Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-24
2004-07-06
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06759195
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods and materials useful for performing reverse transcription-polymerase chain reaction in prokaryotic cells.
2. Description of the Related Art
The first comprehensive studies of cellular response resulted from the development of two-dimensional gel electrophoresis and a complementary method for quantitatively measuring protein levels (O'Farell, P. H., “High Resolution Two-Dimensional Electrophoresis of Proteins”,
Journal of Biological Chemistry
, 250(10):4007-21 (1975); and Pederson el al., in 1978). In parallel, Casadaban created transcriptional fusion proteins to assist in the study of gene regulation, which has been particularly useful for analyzing genes whose products are difficult to characterize (Casadaban, M. J., “Regulation of the Regulatory Gene for the Arabinose Pathway, araC”,
Journal of Molecular Biology
, 104:557-66 (1976)). Both of these techniques have since been further refined (See Casadaban, M. J. et al., “Lactose Genes Fused to Exogenous Promoters in One Step Using a Mu-lac Bacteriophage: In vivo Probe for Transcriptional Control Sequences”,
Proceedings of the National Academy of Sciences
, 76(9):4530-33 (1979); and see Kenyon, C. J. et al., “DNA-damaging Agents Stimulate Gene Expression at Specific Loci in
Escherichia coli”, Proceedings of the National Academy of Sciences
, 177(5):2819-23 (1980)).
More recently, detection of transcriptional regulation has been further simplified due to such bioluminescent reporter proteins as luciferase (Rupani, S. et al, “Characterization of the Stress Response of a Bioluminescent Biological Sensor in Batch and Continuous Cultures”,
Biotechnology Progress
, 12:387-92 (1996); and VanDyk, T. K. et al.,“Rapid and Sensitive Pollutant Detection by Induction of Heat Shock Gene-bioluminescence Gene Fusions”,
Applied Environmental Microbiology
, 60:1414-20 (1994)) and green fluorescent protein (Gill, R. T. et al., “Physiological Effects of DTT Addition to
E. coli
Including Growth Rate, Specific Oxygen Uptake, Heat Shock Protein Expression, and Specific Activity of Recombinant Protein”,
Biotechnology and Bioengineering
, 59:248-59 (1998)).
In the mid-1980's, Kohara et al. developed a restriction map of 3400 &lgr; bacteriophage clones containing segments of the
E. coli
chromosome (Kohara et al., “The Physical Map of the Whole
E. coli
Chromosome: Application of a New Strategy for Rapid Analysis and Sorting of a Large Genomic Library”,
Cell
, 50:495-508 (1987)). This set was originally developed to not only map the location of
E. coli
genes but also to map and clone the gene or genes that is (are) induced in response to a certain external or internal signal(s). According to Kohara et al., their identified clones could be exploited for the isolation of any desired
E. coli
genes if their map positions were known.
Using the Kohara set of overlapping &lgr; phage clones, Chuang el al. later demonstrated that global gene regulation in
E. coli
could be analyzed using single stranded reverse transcribed complementary DNA (hereinafter cDNA), in which radiolabeled cDNA was hybridized with the Kohara clones (Chuang, S. el al., “Global Regulation of Gene Expression in
Escherichia coli”, Journal of Bacteriology
, 175:2026-36 (1993)). These clones, containing the entire
E. coli
genome, were used to map the location of cDNA homologs. Through follow-up Southern blotting (Southern, E. M., “Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis”,
Journal of Molecular Biology
, 98:503-17, (1975)), Chuang et al. identified 26 new heat shock genes for
E. coli
(Chuang, S. et al., “Global Regulation of Gene Expression in
Escherichia coli”, Journal of Bacteriology
, 175:2026-36, (1993)). While this technique was a significant improvement over the previous methodologies of two-dimensional electrophoresis or transcriptional fusions for analyzing global genetic regulation, the messenger RNA (hereinafter mRNA) signal to total RNA noise ratio remained small.
