Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-08-17
2001-05-15
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S007800, C435S091100, C435S091200, C436S501000, C536S026110, C536S027400, C536S028500, C536S077000, C536S078000
Reexamination Certificate
active
06232067
ABSTRACT:
FIELD OF THE INVENTION
Embodiment sof the invnetion are in the field of polynucleotide analysis throught the use of multiple hydrization probes.
BACKGROUND
The cells that constitute different tissues in an organism, although having the same genomic DNA, differ significantly from one another with respect to the specific genes that are expressed and the levels of expression. Similar differences in gene expression can be observed when comparing cells that are obtained from a healthy organism and corresponding cells from an organism manifesting a disease state. Other examples of variations in gene expression include changes induced by exposing a cell to a pharmaceutical compound or toxin. It is of interest to provide methods for analyzing changes in gene expression. Methods of analyzing gene expression find wide use in both research and diagnostics.
SUMMARY
Embodiments of the invention described herein relate to methods of analyzing an individual polynucleotide or a polynucleotide mixture comprising multiple diverse polynucleotides, typically a cDNA mixture formed from an RNA population of interest. Not only is the analysis of RNA populations of major interest in research, such analysis may be used to predict, diagnose, or treat a variety of diseases. Various embodiments of the invention permit the simultaneous analysis of a large number of different mRNA molecules that form a given mRNA population. Various embodiments of the invention also permit the convenient isolation of polynucleotides of interest identified through the subject analytical techniques.
In accordance with the description of the invention provided herein, the identity of a particular polynucleotide of interest may be ascertained by producing a short identifier sequence based on the nucleotide sequence information obtained from (1) the recognition site of a restriction endonuclease used to generate a restriction fragment from the polynucleotide of interest, and (2) the hybridization of: (i) a terminus probe of known sequence, and (ii) an internal fragment probe of known sequence, at adjacent positions on the polynucleotide of interest. Polynucleotide sequence databases may be conveniently searched for previously identified polynucleotide sequences that match or partially match the identifier sequence. Alternatively, the subject methods may be used to “fingerprint” complex polynucleotide populations without the need to generate identifier sequences. The identification sequences may also be used to develop oligonucleotide primers (or probes) to isolate the polynucleotides from which the identifier sequence is derived.
In preferred embodiments of the invention, representative restriction fragments for analysis are joined to adapters prior to contacting either terminus probes or internal fragments probes. Multiple identifier sequences may be obtained in parallel, thereby permitting the rapid characterization of a large number of polynucleotides. The terminus probe and internal fragment probe as used in the subject methods may each be identified by a “marker” that is correlated with the known base sequence of the probe oligonucleotide so as to facilitate the rapid characterization of a large number of diverse polynucleotides in parallel. Parallel analysis of multiple diverse polynucleotides may be carried by using ordered arrays of oligonucleotides (terminus probes) such that the position of the oligonucleotides in the array serve as markers to identify the base sequence of the oligonucleotide in the array.
In one embodiment of the invention, methods are provided for analyzing diverse polynucleotide mixtures such as a cDNA mixture generated from an RNA population. Restriction fragments are formed by digesting the polynucleotide population for analysis with a restriction endonuclease. Preferably, representative restriction fragments are generated from the different cDNA molecules in the mixture in such a way that only a single restriction fragment is recovered for each polynucleotide analyzed. By employing representative restriction fragments, quantitative (or semi-quantitative) measurements of the relative amounts of different polynucleotides in a polynucleotide mixture for analysis may be greatly facilitated. Adapters may be ligated to the termini of the representative restriction fragments so as to produce a set of adapter-modified representative restriction fragments. The representative restriction fragments may then be optionally amplified in a nucleic acid amplification reaction employing primers specific for the adapters, thereby producing an amplified set of adapter-modified representative restriction fragments. The amplified set of adapter-modified representative restriction fragments (or a corresponding non-amplified set) may then be contacted under nucleic acid hybridization conditions with marked terminus probes so that hybridization may take place between each of the different adapter-modified representative restriction fragments and each terminus probe present so as to permit hybridization of the probe to complementary strands of the matching adapter-modified representative restriction fragments. Terminus probes may be marked by virtue of their location on an oligonucleotide array. An oligonucleotide array comprising a plurality of oligonucleotide features, wherein each feature of the array is a terminus probe, may be used to analyze a plurality of polynucleotides in parallel. After hybridization with the terminus probe, the adapter-modified representative restriction fragments that have hybridized to the array are contacted (under nucleic acid hybridization conditions) with a labeled probe solution comprising at least one internal fragment probe. The internal fragment probes may be marked, preferably with a fluorescent dye, so as to identify array sites at which hybridization has occurred. Solutions containing multiple differentially labeled internal fragment probes may be used to simultaneously test different internal fragment probes for hybridization to multiple different representative restriction fragments in parallel. Labeled probes that have hybridized to adapter-modified representative restriction fragments at positions adjacent to the terminus of the array feature oligonucleotides are subsequently joined (e.g., by ligase catalyzed ligation), thereby covalently attaching the internal fragment probe to the array. The location of the joined terminus probe or probes on the array may subsequently be identified, thus serving to identify which of the oligonucleotide features (terminus probes) are complementary to a given representative restriction fragment. Sequence information from the internal fragment probe and the terminus probe ligated to the internal fragment probe may be used to obtain an identifier sequence corresponding to the restriction fragment that hybridized to the array at a given feature. Alternatively, arrays of terminus probes may be used to “fingerprint” complex polynucleotide populations with or without the generation of identifier sequences.
Other embodiments of the invention include oligonucleotide arrays comprising features that are complete sets of terminus probes. Embodiments of the subject arrays include arrays that comprise multiple subarrays, wherein at least two of the subarrays comprise the same set of terminus probes; preferably each set of terminus probes in each subarray is a complete set of terminus probes. Each subarray may be organized so as to provide for the addition of oligonucleotide probe solution to one array subunits without having the solution contacting the other subunits.
In another embodiment of the invention, terminus probes and internal fragment probes are hybridized at adjacent positions on an adapter-modified representative restriction fragment (or adapter-modified restriction fragments) and subsequently joined to each other prior to the step of immobilization on a sorting array. The use of sorting arrays permits the step of joining terminus probes and internal fragment probes to take place in solution rather than on an array. In embodiments of the inve
Hunkapiller Michael W.
Richards John H.
Bortner Scott R.
Jones W. Gary
Taylor Janell E.
The Perkin-Elmer Corporation
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