Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
2001-01-27
2003-09-09
Moran, Marjorie (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C435S006120, C435S091200, C536S024330
Reexamination Certificate
active
06618679
ABSTRACT:
BACKGROUND OF THE INVENTION
Functional genomics is a rapidly growing area of investigation, which includes research into genetic regulation and expression, analysis of mutations that cause changes in gene function, and development of experimental and computational methods for nucleic acid and protein analyses. The Human Genome Project has been the major catalyst driving this research; it has been through the development of high-throughput technologies that it has been possible to map and sequence complex genomes. However, while the nucleic acid sequence information elicited by these technologies represents the “structural” aspects of the genome, it is the interworkings of the genes encoded therein, and the gene products derived from these sequences, that will give a meaningful context to this information. In particular, gene expression monitoring can be utilized to examine groups of related genes, interlocking biochemical pathways, and biological networks as a whole.
This rapidly growing set of cloned human genes provides a plethora of candidate drug targets for testing against complex chemical libraries. In order to efficiently test the impact(s) of a large number of putative drug compounds on the expression profile of one or more sets of genes, methods are needed that are sensitive, quantitative, extremely rapid, and adaptable to automation, in order to be cost-effective. Present day technologies do not meet these demands. The present invention addresses this need by providing novel methods for analyzing gene expression, systems for implementing these techniques, compositions for preparing a plurality of amplification products from a plurality of mRNA target sequences, and related pools of amplification products.
SUMMARY OF THE INVENTION
The present invention provides methods for analyzing gene expression. The methods include obtaining a plurality of cDNA target sequences, and multiplex amplifying these sequences, a process which involves combining the plurality of target sequences with a plurality of target-specific primers and one or more universal primers, to produce a plurality of amplification products. The target sequences are obtained in any of a number of manners, such as by performing reverse transcription on a set of mRNA molecules. The mRNA molecules are optionally derived from cells, organisms, or cell cultures, which are optionally exposed to one or more specific treatments that potentially alter the biological state of the cell, organism, or cell culture.
Target-specific primers for use in the methods of the present invention include oligonucleotides comprising a first sequence that is derived from a target gene of interest and positioned within a 3′ region of the oligonucleotide, and a second sequence that is complementary to a universal primer and positioned within the 5′ region of the oligonucleotide. The target specific primers can be categorized as forward primers or reverse primers, depending upon the relative orientation whether the primer versus the polarity of the nucleic acid sequence (e.g., whether the primer binds to the coding strand or a complementary (noncoding) strand of the target sequence).
The universal primers used in the methods of the present invention are sequences common to a plurality of target-specific primers, but preferably not present in the template nucleic acid (i.e., the plurality of target sequences). As such, a universal primer typically does not hybridize to the target sequence template during a PCR reaction. However, since the universal primer sequence is complementary to a portion of one or more target-specific primers used in the present invention, the universal primer can initiate polymerization using a target-specific primer-amplified product as a template. In some embodiments of the present invention, multiple universal primers having sequences distinct from one another are utilized; these universal primers are then called “semi-universal” primers. As one example, a plurality of semi-universal primers can include primer sequences that are complementary to one or more forward target-specific primers, one or more reverse target-specific primers, or a combination thereof.
Optionally, the multiplex amplification process involves simultaneously amplifying a plurality of cDNA molecules in the same reaction mixture. This can be achieved, for example, by employing one or more target-specific primer pairs (where each pair comprising a forward target-specific primer and a reverse target-specific primer) and one or more universal primer pairs, (also comprising pairs of forward and reverse universal primers). In some embodiments of the present invention, the multiplex amplification involves providing the universal primer in an excess concentration relative to the target-specific primer.
In some embodiments of the methods of the present invention, the length of one or more of the universal primers or target-specific primers is altered prior to combination in the multiplex amplification step. This alteration in length can be achieved, e.g., by adding nucleotides to the end of the primer sequence, inserting nucleotides within the primer sequence, incorporating a non-nucleotide linker within the primer sequence, or cleaving a cleavable linkage within the primer sequence. As one example, alteration of the length of a target-specific primer is achieved by inserting nucleotides between the universal sequence portion (i.e., that sequence complementary to the universal primer sequence) and the target-specific sequence of the primer.
One or more of the nucleic acid sequences used as universal primers and target-specific primers in the methods of the present invention can optionally include a cleavable linkage or a non-nucleotide linker as a sequence element. This non-nucleotide linker can include, e.g., non-cleavable linkages, alkyl chains, or abasic nucleotides. Furthermore, the nucleic acid sequences used as universal primers and target-specific primers in the methods of the present invention can optionally include one or more labels. Labels for use in the methods of the present invention can include, e.g., a chromaphore, a fluorophore, a dye, a releasable label, a mass label, an affinity label, a friction moiety, a hydrophobic group, an isotopic label, or a combination thereof. The same label can be incorporated into disparate primers used in a multiplexed amplification; alternatively, unique labels or combination of labels can be associated with each member of the plurality of primers.
Furthermore, the multiplex amplification optionally includes a reference sequence that contains a region homologous to at least one member of the plurality of target-specific primers. The reference sequence (or sequences) can be endogenously present in the cDNA containing the target sequence, or it can be exogenously added to the cDNA sample.
One or more members of the plurality of amplification products are separated by any of a variety of techniques known to those of skill in the art. In a preferred embodiment of the present invention, the members are separated using one or more separation techniques, such as mass spectrometry, electrophoresis (using, for example, capillary electrophoresis, microcapillary electrophoresis, agarose and/or acrylamide gel platforms), chromatography (e.g., such as HPLC or FPLC), or various microfluidic techniques.
The one or more members are detected by any of a number of techniques, thereby generating one or more sets of gene expression data. For example, in a preferred embodiment, the amplification products are separated and detected by performing HPLC followed by mass spectroscopy.
Detection is performed, for example, by measuring the presence, absence, or quantity/amplitude of one or more properties of the amplification products. Example properties of the amplification products include, but are not limited to, mass, light absorption or emission, and one or more electrochemical properties. In embodiments in which one or more of the primers includes a label, the inherent property can be dependent upon the identi
Loehrlein Christine
Monforte Joseph
Pollart Dan
Shaler Thomas
Stephens Kathy
Althea Technologies Inc.
Horne Angela P.
Moran Marjorie
Quine Intellectual Property Law Group P.C.
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