WO2005093065A1 - Improved method of isolating nucleic acids - Google Patents

Improved method of isolating nucleic acids

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Publication number
WO2005093065A1
WO2005093065A1 PCT/EP2005/002667 EP2005002667W WO2005093065A1 WO 2005093065 A1 WO2005093065 A1 WO 2005093065A1 EP 2005002667 W EP2005002667 W EP 2005002667W WO 2005093065 A1 WO2005093065 A1 WO 2005093065A1
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ph
buffer
rna
nucleic
solid
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PCT/EP2005/002667
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French (fr)
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Marcellinus Beld
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Roche Diagnostics Gmbh
F. Hoffmann-La Roche Ag
Academic Medical Center At The University Of Amsterdam
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00Processes for the preparation of sugar derivatives
    • C07H1/06Separation; Purification
    • C07H1/08Separation; Purification from natural products

Abstract

The present direction is directed to a method and a kit for the isolation of nucleic acid from a sample containing cells or complex biological material, said method, comprising the steps (i) mixing said sample with a suspension comprising a buffer and a solid support, said suspension having a pH value below pH 5.0 characterized in that said buffer causes lysis of said cells or said biological matrial contained in said sample, further characterized in that subsequent to lysis, the nucleic acid is bound to said solid support, (ii) washing the generated nucleic acid/solid support complex with an appropriate washing solution, and (iii) eluting the nucleic acid from said solid support with an appropriate elution solution.

Description

Improved method of isolating nucleic acids

The present invention relates to the technical field of nucleic acid purification. The nucleic acid purified according to the invention may be used for a variety of analytical test methods such as (RT-)-PCR.

Prior art background

There are several methods known in the art for the isolation and purification of nucleic acids from different samples and complex biological materials.

One standard method well known and very often used in the art for isolating nucleic acids is the use of fractionated silica particles according to the standard protocol as described by Boom, R., et al. in EP 389 063 and J. Clin. Microbiol. 28 (1990) 495-505. In principle, the sample is lysed with a chaotropic agent in the presence of a solid support, which is capable of absorbing any kind of released nucleic acid. More specifically, the method utilizes lysing and nuclease-inactive properties of the chaotropic agent guanidine thiocyanate (GuSCN) combined with the nucleic acid binding properties of silica particles (sc). The lysis buffer inactivates virus and is a safe sample transport medium. RNA and DNA in single and double-stranded forms are simultaneously purified and are free from nucleases (RNAses, DNAses). The purification of total NA can be achieved in 60 to 90 minutes and recoveries are usually between 50 (RNA) and 100% (DNA). The disclosed method is worldwide the most used purification method and has recently also become commercially available. The isolated nucleic acid may subsequently become subjected to different kinds of analytical tests such as PCR in case of DNA or RT-PCR in case of RNA.

In some cases, it is desirable to isolate nucleic acid from sample material such as feces, which has been proven to be a very difficult source. Feces samples are analyzed for the presence of microbial pathogens (viruses, bacteria, parasites, and fungi), or for the detection of tumor cell DNA. Many gastrointestinal diseases, such as severe diarrhea, are caused by vifuses, bacteria, or parasites and may be diagnosed by the use of feces samples. Feces samples however, are known to contain a variety of substances and inhibitors that make feces one of the most troublesome clinical specimens to isolate and analyze. For viral research, feces samples are suspended in virus transport medium (Nutrient broth no. 2 from Oxoid, supplemented with 500 IU penicillin, 500 μg of streptomycin, and 3 μg of amphotericin B per mL) for transport and storage.

In some instances, casein has already been used as an additive in order to improve the performance of enzymes conventionally used in molecular biology. For example, WO 99/61454 discloses that the addition of total casein (the unpurified form) in a lysis buffer purifies not only the nucleic acids but also the active form of casein which relieves inhibition of modifying enzymes like restrictions enzymes, T4 ligase, and DNA polymerases (Boom, R., et al., J. Clin. Microbiol. 37 (1999) 615- 619).

