Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-08-24
2002-01-15
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120
Reexamination Certificate
active
06338954
ABSTRACT:
The present invention is concerned with a method for generating, in a non specific manner, multiple copies of RNA from a pool of mRNA's. Such a method is of particular importance in techniques for screening the differences in expression in given cell types or in cells under specific conditions.
In cells of higher organisms only some 15% of the genes present (each cell contains about 100,000 genes) is expressed. Gene expression varies between different cell types and between different stages of development of a given cell and is crucial to all biological processes, such as aging, cell differentiation, and infectious or other disease states. Thus the identification of genes that are differentially expressed in cells under different conditions is of prime interest in cellular biology.
To be able to analyze the mRNA content derived from only a few cells a method is needed to amplify the mRNA present in the cell(s) under investigation. Much effort has already been put in methods to examine the mRNA population of a cell. This has lead to the development of techniques to label nucleic acid material starting from the mRNA population of a cell aimed at the identification of genes that are differentially expressed in cells under various conditions. One method for screening differences in gene expression is a method known as Differential Display (Liang and Pardee, Science, Vol 257, 967-971, 1992; U.S. Pat. No. 5,262,311 which issued on Nov. 16, 1993). With the method of Liang and Pardee mRNA is first transcribed into cDNA and amplified using the Polymerase Chain Reaction (PCR). A set of oligonucleotide primers is used, the first of which is anchored to the polyadenylated tail of a subset of mRNA's, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The method is used with different pairs of alterable sequences aiming at the amplification of as many mRNA's as possible from the cells under investigation. The PCR products are labeled using tracer amounts of labeled (radioactive) nucleotides.
An improvement on the Differential Display method of Liang and Pardee was disclosed in U.S. Pat. No. 5,589,726. The method described in U.S. Pat. No. 5,589,726 differs from the method of Liang and Pardee in that it uses longer primers (22-30 nucleotides compared to the 9-14 base primers originally described by Liang and Pardee).
Another alleged improvement over the Differential Display technique as originally disclosed by Liang and Pardee is disclosed in WO 97/37045. In this application a method is disclosed that, again, is based on PCR: This method uses an oligo-dT primer rather than an anchored primer. Thus, after the reverse transcription step only one cDNA population covering all possible mRNA sequences is created. The cDNA thus obtained is titrated into the PCR process by running several IPCR reactions at decreasing concentrations of cDNA. This serves to calibrate the method and to protect it against false negatives. The PCR reaction may be performed with anchored primers again.
Yet another method for “expression profiling” of mRNA's is disclosed in U.S. Pat. No. 5,514,545. With this method mRNA in a single cell can be characterized by microinjecting into a cell a first amplification oligonucleotide comprising oligo-dT and the sequence of a bacteriophage promoter such as T7, T3 or SP6, reverse transcriptase and nucleotides to synthesize a first strand of cDNA from the mRNA in the cell. From the first strand of cDNA double stranded cDNA is synthesized. Since this double stranded cDNA includes a functional promoter aRNA (anti-sense RNA) can now be synthesized therefrom using an RNA polymerase. The aRNA is now reamplified using random hexanucleotide primers with a reverse transcriptase to form first strand cDNA.
With all the above techniques cDNA is made starting with a primer using the mRNA as a template. However, the enzyme that is used for this reaction (reverse transcriptase) is hampered in the cDNA synthesis by structures in the mRNA. As a result the prior art methods are selective for mRNA's with little or no structure. This effect is further enhanced if the synthesized cDNA is amplified further, for instance by PCR. Due to the aforementioned it is common practice to use a large sample amount in these type of expression profiling analysis. Thus this technical threshold does not allow the analysis of only a few cells isolated on a cell sorter or a few cells isolated via micro dissection from a glass slide after microscope identification and selection.
The solution to the problem is the use of a non-selective poly A mRNA labeling and amplification method, i.e. a method not encompassing cDNA synthesis.
The present invention provides such a method. The present invention is directed to a method for amplifying RNA by creating, in a non specific manner, multiple RNA copies starting from nucleic acid containing starting material comprising a pool of mRNA's each mRNA comprising a poly-A tail, wherein the material is contacted simultaneously with an oligonucleotide comprising an oligo-dT sequence, the sequence of a promoter recognized by a RNA polymerase and a transcription initiation region which is located between the oligo-dT sequence and the sequence of the promoter, and further with an enzyme having reverse transcriptase activity, an enzyme having RNase H activity and an enzyme having RNA polymerase activity and the necessary nucleotides and the resulting reaction mixture is maintained under the appropriate conditions for a sufficient amount of time for the enzymatic processes to take place.
This will lead to the formation of multiple anti-sense RNA copies of the mRNA's present in the reaction mixture. The method of the present invention does not involve the production of cDNA intermediates; RNA is copied directly from the mRNA present in the material under investigation. The method of the present invention does not need a cDNA as a basis for the amplification of the RNA. The RNA is synthesized by an RNA polymerase, directly from the mRNA template. The activity of the RNA polymerase is independent from any secondary structures present in the mRNA and thus there are no differences in the way the different mRNA's are amplified depending on structures in the mRNA's. The copies made represent the original mRNA population as present in the starting material.
The oligonucleotides used with the method of the invention comprise an oligo-dT sequence which will hybridize to the poly- and enylated tail at the 3′ end of the mRNA's. The oligonucleotides further comprise the sequence of a promoter recognized by an RNA polymerase and a transcription initiation region which is located between the oligo-dT sequence and the sequence of the promoter. The promoter may be the promoter for any suitable RNA polymerase. Examples of RNA polymerases are polymerases from
E. coli
and bacteriophages T7, T3 and SP6. Preferably the RNA polymerase is a bacteriophage-derived RNA polymerase, in particular the T7 polymerase.
The oligonucleotide may be blocked at its 3′ end. If the oligonucleotide is not blocked at its 3′ end, it is extendible by the reverse transcriptase. However, the cDNA that would thus be generated will not be a part of the amplification mechanism (as it is with prior art methods). It even interferes with the other enzymatic reactions. The extension of the oligonucleotide is only with a very limited number of nucleotides because if the promoter is made double stranded the transcription on the mRNA template by the RNA polymerase will start immediately. This transcription will “push” the RT from the RNA template and extension (i.e. cDNA synthesis) of the oligonucleotide can no longer occur. The oligonucleotide can be blocked at its 3′ end to prevent any extension therefrom by the reverse transcriptase along the RNA template; the reverse transcriptase will not be able to start extension of the 3′ end of the oligonucleotide and no cDNA is synthesized. The reverse transc
Horlick Kenneth R.
PamGene B.V.
Strzelecka Teresa
LandOfFree
Method for the non-specific amplification of nucleic acid 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 for the non-specific amplification of nucleic acid, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for the non-specific amplification of nucleic acid will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2861526