Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-19
2002-12-31
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S024330
Reexamination Certificate
active
06500620
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates generally to the field of nucleic acid biology. More specifically, the invention provides methods and compositions for high-throughput amplification, detection and comparison of gene expressions in biological samples for diagnostic and therapeutic applications.
BACKGROUND
Detection of small quantities of genetic materials represents a major challenge in biological research and clinical diagnosis. Polymerase chain reaction (PCR) provides a powerful tool for in vitro amplification of specific polynucleotide sequences, such as genomic DNA, single stranded cDNA or mRNA, with high sensitivity and specificity. One application of this is the amplification of target gene sequences in biological samples from, for example, environmental, food and medical sources, etc. to allow identification of causative, pathogenic, spoilage or indicator organisms present in the sample.
The basic PCR technique, as described in U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,800,159 (the disclosures of which are incorporated herein by reference), typically involves using two oligonucleotide primers capable of hybridizing to specific nucleic acid sequences flanking a target sequence of interest. By repeating multiple cycles of template denaturation, primer annealing and strand elongation, an exponential duplication of the target sequence can be obtained.
A major technical problem with standard PCR methods is contamination. While PCR provides a sensitive way for detecting and amplifying small amounts of target polynucleotides, it can amplify non-specific nucleic acid sequences, therefore creating false positive products in the final detection and assay. In a standard solution-phase PCR, in which primers bind to templates and initiate nascent strand synthesis in solution, reaction mixtures and products often need to be transferred several times for final detection and assay, increasing the chances for contamination.
Kohsaka and Carson (1994)
J Clin. Lab Anal
8:452-455 describes a solid-phase PCR approach to allow amplification and detection of a target gene sequence in the same microwell without transfer. One of the two oligonucleotide primers is covalently attached to the wells of a microtiter plate, the other primer remains in solution. The immobilized primer binds to template and initiates the extension of a nascent complementary strand. The newly synthesized strand remains attached to the plate after removal of the template by denaturation and, at the completion of PCR, can be detected with a labeled probe. The solid-phase PCR approach has also been used for a quantitative determination of the target nucleic acid by adding a known amount of an internal competitive DNA template prior to amplification. See, for example, U.S. Pat. No. 5,747,251, the disclosure of which is incorporated herein by reference.
The solid-phase PCR of Kohsaka et al. is limited to detecting one single target polynucleotide in a well on a 96-well microtiter plate. The quantitative solid-phase PCR using competitive template is limited to detecting target from one species or tissue. Furthermore, the amplified products that are attached to plate must be single-stranded in order to be detected by hybridizing with labeled probes, therefore limiting the sensitivity of the detection.
U.S. Pat. No. 5,641,658 (Adams et al.) describes a method for amplifying nucleic acid with two primers bound to a single solid support. The method requires selection of two primers flanking a target sequence and immobilization of both primers onto a solid support. The primer pairs are used to detect and amplify the target polynucleotide on the support. The amplified products are fixed on the support, and two adjacent strands, if reasonably distanced from each other, can further hybridize together to form a “loop.” While the two-primer amplification system is promised to be sensitive in detecting the presence or absence of particular target nucleic acid in a sample, the use of two immobilized primers for each target requires a careful arrangement of the primers on the support so that the primer array would allow the formation of the loops and yet would not interfere the amplification of additional strands. In other words, the methods may not be ideal for a high density, high throughput assay.
The pattern of gene expression in a particular biological sample provides significant insights into the molecular fundamentals of almost all biological function and activities. A number of methods are known in the art for detecting and comparing gene expression levels in different biological sources. One standard method for such comparisons is the Northern blot. In this technique, RNA is extracted from the sample and loaded onto any of a variety of gels suitable for RNA analysis, which are then run to separate the RNA by size, according to standard methods (see, e.g., Sambrook, J., et al., Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2nd ed. 1989)). The gels are then blotted (as described in Sambrook, supra), and hybridized to probes for RNAs of interest.
Sutcliffe, U.S. Pat. No. 5,807,680, teaches a method for the simultaneous identification of differentially expressed mRNAs and measurement of their relative concentrations. The technique, which comprises the formation of cDNA using anchor primers followed by PCR, allows the visualization of nearly every mRNA expressed by a tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA.
Another group of techniques employs analysis of relative transcript expression levels. Four such approaches have recently been developed to permit comprehensive, high throughput analysis. First, cDNA can be reverse transcribed from the RNAs in the samples (as described in the references above), and subjected to single pass sequencing of the 5′ and 3′ ends to define expressed sequence tags for the genes expressed in the test and control samples. Enumerating the relative representation of the tags from the different samples provides an approximation of the relative representation of the gene transcript within the samples.
Second, a variation on ESTs has been developed, known as serial analysis of gene expression, or “SAGE,” which allows the quantitative and simultaneous analysis of a large number of transcripts. The technique employs the isolation of short diagnostic sequence tags and sequencing to reveal patterns of gene expression characteristic of a target function, and has been used to compare expression levels, for example, of thousands of genes in normal and in tumor cells. See, e.g., Velculescu, et al., Science 270:368-369 (1995); Zhang, et al., Science 276:1268-1272 (1997).
Third, approaches have been developed based on differential display. In these approaches, fragments defined by specific sequence delimiters can be used as unique identifiers of genes, when coupled with information about fragment length within the expressed gene. The relative representation of an expressed gene within a cell can then be estimated by the relative representation of the fragment associated with that gene. Examples of some of the several approaches developed to exploit this idea are the restriction enzyme analysis of differentially-expressed sequences (“READS”) employed by Gene Logic, Inc., and total gene expression analysis (“TOGA”) used by Digital Gene Technologies, Inc. CLONTECH, Inc. (Palo Alto, Calif.), for example, sells the Delta™ Differential Display Kit for identification of differentially expressed genes by PCR.
Fourth, in preferred embodiments, the detection is performed by one of a number of techniques for hybridization analysis. In these approaches, RNA from the sample of interest is usually subjected to reverse transcription to obtain labeled cDNA. The cDNA is then hybridized, typically to oligonucleotides or cDNAs of known sequence arrayed on a chip or other surface in a known order. The location of the oligonucleotide to which the labeled cDNA hybridizes provides sequence i
Hu Qianjin
Peng Zaoyuan
Yu Zailin
Horlick Kenneth R.
Mergen Ltd.
Morrison & Foerster / LLP
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