However, all the methods disclosed above have the disadvantage that for some sample material such as feces or sputum, isolation of nucleic acids is only possible with pure yield and suboptimal quality. In particular, recovery of single stranded RNA is suboptimal, characterized in that the RNA is not recovered or partly recovered (< 20%) or maybe (partly) degraded.

As a consequence, the technical problem underlying the invention was to provide a fast and easy method for isolating RNA resulting in high yield and good quality from any kind of sample. In this context, it was also an object of the invention to improve the result of an RT-PCR reactions with RNA obtained from any arbitrary sample.

Brief description of the invention

Therefore, in a first major aspect, the present invention is directed to a method for the isolation of nucleic acid from a sample containing cells or complex biological material, said method, comprising the steps

a) contacting said sample with a suspension comprising a buffer and a solid support, said suspension having a pH value below pH 5.0, characterized in that said buffer causes lysis of said cells or said biological material contained in said sample and makes said nucleic acid to be isolated accessible for direct binding onto said solid support, b) binding said nucleic acid to said solid support, c) washing the generated nucleic acid/solid support complex with an appropriate washing solution, and d) eluting the nucleic acid from said solid support with an appropriate elution solution.

In particular, the inventive method is directed to the isolation of single stranded RNA.

It has been discovered by the inventors that the present inventive method is particularly applicable for nucleic acid isolation from a variety of different sample types such as feces, sputum, nasal swab, throat washes and bronchoalveolar lavage (BAL), spinal fluid or soil.

For performance of the inventive method, any kind of nucleic acid binding solid support. Particular examples not limiting the scope of the invention are silica particles, diatoms, glass particles and magnetic glass particles.

Also for performance of the inventive method, any kind of lysis buffer and elution buffer known in the art may be used. Preferably, such lysis buffer contains a chaotropic salt such as guanidinium-iso-thio-cyanate.

In a particular aspect of the invention, an RNA isolated according to the inventive method may be subjected to a reverse transcriptase reaction to generate a cDNA. In this regard, it has been proven to be advantageous, in case the reverse transcriptase reaction is carried out in the presence of alpha-Casein.

In another aspect, the invention is directed to a suspension comprising a buffer and a solid support, said suspension having a pH value below pH 5.0, characterized in that said buffer causes lysis of cells contained in said sample, further characterized in that said solid support is capable of binding nucleic acid. Said buffer preferably comprises a chaotropic salt, whereas said solid support is selected from a group consisting of silica particles, diatoms, glass particles and magnetic glass particles.

In a final aspect, the invention is directed to kits for performing the inventive method disclosed above. Detailed description of the invention

The present invention is particularly applicable and useful for the isolation of nucleic acid from a sample, which contains mammalian or bacterial cells or cellular components or any other complex biological material. Subsequently, the nucleic acid may become subjected to any kind of analytical test method such as for example but not limiting PCR, RT-PCR or sequencing.

In general, the present invention is directed to a method for the isolation of nucleic acid from a sample containing cells or complex biological material, said method, comprises the following steps treating said sample with a buffer causing lysis of cells contained in said sample and making all nucleic acids accessible for direct further treatments, said buffer having a pH value below pH 5.0, preferably below pH 4.7 and most preferably below pH 4.3, - either at the same time or subsequently contacting said sample or lysate with a solid support under acidic conditions, characterized by a pH value below pH 5.0, preferably below pH 4.7 and most preferably below pH 4.3, further characterized in that under these conditions, the nucleic acid is bound to said solid support, - washing the generated nucleic acid/solid support complex with an appropriate washing solution, and eluting the nucleic acid from said solid support with an appropriate elution solution.

