Isolation of nucleic acid

Details Isolation of nucleic acid Isolation of nucleic acid

2002-04-29

2004-09-21

Whisenant, Ethan (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091510, C536S025320, C536S025400

Reexamination Certificate

active

06794140

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method for isolating RNA from a sample and to a kit for use in such a method.
BACKGROUND OF THE INVENTION
The process by which RNA is separated from other molecules, in particular other cellular components such as proteins, carbohydrates, lipids and DNA is widely known and described in the literature. It is a key process in the understanding of gene function and structure and drug development. Furthermore nucleic acid based diagnostic procedures for detecting RNA viruses such as HCV and HIV absolutely require the viral genomic RNA to be isolated in an intact and relatively pure form. In order to separate RNA in an intact and pure form from other biological material it is necessary to rapidly inactivate all ribonucleases that are present and separate the RNA based on a biophysical property unique to the RNA. However, due to the similar physical characteristics of RNA and DNA, RNA preparations are frequently if not always contaminated with DNA, leading to major difficulties in the analysis of results.
Currently mRNA is separated from genomic DNA by using a method based on oligo (dT) interacting with the poly A tail (Aviv and Leder., Proc. Natl. Acad. Sci. USA. 69, 1408-1412 (1972). However, A-rich DNA sequences are co-purified with this method leading to mRNA contaminated with A-rich DNA. Alternatively, nucleases that are specific for either RNA or DNA can be employed. Such highly purified enzymes are costly to use and frequently require removal before the nucleic acid can be used. For example RNase free, DNase must be removed by phenol extraction or heat inactivation otherwise it will destroy for example PCR primers or other DNA based reagents in all post-nuclease reactions. Another method is to use a mixture of phenol, chloroform, isoamyl alcohol (50:49:1); whereby DNA preferentially partitions into the organic phase whilst RNA remains in the aqueous phase. This method at best still leaves a significant amount of DNA contaminating the RNA and hence is of limited practical use. Another method is the TRI REAGENT™ (Molecular Research Centre, Inc) that allows the simultaneous separation of DNA, RNA and proteins. However, it requires careful separation of different phases from each other and subsequent centrifugation steps whilst not assuring complete separation of DNA from the RNA due to the difficulty of pipetting small volumes of liquid without cross-contamination. Another method is anion exchange chromatography which can separate RNA from DNA but the expense and difficulty of setting up the column as well as the restriction to purifying only small nucleic acids precludes its use from the majority of laboratories. Numerous methods to purify RNA are described in general texts (Jones et al., (1994) in RNA Isolation and Analysis. Bios. Oxford., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, CSH.).
SUMMARY OF THE INVENTION
The present invention provides a preparative method for isolating RNA comprising an oligo- or polynucleotide from a sample, which method comprises:
(a) treating the sample with a reactant capable of covalently modifying the 2′-OH position of the ribose rings of the RNA under conditions so that a proportion of the 2′-OH positions of the ribose rings bear a substituent; and
(b) preparing isolated RNA therefrom by separating material containing the substituent from the sample on the basis of a property of the substituent.
The RNA may be mRNA, tRNA, rRNA, viral RNA, viroid RNA, synthetic RNA such as chemically synthesised or in vitro transcribed forms, or any other form of RNA, such as hnRNA. The RNA may be a mixture of different types of RNA and may be in single- or double-stranded form, linear or circular and contain internal regions of secondary and tertiary structure such as is commonly found in tRNA. According to the present invention an oligonucleotide generally has a sequence of up to about 80 bases and a polynucleotide generally has a sequence length of more than about 80, preferably more than about 100 bases. A preferred length for a polynucleotide is at least 1000 bases.
The mRNA may or may not have a cap and/or poly A tail. The RNA used in the present invention is preferably naturally-occurring. A naturally-occurring RNA according to the present invention typically comprises a nucleotide sequence which is found in nature and which has a structural function or generally encodes a polypeptide having biological activity, or such a nucleotide sequence which is modified, for example to alter in some way the biological activity of the polypeptide encoded thereby. Whilst the naturally-occurring RNA is preferably obtained by transcription from a suitable RNA or DNA template, itself usually naturally-occurring, in some cases the naturally-occurring RNA can be obtained synthetically. RNA according to the present invention does not encompass simple homopolynucleotides (poly A, poly U, poly G and poly C) which can be generated synthetically but are biologically non-functional.
The naturally occurring RNA can be derived from a biological material such as bacteria, viruses such as those causing infection in humans, animals or plants, viroids, or cells such as fungal, animal and plant cells.
An important aspect of this invention is modification of mRNA, rRNA and viral RNA since they are of major scientific and clinical interest and serve as a good example of the problems encountered when manipulating RNA. The invention further provides methods for obtaining intact full-length copies of mRNA, rRNA, viral RNA and other types of RNA isolated from cellular sources or extracellular fluids that demonstrate increased stability in conditions that would otherwise destroy a major fraction of the unmodified RNA.
Measuring the Percentage Modification of RNA
Due to the polymeric nature of RNA, it is difficult to measure its molecular weight above 100 nucleotides using mass spectrometry because a large amount of RNA degradation occurs during the analytical process. However, RNA (tRNA) up to 142 nucleotides (Nordhoff et al., (1993) Nucleic Acids Res. 21:3347; Gruic-Sovulj et al., (1997) Nucleic Acids Res. 25:1859; Tolson and Nicholson (1998) Nucleic Acids Res. 26:446) and double stranded DNA up to 500 base-pairs (Bai et al. (1995) Rapid Comm. Mass Spectrom. 9:1172; Taranenko et al., (1998) Nucleic Acids Res. 26:2488; Ausdall and Marshall (1998) Anal. Biochem. 256:220) have been measured using MALDI mass spectrometry (for reviews see; Smith (1996) Nat. Biotech. 14:1084; Murray (1996) J. of Mass Spectrom. 31:1203. Phosphate (Schuette et al., (1995) J. Pharm. Biomed. Anal. 13:1195; Sinha et al., (1994) Nucleic Acids Res. 22:3119) and chemically modified oligonucleotides (Potier et al., (1994) Nucleic Acids Res. 22:3895) have also been measured using mass spectrometry.
Although there is a molecular weight limitation to a few hundreds of nucleotides when using mass spectrometry, it provides a simple, automated means to accurately determine the exact molecular weight and therefore the percentage modification of a polynucleotide. Optimisation relies on a number of factors such as the type of mass spectrometry being carried out (electro-spray, MALDI-TOF etc), the method used to purify the modified RNA from the modification reaction, the size of the polynucleotide, the ionisation matrix used, the method used to remove cations from the RNA, positive or negative ion mode and the voltage strength used (Fenn et al., 1989) Science 246:64). Capillary high performance liquid chromatography can be used prior to mass spectrometry of RNA because desalting and other purification steps are not required prior to ionisation (Taniguchi and Hayashi (1998) Nucleic Acids Res. 26:1481).
To measure the molecular weight and hence the percentage modification of polynucleotides consisting of thousands of nucleotides requires a different approach. In certain situations where it is preferable to measure the percentage modification of the polynucleotide using more precise means a degradative step may be employed followed by an analyti

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