One year later, Wong et al. applied a random arbitrary primed PCR amplification step, after reverse transcription of total RNA to detect a stress induced gene in
Salmonella typhimurium
(Wong, K. K. et al., “Stress-inducible Gene of
Salmonella typhimurium
Identified by Arbitrarily Primed PCR of RNA”,
Proceedings of the National Academy of Science U.S.A.
, 91:639-43 (1994). While this technique did improve the level of mRNA signal, the signal to noise ratio did not change due to “random” amplification of RNA templates. In addition, due to the use of sequencing gels for transcript identification, this technique did not permit quantification at the genomic level.
An innovative refinement of these two methods was recently reported by de Saizieu et al., in which non-radioactively labeled total prokaryotic RNA was hybridized directly to an oligonucleotide array synthesized and bonded to a silicon chip (de Saizieu, A. et al., “Bacterial Transcript Imaging by Hybridization of Total RNA to Oligonucleotide Arrays,
Nature Biotechnology
, 16(l):45-8 (1998). This analysis, which allowed quantification for a large subset of transcribed genes, additionally required scanning confocal microscopy as RNA levels were detected as unamplified transcripts.
In the meantime, differential display techniques based on polymerase chain reaction (hereinafter PCR) amplification have advanced rapidly in eukaryotic systems due to mRNA polyadenylation that exclusively occurs in eukaryotic organisms. Reverse transcription-polymerase chain reaction (hereinafter RTPCR) (Liang, P. et al., “Differential Display of Eukaryotic Messenger RNA by Means of the Polymerase Chain Reaction”,
Science
, 257:967-71 (1992)) and random arbitrary-primed polymerase chain reaction (hereinafter RAP-PCR) (Welsh, J. et al., “Arbitrarily Primed PCR Fingerprinting of RNA,
Nucleic Acids Research
”, 20:4965-70 (1992)) specifically were developed in response to the problem of obtaining a small mRNA signal ratio to total RNA noise ratio. These techniques enhanced the mRNA signal to total RNA noise ratio via differential display experiments. With these techniques, the identification of differentially expressed genes among the mRNA for a pair of eukaryotes was carried out, with subsequent recovery of the cDNA and genomic clones for each eukaryote (Liang et al. 1992).
Additional developments in eukaryotic-based RTPCR/RAP-PCR included the use of random arbitrary primers or “motif” primers, the use of sense and antisense oligonucleotide primers, usually degenerate in sequence, which were designed to amplify cDNA templates encoding proteins using particular structural motifs, and amplification products were displayed using agarose gel electrophoresis and ethidium bromide fluorescence staining (Donohue, P. et aL,
Differential Display Methods and Protocols
, Chapter 3: “Differential Display Using Random Hexamer-Primed cDNA, Motif Primers, and Agarose Gel Electrophoresis, 85: 25-35, 25-6 (Peng Liang and Arthur B. Pardee ed., 1997).
Pardee et al, U.S. Pat. No. 5,262,311, disclose a method of isolating mRNAs as cDNAs by employing a polymerase amplification method using two specific primers. This method is used in eukaryotic organisms and is known as differential display. Differential display involves amplifying partial cDNA sequences from subsets of mRNAs by reverse transcription and the polymerase reaction, then displaying these sequences on a sequencing gel.
McClelland et al., U.S. Pat. No. 5,487,985, disclose an arbitrarily primed polymerase chain reaction (hereinafter AP-PCR) for a method of generating a set of discrete DNA amplification products characteristic of a genome as a “fingerprint”. This method is suitable for the identification of bacterial species, bacterial strains, mammals, and plants and utilizes a single-stranded DNA primer.
Villeponteau et al., U.S. Pat. No. 5,580,726, disclose a method and kit for enhanced differential display for eukaryotic organisms which comprises using first oligonucleotide pr
Bentley William E.
Gill Ryan
Fredman Jeffrey
Fuierer Marianne
Hultquist Steven J.
University of Maryland Biotechnology Institute
Yang Yongzhi
LandOfFree
Method of differential display of prokaryotic messenger RNA... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of differential display of prokaryotic messenger RNA..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of differential display of prokaryotic messenger RNA... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3238680