In most cases, the inventive method comprises at least the following 3 steps:

In step a), the sample is contacted with a suspension comprising a buffer and a solid support in such a way that the suspension has a pH value below. pH 5.0 and preferably below pH 4.7 and most preferably below pH 4.3. The buffer and facultative subsequent enzymatic treatment with proteases or lyticases causes lysis of essentially all cells, which are present in that sample. In addition, the buffer causes breakage of cell debris into smaller pieces to such an extend that substantially all nucleic acids becomes available for further manipulating steps. In the case of the present invention, the liberated nucleic acid is subsequently bound to the solid support present in that suspension.

Moreover, the present invention is also applicable when no lysis is required, e.g. if cells are lysed by apoptosis. if free nucleic acids are present in the sample, or in cases where nucleic acids from "cell free" samples like serum, plasma or "theoretically" sputum are to be isolated.

Step b) comprises washing the generated nucleic acid solid support complex with an appropriate washing solution. This washing step maybe carried out not only once but several times with either the same or different washing solutions.

Step c) comprises eluting the nucleic acid from the solid support with an appropriate elution solution. The elution step as well may be repeated several times, which is preferably done with the same elution solution.

In principle, all kinds of different nucleic acids can be isolated according to the invention. This includes double stranded DNA, single stranded DNA, double stranded RNA and single stranded RNA. The present invention however, is especially applicable for the isolation of single stranded RNA such as single stranded viral RNA, mRNA, precursor mRNA or ribosomal RNA .

The source of the sample containing cells or biological complex material maybe very different.

For example, the sample maybe derived for feces, sputum, nasal swab, throat washes, bronchoalveolar lavage (BAL), or spinal fluid. Moreover, the source of the sample is not restricted to body material, since the sample may also be soil as an example for another complex biological material.

In a preferred embodiment, the sample is feces, which is an extremely difficult sample source for which most if not all conventional methods of nucleic acid purification that are known in the art have failed to provide yields in a quantitatively satisfying manner. Depending on the origin of the sample, the present invention is especially applicable for the isolation of nucleic acids of different origin. In case of feces, the invention is particularly applicable for the isolation of nucleic acids from gastroenteric viruses. In case biopsy material, the present invention is especially applicable for the isolation of mRNA specifically expressed in tumor cells thereby enabling for methods of detecting disseminated tumor cells. The following table 1 summarizes preferred embodiments for the isolation of different viral nucleic acids with respect to the preferred clinical indication and the group of viruses that may become detected.

Table 1;

Clinical specimen Group of viruses ΝA

Feces Gastro-enteritis viruses Astroviruses ss-RΝA Adenoviruses ds-DΝA Caliciviruses ss-RΝA Coronaviruses ss-RΝA Enteroviruses ss-RΝA Norwalk like viruses ss-RΝA Parvoviruses ds-DΝA Rotaviruses ds-RΝA

Sputum/BAL/nasal swab/ throat Respiratory viruses washes Flu-A ss-RΝA Flu-B ss-RΝA RSN ss-RΝA Adenoviruses ds-DΝA SARS ss-RΝA Metapneumoviruses ss-RΝA Coronaviruses ss-RΝA Parainluenza 1,2,3,4 ss-RΝA

Cerebrospinal fluid Νeurotropic viruses Enteroviruses ss-RΝA HSN ds-DΝA

The inventive method can be performed using any kind of solid support, which is capable of unspecific binding of nucleic acids. Examples for these are silica particles, which are appropriately size fractionated, diatoms, as disclosed in Boom, R., et al, J. Clin. Microbiol. 28 (1990) 495-505) glass particles or magnetic glass particles which can be used for automated nucleic acid isolation (for example Roche Diagnostics Catalog No. 3038505). Similarly, also the buffer system, and in particular, the lysis buffer can be chosen arbitrarily from methods known in the art. For example, lysis of cells can simply be obtained with a heated solution comprising ProteinaseK and 1 % SDS using different kinds of protocols. Yet, in context of the present invention it is preferred if the lysis buffer comprises a chaotropic salt such as sodium iodate, urea, or potassium chloride. Highly preferred as a chaotropic salts are Guanidinium chloride (Gu-HCl), or guanidinium-iso-thio-cyanate (GU-SCN). Preferred chaotropic salt concentrations are in the range of 1.5 to 5 mol and most preferably in the range between 1.8 and 2.2 mol.

