Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-30
2001-07-10
Jones, W. Gary (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S006120, C435S007800, C435S091100, C435S091200, C435S091500, C536S026110, C536S027400, C536S028500, C536S077000, C536S078000, C514S100000
Reexamination Certificate
active
06258539
ABSTRACT:
FIELD OF THE INVENTION
Embodiments of the invention are in the field of polynucleotide analysis through the use of hybridization and restriction analysis.
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. Differences in gene expression can also 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, a toxin, or some other environmental variable. 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. Additionally, variations exist at the genomic level between cells from different members of the same species, i.e., polymorphisms. Genetic polymorphism may give rise to gene expression difference between species members. Furthermore, polymorphisms may also give rise to phenotypic differences irrespective of the effect of the polymorphisms on transcription. Thus it is also of interest to provide techniques for analyzing nucleotide sequences, including the detection and monitoring of polymorphisms. Moreover, in many instances, it may be of interest to obtain both gene expression data and sequence information in the same experimental procedure. Various embodiments of the invention provide convenient techniques for addressing one or more of these needs for genetic analysis.
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. DNA (including genomic DNA preparations) may also be analyzed by the subject methods. 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 (i) the recognition site of a restriction endonuclease used to generate a restriction fragment from the polynucleotide of interest, and (ii) the hybridization of a terminus probe of known sequence. In some embodiments of the invention sequence information from other steps such as chain extension sequencing or selective nucleic acid amplification may be used to obtain additional information to produce an identifier sequence. Furthermore, the base sequence information contained within identifier sequences may be used to detect, discover, or compare polymorphic sequences, e.g., SNPs (single nucleotide polymorphisms). 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 identifier sequences may also be used to develop oligonucleotide primers (or probes) to isolate the polynucleotide from which a specific identifier sequence is derived. Multiple identifier sequences may be obtained in parallel, thereby permitting the rapid characterization of a large number of polynucleotides.
In preferred embodiments of the invention, representative restriction fragments for analysis are joined to adapters prior to contacting terminus probes. The terminus probes used in the subject methods may 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, the restriction fragments are representative restriction fragments, i.e., restriction fragments 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. One or more 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 adapter-modified 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. Selective amplification primers may be used as to reduce the complexity of the mixture of adapter-modified restriction fragments brought into contact with the terminus probes. 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 terminus probes so that hybridization may take place between each of the different adapter-modified representative restriction fragments and each terminus probe present, thereby permitting 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 may optionally be extended in a primer extension reaction (e.g., minisequencing), from the template of the hybridized adapter-modified restriction fragment. The array location of adapter-modified restriction fragments that have hybridized to an array of terminus probes may be detected by a variety of methods, such as detectably labeling the fragments before hybridization or through the use of labeled chain terminators in the optional primer extension reaction step. Alternatively, the fragments may be labeled through the use of fluorescently labeled adapters or labeled amplification primers. Thus the probes on the array that have been hybridized to adapter-modified restriction fragments may be determined, thereby serving to identify which of the oligonucleotide features (terminus probes) are complementary to a given representative restriction fragment. Sequence information from the terminus probe hybridized to the adapter-modified restriction fragment 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
Hunkapiller Michael W.
Richards John H.
Bortner Scott R.
Jones W. Gary
Taylor Janell E.
The Perkin-Elmer Corporation
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