Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-09
2002-04-30
Siew, Jeffrey (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091200, C435S285200, C435S287200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06379897
ABSTRACT:
FIELD OF TEE INVENTION
The present invention presents methods for gene expression monitoring that utilize microelectronic arrays to drive the transport and hybridization of nucleic acids. Procedures are described for generating mRNA expression samples for use in these methods from populations of cells, tissues, or other biological source materials, that may differ in their physiological and/or pathological state. Provided in the invention are methods for generating a reusable nucleic acid transcript library from mRNA in a sample of biological material. In order to improve gene expression monitoring on the microelectronic arrays, the transcripts are amplified to produce sample nucleic acid amplicons of a defined length. Because multiple sample amplicons may be selectively hybridized to controlled sites in the electronic array, the gene expression profiles of the polynucleotide populations from different sources can be directly compared in an array format using electronic hybridization methodologies. Also provided in the invention are methods for detecting the level of sample amplicons using electronically assisted primer extension detection, and utilizing individual test site hybridization controls. The hybridization data collected utilizing the improved methods of the present invention will allow the correlation of changes in mRNA level with the corresponding expression of the encoded protein in the biological source material, and thus aid in studying the role of gene expression in disease.
BACKGROUND OF THE INVENTION
The human genome contains approximately 100,000 genes. These genes are expressed at vastly different levels; the majority of species, over 90%, are present at low abundance, i.e. at five to fifteen copies per cell, while a few high abundance genes are expressed at thousands of copies per cell. In addition to the different levels of basal expression, gene expression is modulated in response to cell state, cell type, extracellular environment, disease, etc. Thus, information on changes in the levels of genes will enable a greater understanding of the pathological and/or physiological state of the organism under conditions of interest.
A number of methods currently exist for analyzing the expression levels of different messenger RNA (mRNA) species. Subtractive hybridization was used early in the history of monitoring of gene expression to analyze differences in levels of gene expression in different cell populations (Scott, et al.). This technique is not sufficiently sensitive to detect messages present at low levels in a polynucleotide population. Representational difference analysis is a more recent modification that includes amplification after subtraction, in order to detect mRNAs that are expressed at low levels (Hubank and Schatz). While this method allows identification of differentially expressed messages that are present at low levels, the amplification step makes quantification difficult.
Adaptations of the polymerase chain reaction (PCR) have proven valuable in the field of gene expression. Reverse transcription coupled with competitive PCR (Competitive RT-PCR) involves co-amplifying a known amount of an exogenous RNA competitor with the target mRNA sequence (Gilliland, et al.). The amount of target is extrapolated from a titration curve based on the concentration of competitor. The difficulties with this technique lie in the limited dynamic range of the assay and the tedium of constructing separate competitors for each target of interest.
Real-time PCR is a powerful approach for gene expression monitoring. The original method detected accumulation of double stranded species during amplification using ethidium bromide and an adapted thermocycler (Higuchi, et al.); detection of non-specific products was a drawback that was subsequently overcome by designing of probes that generate signal only if the target of interest is amplified (Holland, et al.; Lee, et al.). This approach requires that the linear ranges of amplification are similar for abundant internal controls and endogenous target mRNAs that may be present at much lower levels. In addition, primer design is critical and requires special software programs for optimal efficiency.
Differential display PCR (dd-PCR) is also a PCR-based method that has been adapted for monitoring gene expression. The original protocol used sets of random, anchored primers to amplify all mRNAs in two different cell populations; differences in levels are visualized by separating the PCR product on denaturing polyacrylamide gels (Liang and Pardee). Many variations on this original technique have been devised. In general, however, the PCR-based amplification of these methods results in a lack of quantitative correlation of band intensity with message abundance, variable reproducibility, and a high level of false positives. Results generated by dd-PCR must therefore be confirmed by other methods.
Serial analysis of gene expression (SAGE) is another technique for gene expression monitoring. Short sequence tags that uniquely identify the mRNA transcripts in a given cell population are isolated, concatenated, cloned and sequenced (Velculescu, et al.). The frequency of any given tag reflects the abundance of the corresponding transcript. This technique, while powerful, is rather complicated, requires generation and analysis of large amounts of sequence data, and the amplification event can skew quantitation.
The most recent developments in the field are in the area of microarrays (Schena, et al.; DeRisi, et al.; Zhao, et al.). Gene-specific probes are individually arrayed on a solid matrix and incubated with labeled cDNAs from control and experimental populations. Comparison of the intensity of probe hybridization with cDNA targets from the distinct samples reveals differences in expression of the corresponding mRNAs. Because these arrays are hybridized passively in a low stringency buffer, differences in availability of a relevant target sequences to the complimentary probes on the array may not be uniform. In addition, hybridization characteristics of each probe will vary, due to T
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considerations and the affinity of probe-target interactions. Therefore, while these high-density microarrays offer high-throughput, the hybridization kinetics may not be optimal for all different probe-target combinations.
Although great strides have been made in methods to detect alterations in gene expression, each of the procedures has drawbacks as well as advantages, as indicated above. All of the above approaches are either time consuming, complicated, labor intensive, or a combination of all three. Rapid, sensitive approaches that allow simultaneous monitoring of multiple mRNAs are still needed.
SUMMARY OF THE INVENTION
The present invention provides a method that allows efficient electronic hybridization of amplified nucleic acids generated from target mRNAs to complementary probes in a microarray format. The use of electric fields to transport and drive hybridization of nucleic acids allows the rapid analysis of polynucleotide populations. Utilizing electronic hybridization devices, such as those described in U.S. Pat. No. 5,605,662, hybridization assays may be accomplished in as little as 1-5 minutes. Additionally, because each site on the microarray is individually controlled, targets from different samples can be analyzed on the same matrix under optimized conditions, an aspect unique to this technology. By improving the use of electronic hybridization methods and devices in gene expression monitoring applications, the disclosed methods will dramatically increase the ability of those in the art to rapidly generate gene expression information with a minimum of sequence-specific optimization.
The methods of the invention facilitate the use of electronically hybridized gene expression monitoring for both research and clinical applications in several ways. First, through the use of shortened amplicons of uniform size, the methods of the invention allow the rapid, simultaneous monitoring of dozens of genes in comparative and quantitative pr
Heller Michael J.
Kahl Brenda F.
Wang Ling
Weidenhammer Elaine M.
Xu Xiao
Nanogen Inc.
Siew Jeffrey
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