Similarly, washing and elution buffers can be chosen arbitrarily from to methods known in the art. The washing buffer may comprise ethanol or isopropanol in various concentrations. Preferably, the washing buffer may also comprise a chaotropic salt, which can be the same as the chaotropic salt of the respective lysis buffer.

The elution buffer may also contain various types of alcohol but also may simply be a TE-buffer based on low concentrations of Tris, borate and EDTA, or even just water.

As it has been discovered by the inventors of the present invention, it is crucial that the sample providing the nucleic acid is treated with a buffer having a pH value below pH 5.0, preferably below 4.7, and most preferably below pH 4 and, equally important, binding to the solid support occurs in an environment having a pH value below pH 5.0, preferably below 4.7, and most preferably below pH 4.3. There exist different alternative embodiments to prepare such a suspension.

In one embodiment, an excess volume of buffer (which maybe a lysis buffer) with a low pH is mixed with a small volume of solid support particles having a more neutral pH.

Alternatively, the solid support particles may first become suspended in a buffer having a low pH and subsequently are mixed with the sample that is present in a buffer system of more neutral pH. In both cases it is possible to obtain a buffer / solid support suspension having an acidic pH value below pH 5.0 or even lower, which maybe used for the desired isolation of nucleic acid. In a different embodiment, the sample is first contacted with the buffer (and in particular with a lysis buffer) having a low pH value. Subsequently, the (lysed) sample is contacted with an acidic suspension comprising the solid support under conditions characterized in that nucleic acids are bound to said solid support.

In another embodiment of the invention, the sample is also lysed with a buffer having an acidic pH value, but the solid support may be packed in a column. The column is equilibrated appropriately with an acidic buffer, preferably with the same buffer, resulting in an acidic pH value below pH 5, preferably below 4.7, and most preferably below pH 4.3

In a second aspect, the present invention is directed to the specific method for synthesizing a cDNA, characterized in that the template single stranded RNA has been isolated according to one embodiment of the inventive method disclosed above. The reverse transcriptase reaction is either performed by means of using a specific primer in order to obtain a specific cDNA or by means of using random primers in order obtain a pool of RNA's from the sample. In addition, it is also possible to use an oligo-dT primer in order to reverse transcribe the pool of mRNA present in the sample.

The reverse transcription reaction may be followed by a PCR amplification reaction, which results in a typical 2-step RT-PCR. Alternatively, using appropriate thermostable enzymes known in the art, it also within the scope of the invention if a one step RT-PCR starting from an RNA isolated according to the inventive method is performed.

In all cases of reverse transcriptase reactions that have been investigated by the inventors so far, it turned out that alpha-Casein at a concentration of 50-600 μg/ml, preferably 250-500 μg/ml and most preferably 350-450 μg/ml is a useful additive which enhances the performance of the reverse transcription reaction.

In still another aspect, the present invention is directed to a suspension, which comprises a buffer and a solid support, which are useful for carrying out the inventive method. Suspensions comprising a buffer and a solid support according to the present invention have a pH value below pH 5.0, preferably below pH 4.7 and most preferably below pH 4.3. Moreover, it is also preferred if the lysis buffer comprises chaotropic salt. Preferably, the chaotropic salt is selected from a group consisisting of guanidinium- iso-thiocyanate, guanidinium chloride, sodium iodate, urea, or potassium chloride. The final concentration of the chaotropic salt within a suspension comprising the lysis buffer and the solid support should be between 1.0 and 6 mol, and advantageously 1.5 to 5 mol. Most advantageously, the concentration is in the range between 1.8 and 2.2 mol. In addition, it turned out to be advantageous if the Triton X100 is present in the buffer in a concentration between 1 and 10 %, preferably between 1.5 and 3%.

