Nucleic acid detection methods using universal priming

Details Nucleic acid detection methods using universal priming Nucleic acid detection methods using universal priming

2001-02-07

2004-11-02

Whisenant, Ethan (Department: 1634)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091100, C436S094000, C536S023100, C536S024300, C536S024330

Reexamination Certificate

active

06812005

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to providing sensitive and accurate assays for gene detection, genome-wide gene expression profiling and alternative splice monitoring, with a minimum or absence of target-specific amplification.
BACKGROUND OF THE INVENTION
The detection of specific nucleic acids is an important tool for diagnostic medicine and molecular biology research. Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses, in probing the expression of normal and mutant genes and identifying mutant genes such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, and for exploring homology among genes from different species.
Ideally, a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 (1993)).
Specificity, in contrast, remains a problem in many currently available gene probe assays. The extent of molecular complementarity between probe and target defines the specificity of the interaction. reaction temperature, and in the length of the probe may alter or influence the specificity of the probe/target interaction.
Genes in higher eukaryotes contain introns, which are removed during RNA processing to generate mature functional mRNAs. In most cases, the removal of introns is efficient, and thus these splicing events are constitutive. However, many transcripts are alternatively processed to generate multiple mRNAs from a single mRNA precurson(pre-mRNA) through the use of different 5′ or 3′ splice sites, exon inclusion or exclusion, and intron retention. The complexity of gene expression is further increased in many cases by coupling alternative splicing with alternative promoters and the use of alternative polyadenylation sites. Based on comparison among expressed sequence tags (ESTs) in databases, it is estimated that as many as 30% of the genes in humans exhibit alternative splicing (Gelfand, M. S., Dubchak, I., Dralyuk, I., & Zorn, M. (1999). ASDB: database of alternatively spliced genes.
Nucleic Acids Research
27:301-302.). Considering that one transcript often gives rise to more than two isoforms, the number of alternatively spliced mRNAs may surpass the total number of genes that are expressed in a higher eukaryotic organism. Because alternatively spliced transcripts may encode protein isoforms that have distinct functions, it becomes a major challenge in functional genomics to relate a biological function not only to the expression of specific genes but also to their isoforms resulting from post-transcriptional processing. This is particularly relevant to cancer research as molecular alterations during malignancy may result from changes not only in gene expression but also in RNA processing.
The functional consequences of alternative splicing plays a vital role in biology and medicine, with a number of well-known examples being illustrative.
Epithelial cells secrete acidic Fibroblast Growth Factor (aFGF), which binds and activates its receptor FGFR2 on the cell surface of fibroblasts. Conversely, fibroblasts secrete Keratinocyte Growth Factor (KGF), which binds and activates KGFR on epithelial cells. Interestingly, FGFR2 and KGFR are generated from the same pre-mRNA by alternative splicing (Miki T., et al., (1992). Determination of Ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene.
Proc. Natl. Acad. Sci. USA
89:246-250). Such cell-specific alternative splicing must be tightly regulated because cells expressing both a growth factor and its specific receptor will be transformed to uncontrolled growth.
A number of apostolic regulators such as Bcl-x, Ced-4, and Caspase-2 (Ich. 1 ) have two isoforms generated by alternative splicing (reviewed by Jiang, Z. H., Zhang, W. J., Rao, Y., & Wu, J. Y. (1996) Regulation of Ich-i pre-mRNA alternative splicing and apoptosis by mammalian splicing factors.
Proc. Natl. Acad. Sci
., 95:9155-9160). In each case, one form promotes programmed cell death and the other prevents cell death. Thus, alternative splicing provides a life or death choice in determining and regulating the ratio of these isoforms.
CD44 is an important cell surface molecule involved in tissue-specific targeting of T cells, B cells, and macrophages in the immune system as well as in cell adhesion and signal transduction. The transcript has 10 alternative exons, which are included/excluded in combination to generate numerous isoforms. Alterations in CD44 splicing are among the best tumor markers (reviewed by Goodison, S. & Tarin, D. (1998). Current status of CD44 variant isoforms as cancer diagnostic markers.
Histopathology
32:1-6). CD44 alternative splicing appears to be regulated by cytokines and by oncogenic activation, and the inclusion of a specific exon (v6) was shown to cause tumor metastasis in a model system (Gunthert et al., 1992. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells.
Cell
65:13-24).
AML1 is a transcription factor required for granulocyte differentiation. The protein contains an N-terminal DNA binding and protein dimerization domain, and a C-terminal transcriptional activation domain. In 20% of acute myelogenous leukemia (AML) patients, the N-terminal sequence of AML1 is fused to sequences from other chromosomes via chromosome translocation. However, in many AML cases, no chromosome translocation is detected, but a change in alternative splicing of AML1 pre-mRNA appears instead. Alternative splicing results in a truncated version of AML1, which was shown to suppress granulocyte differentiation (Tanaka, T. et al., (1995). An Acute myeloid leukemia gene, AML1, regulates hemopoietic myoloid cell differentiation and transcriptional activation antagonistically by two alternative spliced forms.
EMBO J
. 14:341-350.) Thus, some fraction of AML cases may be triggered by a malfunction in splicing control and regulation.
In conclusion, alternative splicing is associated with important biological events, and in many cases, the pattern or alteration of alternative splicing may be markers for specific diseases and/or targets for disease prevention and intervention.
Alternative RNA splicing is widespread in higher eukaryotic cells and plays a vital role in gene expression. However, detection and analysis of alternative splicing currently rely on RNase protection and RT-PCR assays, which are labor intensive, inefficient, and low scale, especially in the era of functional genomics. and RT-PCR assays, which are labor intensive, inefficient, and low scale, especially in the era of functional genomics.
Accordingly, it is an object of the invention to provide a very sensitive and accurate approach for genome-wide gene expression profiling and alternative splice monitoring with a minimum or absence of target-specific amplification.
SUMMARY OF THE INVENTION
In one aspect the invention provides a method of detecting a first target sequence comprising a poly(A) sequence in a sample. The method includes hybridizing a first probe to the target sequence to form a first hybridization complex. The first probe comprises an upstream universal priming site (UUP), an adapter sequence, a first target-specific sequence, and a downstream universal priming site (DUP). The poly(A) sequence remains single-stranded. The method further includes contacting the first hybridization complex with a support comprising a poly(Y) sequence, such that the poly(A) sequence hybridizes with the poly(T) sequence. In addition, the method includes removing unhybridized first probe

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