, surfactant composition, temperature and time. The present invention allows sensitive, selective detection of nucleic acid targets, while preserving the intactness of the adsorbed polymer siliceous surface. The buffer composition and method of the invention overcome the problem
Collins Patrick J.
Holcomb Nelson R.
Lefkowitz Steven M.
Shannon Karen W.
Chakrabarti Arun
Fredman Jeffrey
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