Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-22
2001-05-15
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100
Reexamination Certificate
active
06232068
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to methods and compositions for detecting, monitoring and/or quantifying gene expression using nucleic acid arrays, in particular, by using anti-heteronucleic acid antibodies to detect RNA-DNA duplexes on microarrays.
2. BACKGROUND
Modern biology teaches the importance of genes and gene expression to processes of health and disease. New individual genes causing or predisposing to conditions or diseases are now reported almost daily. Additionally, it is commonly understood that observing and measuring patterns of gene expression in health and disease will contribute immensely to further understanding of these states. Therefore, any observational method that can rapidly, accurately, and economically observe and measure the presence or expression of selected individual genes or of whole genomes will be of great value. Of even more value will be methods that can directly and quantitatively be applied to RNA typically derived directly from biological samples.
2.1 Methods for Monitoring Gene Expression
Current observation and measurement methods suffer from one or more disadvantages that render them unnecessarily inaccurate, time consuming, labor intensive, or expensive. Such disadvantages flow from requirements for, e.g., prior knowledge of gene sequences, cloning of complex mixtures of sequences into many individual samples each of a single sequence, repetitive sequencing of sample nucleic acids, electrophoretic separations of nucleic acid fragments, and so forth.
For example, observation techniques for individual mRNA or cDNA molecules, such as Northern blot analysis, RNase protection, or selective hybridization to arrayed cDNA libraries (see Sambrook et al.,
Molecular Cloning—A Laboratory Manual
, Cold Spring Harbor Press, New York (1989)) depend on specific hybridization of a single oligonucleotide probe complementary to the known sequence of an individual molecule. Since a single human cell is estimated to express 10,000-30,000 genes (Liang et al., 1992, Science, 257:967-971), most of which remain unknown, single probe methods to identify all sequences in a complex sample are prohibitively cumbersome and time consuming.
Similarly, traditional nucleic acid sequencing (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA, 74:5463-5467), sequencing by hybridization (“SBH”) using combinatorial probe libraries (Drmanac et al., 1993, Science 260:1649-1652; U.S. Pat. No. 5,202,231, Apr. 13, 1993 to Drmanac et al.), or classification by oligomer sequence signatures (Lennon et al., 1991, Trends Genetics 7:314-317), and positional SBH (Broude et al., 1994, Proc. Natl. Acad. Sci. USA 91:3072-3076) also require that samples be arrayed into purified clones, making the methods inappropriate for complex mixtures.
Several approaches have been described that attempt to characterize complex mixtures of nucleic acids without cloning, all of which at least require electrophoretic separation and/or traditional sequencing. A basic approach is that of differential display (Liang et al., 1992, Science 257:967-71; Liang et al., 1995, Curr. Op. Immunol. 7:274-280), which uses the polymerase chain reaction (“PCR”) with an oligo (dT) primer and a degenerate primer designed to hybridize within a few hundred bases of the cDNA 3′-end. The resulting DNA subsequences of varying length are electrophoretically separated to yield a pattern of, preferably, 100-250 bands. This approach, at best providing only qualitative “fingerprints” of gene expression, suffers from well-known problems, including a high false positive rate, migration of multiple nucleic acid species within a single observed band, and non-quantitative results. Further, putative gene identification depends on purification and traditional sequencing of the components in electrophoretic bands.
Additionally, approaches have been described which attempt to improve differential display, but without obviating the need for traditional sequencing and/or electrophoretic separation. For example, a method described in European Patent Application 0 534 858 A1 (published Mar. 31, 1993), is directed to applying differential display to gDNA samples by using restriction endonuclease (“RE”) digestion together with PCR employing phasing primers in order to reduce the complexity of such samples to levels electrophoretically observable. The multiple phasing primers divide the gDNA samples in multiple pools of lower complexity, which are electrophoretically separated to yield qualitative “fingerprints.”
Other methods improving on differential display include the following, all of which are similarly limited to generating electrophoretic “fingerprints.” One such improvement is described in U.S. Pat. No. 5,459,937 (Oct. 17, 1995). This method generates multiple pools of lower complexity by using sequential rounds of PCR applied to 3′-end fragments of cDNAs. The 3′-end fragments lie between a recognition site for a frequently-cutting RE and the poly(A) tail of the cDNA. Fragments in the multiple pools are finally putatively identified by electrophoretic separation and individual sequencing. Another example of such an improvement is described by Prashar et al., 1996, Proc. Natl. Acad. Sci. USA 93:659-663. Primarily, this reference describes an alternative method for generating similar 3′-end fragments, which lie between a recognition site for a frequently-cutting RE and the poly(A) tail of the cDNA.
Differing from differential display is another class of methods, which observe gene expression by sampling, that is, these methods repetitively sequence nucleic acids in a sample and count the sequence occurrences in order to statistically observe gene expression. Such methods require sequencing and are statistically limited in their ability to discover rare transcripts. An early example of such a method determined and counted expressed sequence tags (“ESTs”), and is described in Adams et al., 1991, Science, 252:1651-1656. Another example is named “serial analysis of gene expression” (Velculescu et al., 1995, Science, 270:484-487). According to this method, cDNA molecules are converted into representative “tags,” which are short oligonucleotides generated from Type IIS RE single-stranded overhangs located at determined distances from the 3′-end of source cDNA. (Type IIS REs cleave a defined distance (up to 20 bp) away from their asymmetric recognition sites (Szybalski, 1985, Gene 40:169). Approximate, putative identification of the source of a tag requires sequencing the tag and using the sequence and location information to look up possible source sequences in a nucleic acid sequence database.
Other methods for gene and gene-expression measurement, although unrelated to differential display, still, e.g., require electrophoretic separation. Such a method is described in PCT Publication WO 97/15690. According to this method “signals” are generated that represent the length of a nucleotide sequence between defined subsequences in a target nucleic acid. The defined subsequences are preferably restriction endonuclease sites or oligomer binding sites. These signals can then be compared to results of computer simulated signal generation experiments using computer databases of nucleic acid sequences. By this comparison, particular DNA sequences in the database can be identified as present in a sample, since they are predicted to generate signals which are also observed. The length information of the signals of this method is observed electrophoretically.
Many of the methods previously described for the analysis of complex mixtures of nucleic acids require electrophoretic separation, possibly together with nucleic acid cloning and sequencing. These procedures can be unnecessarily labor intensive, slow, and uneconomical. Recently new approaches have been reported that can, in some implementations, obviate the need for, at least, electrophoretic separation. Such methods depend on hybridization of probe oligonucleotides to recognize short subsequences of from 4 to 20 base pairs on target nucleic acids. The
Baeuerle Patrick
Linsley Peter S.
Campbell Eggerton A.
Pennie & Edmonds LLP
Rosetta Inpharmatics, Inc.
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