Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-08-07
2001-08-07
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S015000, C435S091200, C435S091500, C435S091510, C435S091520, C536S024200, C536S024330
Reexamination Certificate
active
06270966
ABSTRACT:
The present invention relates to a method of detecting and characterizing gene expression in cells, and specifically relates to a method of detecting a population of mRNA molecules by transcribing the mRNA into complementary DNA (cDNA) molecules, restriction cutting the DNA, adding adaptor DNA sequences, amplifying the cDNA molecules by a polymerase chain reaction (PCR) and detecting the amplified cDNA.
BACKGROUND OF THE INVENTION
Mammalian cells each generally express approximately 15% of the total of about 100,000 genes under normal physiological conditions. Gene expression results in about 15,000 individual mRNA species per cell, of which about 1% represent expression unique to a cell type or to a cell's developmental or physiological state. The relatively or almost unique expression of a minority of genes in cells of interest compared with other cells is referred to as differential expression and has been used to classify cells based on their mRNA content. For example, different types of tumor cells may be classified and compared with normal cells based on differential expression of oncogenes.
Because differential expression of genes can be used to characterize cells based on their mRNA content, investigators have developed methods to monitor differential expression of a population of cells. Many methods rely on subtractive RNA hybridization techniques (Lee et al.,
Proc. Natl. Acad. Sci. USA
88: 2825-2830, 1991). More recently, a method of differential display of eukaryotic mRNA following reverse transcription into DNA and amplification by a polymerase chain reaction (PCR) has been developed to visualize subsets of molecules on a gel (Liang, P. and Pardee, A. B.,
Science
257:967-71, 1992). Methods of this type are herein referred to as differential display-polymerase chain reaction (DD-PCR) techniques.
Liang and Pardee described a method of separating and displaying individual mRNA species called the DD-PCR technique (for differential display-polymerase chain reaction). In this method, mRNA isolated from eukaryotic cells is reverse transcribed into cDNA, which is then selectively amplified using a series of primers in PCR. The primer for the first strand synthesis contains an oligo-dT sequence anchored by the addition of two bases at the 3′ end of the primer (e.g., 5′ T
11
CA 3′). The second strand primer used in PCR amplification is either a selected sequence specific for a known gene or any arbitrary oligonucleotide capable of priming a PCR. The amplified sequences correspond to the 3′ end of the expressed genes. PCR amplification is done in the presence of a radiolabeled nucleotide (e.g., ATP labeled at the &agr; position with
35
S) and the amplified radioactive molecules are separated as single stranded molecules on a denaturing DNA sequencing gel. They are then visualized by autoradiography. About 50-100 bands (of up to about 500 bp in size) of the reverse transcribed and amplified mRNA are displayed. This visual display of the differentially expressed subset of genes is called a differential display. The pattern of a differential display is characteristic of the cell type analyzed, the cell physiology when the mRNA was isolated, the species from which the cells were derived and the primers used in PCR amplification. Thus, a differential display can be used to distinguish cell types based on their mRNA content, similar to the characterization of cells by DNA fingerprinting.
By amplifying aliquots of RNA with oligo-dT primers differing in the two additional 3′ bases, the total mRNA can be amplified in twelve subsets (e.g., primer 5′ T
11
CA 3′ to amplify one subset, primer 5′ T
11
GA 3′ to amplify another subset, etc.). Each subset results in its own unique differential display pattern obtained using the same source of mRNA. Bands of interest can be eluted from the gel and used as probes, DNA sequenced or cloned using conventional methods.
The original DD-PCR method has been further refined to include other anchored primers and optimized conditions for PCR (Liang, P., et al.,
Nucleic Acids Res
. 21: 3269-3275, 1993). The optimized anchored oligo-dT primer is degenerate, comprising 5′ T
12
MN 3′, where M can be A, C or G, but not T, and N can be any of the four possible deoxynucleotides (T, A, C or G). Both M and N are essential to anchor the primer to the end of the poly(A) tail of the mRNA, but N lends specificity to the primer. By using a mixture of primers degenerate at the M position, the number of PCR reactions for the differential display of an RNA sample can be decreased to three instead of twelve. DNA-free RNA, either total cellular RNA or mRNA, can be used. Cloning of the amplified fragments is achieved by eluting bands of interest from the display gel, amplifying them again by PCR and cloning them into a vector. An eluted band, reamplified by PCR, can be used to probe RNA blots to identify PCR fragments that hybridize to the RNA of interest to improve the probability of isolating the clones of interest (Utans, U. et al.,
Proc. Natl. Acad. Sci. USA
91: 6463-6467, 1994).
Other modifications of the DD-PCR method include the use of a nondenaturing gel to detect double stranded DNA fragments (Bauer, D., et al.,
Nucleic Acids Res
. 21: 4272-4280, 1993). Dye-labeled primers can be used in place of a radiolabeled primer in the PCR reactions so that the amplified fragments can be detected by using an automated DNA sequencing machine (Bauer, D., et al.,
Nucleic Acids Res
. 21: 4272-4280, 1993; Ito, T. et al.,
FEBS Lett
. 351: 231-236, 1994).
Another modification of the DD-PCR procedure, useful for analyzing in vivo samples, employs hybridization to immobilized RNA or to immobilized plasmid DNA, followed by direct PCR sequencing of the DNA (Mou, et al.,
Biochem. Biophys. Res. Commun
. 199: 564-569, 1994). These hybridization steps selectively display a cDNA of interest instead of displaying the entire population of amplified fragments resulting from the DD-PCR reactions.
In addition to DD-PCR, other methods of RNA amplification using reverse transcription and PCR are known. U.S. Pat. No. 5,104,792 discloses a method of nucleic acid amplification using “universal” primers having identical 5′ end sequences but degenerate 3′ end sequences. The degenerate 3′ sequences anneal to the nucleic acid of interest at random sites, and the 5′ identical ends are used subsequently in sequencing, cloning or other standard molecular genetic manipulations. After two or more rounds of extension with these primers, the 3′ degenerate primers are removed. The sequences are then amplified by PCR using primers in which the 3′ sequence is identical to the 5′ end non-degenerate sequence of the universal primer set.
The published abstract (available from Derwent World Patents Index, Derwent Info Ltd.) of U.S. patent application Ser. No. 7,669,731 discloses a method of detection of RNA sequences using reverse transcription and PCR. This technique specifically amplifies mRNA sequences without amplifying contaminating DNA sequences, allowing for detection of sequences present in the mRNA. In this method, the primer for reverse transcription of mRNA molecules contains a unique, random nucleotide sequence for “tagging” the cDNA strands. Then, a second primer-that anneals to the tagged cDNA at a position upstream of the first primer is used to extend the sequence at a temperature that does not allow hybridization of the first primer.
One method of detecting differential gene expression relies on subtractive hybridization of PCR-amplified cDNA (Hubank, M. and Schatz, D. G.,
Nucleic Acid Res
. 22: 5640-5648, 1994). This method, called representational difference analysis, is a modification of a method used to screen differences in genomic DNA. In this method, mRNA is reverse transcribed into cDNA, which is cut with a restriction enzyme. An adaptor sequence is ligated to the cut ends to serve as a hybridization site for appropriate PCR primers, and the fragments are PCR amplified
Buolamwini John
Weinstein John N.
Johannsen Diana
Knobbe Martens Olson & Bear LLP
Myers Carla J.
The United States of America as represented by the Department of
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