Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-05-11
2004-11-02
Ketter, James (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S007500, C435S006120, C536S023100, C536S025500
Reexamination Certificate
active
06812341
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of RNA isolation, more specifically it concerns a faster, more efficient method for the isolation of messenger RNA (mRNA) from total RNA.
2. Description of Related Art
In eukaryotic organisms, one trait shared by all mRNAs is the presence on the 3′ end of a stretch of tens to hundreds of adenine (A) residues. This has been used as a means for separating these molecules away from rRNA and other non-mRNA species that lack this “polyA tail.” The standard protocol for the selection of polyA
+
RNA is based on the method of Aviv and Leder (1972), wherein a short stretch of DNA consisting solely of thymidine (T) residues (“oligo-dT”) is affixed to an insoluble matrix. The original procedure used oligo-dT covalently linked to cellulose poured into a column. This is then used as a selective immobilization matrix for mRNA in the sample by setting up conditions that favor formation of RNA-DNA double strands. The total RNA sample is applied to the column in an appropriate salt buffer (originally 0.5 M KCl in 10 mM Tris, pH 7.5), encouraging hybridization to the polyA stretches found solely at the 3′ ends of mRNA. The column is then subjected to extensive washing with the application buffer (containing 0.5 M KCl), then a lower-ionic-strength solution (0.1 M KCl), followed by elution of mRNA with 10 mM Tris (pH 7.5). Subsequent modifications on this original procedure have retained the basic process of hybridization to immobilized oligo-dT in approximately 0.5 M salt, but have changed the format from columns to batch procedures to allow the procedure to be performed faster and have used NaCl or LiCl as the salts.
Further changes have been the replacement of cellulose with plastic or glass beads as the immobilization matrix, some of which are impregnated with ferrous material giving them a magnetic quality. This magnetic quality allows such magnetic beads, as they are referred to, to be batch isolated on magnetic stands rather than requiring gravity or centrifugal force to pellet or filter separate. A further wrinkle in the procedure is the use of a biotin-streptavidin linkage in the connection between oligo-dT and bead, where the oligo is biotinylated and the bead is covalently coupled to streptavidin. The hybridization can be performed in solution with this procedure, linking the oligo-dT-mRNA hybrids to the beads in a subsequent step.
This procedure has tended to be an inefficient method for the separation of rRNA from the mRNA. rRNA carryover levels are often high enough to provide the same problems as presented with total RNA, especially for analysis of rare transcripts. In fact, it usually requires two or more passages through a matrix of choice to produce RNA sufficiently low in rRNA levels to provide a useful sample. This requirement for repetitions of the entire selection procedure can lead to several disadvantageous side effects, which can extend the hours required to perform the procedure. Additionally, the representational distribution of various mRNAs may become altered (or more altered), or the unavoidable losses associated with the repeated procedure may reduce the level of the commonly-sought low-abundance messages beyond the limits of detection. While these drawbacks may be tolerable in a research lab, it a major problem in a diagnostic setting where simplicity, speed and reliability are driving characteristics for a viable assay.
Factors affecting the carryover of rRNA have not been thoroughly studied. Remedial measures have been taken with a vague notion of enhancing the “good” interaction in the extended A:T hybrid desired by using low ionic-strength (high-stringency) washes and trying to find more “inert” materials to use as support. Current procedures use washes at lower ionic strength to remove rRNA that is non-specifically bound to the matrix and the oligo dT. To minimize the non-specific binding of rRNA, novel matrix compositions distinct from cellulose have been pursued, using beads made out of synthetic polymers like latex, polystyrene, other plastics, or even glass. A further modification to enable the streamlining of washing routines was the introduction of plastic or glass beads impregnated with magnetite. This enables the capture of all the beads in a microcentrifuge tube by pulling them to the side with a powerful magnet. Supernatants can be aspirated away to the bottom of the tube, leaving very little carryover of each wash.
Isostabilizing agents, such as tetramethylammonium (TMA
+
) and tetraethylammonium (TEA
+
) ions and the amino acid betaine, equalize the hydrogen bonding strength of the G-C and A-T base pairs when used at the appropriate concentrations (Jacobs et al., 1988; Jacobs et al., 1985; Gitschier et al., 1986; Melchior et al., 1973; Rees et al., 1993; Wood et al., 1985; Wozney, 1990). However, while tetramethylammonium chloride (TMAC) and tetraethylammonium chloride (TEAC) have been studied with respect to their effect on thermal transition effects on double-stranded nucleic acid (Golas et al., 1980) and have been employed for hybridization generally (U.S. Pat. No. 5,759,777), they have not been used with a mixed RNA population to facilitate mRNA isolation since some of the problems associated with mRNA isolation with existing techniques were unrecognized.
Thus, there is a need to identify the factors affecting mRNA isolation. With this information, compositions and methods for improved separation of mRNA from other RNA molecules can be developed. Such compositions and methods could facilitate mRNA isolation and purification, for example by increasing the speed of the process or increasing the purity of the end product, which is necessary for scientific research, as well as diagnostic and therapeutic purposes.
SUMMARY OF THE INVENTION
The present invention takes advantage of the discovery that some problems with mRNA isolation stems from rRNA carryover that is based not on rRNA interactions with the targetting molecule, oligo-dT, but on rRNA interactions with the targeted molecule, mRNA. While hybridization between oligo-dT and poly(A) RNA is based solely on A:T base-pairing, interactions between rRNA and mRNA involves both A:T and G:C base-pairing. A G:C base pair is stronger than an A:T or A:U base pair, such that the advantage of exact complementation between A and T residues for long stretches (between the targeting molecule and the targeted molecule) are less significant in the face of stronger G:C base pairing between the targeted molecule, mRNA, and contaminating rRNA, even though the stretches may be localized (or shorter) or less exact. Thus, the invention is based on the additional discovery that mRNA isolation is affected by reducing the hybridization between contaminating rRNA and mRNA through the use of an isostabilizing agent, such as TMAC or TEAC salts or the amino acid betaine, which minimizes any difference in bond strength between A:T and G:C basepairs.
The present invention concerns methods, compositions, and kits that facilitate mRNA isolation by increasing the ease or convenience of mRNA isolation, reducing contamination by other non mRNA molecules, increasing the concentration of mRNA, reducing the amount of time to effect mRNA isolation, reducing degradation of RNA in a sample, or any other way that facilitates mRNA isolation.
Thus, in some embodiments of the present invention, methods for purifying mRNA from a sample are contemplated. The term “purifying” refers to the isolation of mRNA, such that the concentration of mRNA compared to other components increases relative to other components, such as other RNA species. Throughout this application, the terms “mRNA” and “poly(A) RNA” are used synonymously since mRNA from most non-bacterial organisms have a poly(A) tail. Any poly(A) RNA from any species is specifically contemplated for isolation using the methods of the present invention. “Poly(A) RNA” includes any RNA species with consecutive “A” residues toward the 3′ end of an RNA tra
Ambion Inc.
Fulbright & Jaworski L.L.P.
Ketter James
Lambertson David A.
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