Also advantageously, the solid support of a suspension according to the invention is either silica particles, which maybe size fractionated, diatoms, glass particles, or magnetic glass particles.

In a still further aspect, the invention is directed to a kit for performing the inventive method disclosed above. Such a kit may comprise an appropriate buffer and an appropriate solid support for preparing a suspension according to the invention, characterized in that suspension has finally a pH below pH 5.0, preferably below pH 4.7 and most preferably pH 4.3. In one embodiment, the kit comprises a very acidic lysis buffer having a pH below pH 5.0, preferably below pH 4.7 and most preferably below pH 4.3. In another embodiment, it is the pH of the solid support already being present in a concentrated suspension having a low pH value, said value being below pH 5.0, preferably below pH 4.7 and most preferably below pH 4.3. It is also possible, if both the lysis buffer and the solid support components already have a low pH as required.

Finally the present invention is also directed to a kit for performing a reverse transcriptase reaction comprising a DNA polymerase having a reverse transcriptase activity and alpha-Casein. That DNA polymerase maybe a reverse transcriptase such as AMV reverse transcriptase or a thermostable DNA polymerase possessing reverse transcriptase activity such as Tth polymerase.

The following examples, references and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Description of the Figures

Figure 1 Electrophoretic analysis of MS-2 RNA as disclosed in example 1. The feces samples are flanked by marker lambda x Hindlll. Upper panel: different feces samples, unspiked. Lower panel: the same samples as above, spiked with 200 ng MS2 RNA.

Figure 2 Electrophoretic analysis of MS-2 RNA spiked into two feces specimens (upper panel, lower panel) as disclosed in example 2. The feces samples are flanked by marker lambda x Hindlll and 100% recovery MS2 RNA marker. These data suggest that silica particles bind a component of feces, which interferes with ss-RNA binding. Lanes 1, 2: lack of recovery from particles. Lanes 3, : recovery from supernatant. Lanes 5, 6: lack of recovery from particles treated with fresh binding buffer containing MS2. Lanes 7, 8: recovery from supernatant after treatment with fresh binding buffer containing MS2.

Figure 3 Electrophoretic analysis of MS-2 RNA spiked into 4 different feces . specimens A-D using silica particles with a pH of 5.5, 5.3, 5.0 and 4.7.

Figure 4 Electrophoretic analysis of MS-2 RNA spiked into 10 different feces specimens using silica particles. Lanes 1 and 14: marker lambda DNA x Hindlll. Lane 2 and 13: marker ss-MS2 RNA. Lanes 3 to 12: recovery of feces samples supplemented with ss- MS2 RNA according to the Boom method (upper panel) and with low pH according to the invention (lower panel).

Figure 5 Electrophoretic analysis of cDNA reverse transcribed from MS-2 RNA that had been spiked into 10 different feces specimens using silica particles with a pH of 4.7. Lanes 1 and 14: marker lambda DNA x Hindlll. Lane 2: marker ss-MS2 RNA. with MS2 primer. Lane 13: marker ss-MS RNA without MS2 primer. Lanes 2 to 12: reverse transcribed MS2 RNA in the absence (upper panel) or presence of alpha-Casein (lower panel).

Example 1

Low ss-RNA yield and RNAse activity

The prior art method disclosed in EP 0 389 063, termed hereinafter the "Boom- method" was used for the isolation of nucleic acids from 80 randomly chosen feces samples and prior to extraction were spiked with 800 ng of MS2 RNA (Roche Diagnostics Cat. No.0165948) to monitor the efficiency of recovery of said spiked RNA. An impaired recovery of MS2 RNA was found in 16% of 80 randomly chosen feces samples (Table 2, figure 1, upper panel).

Table 2:

Feces sample numbers Impaired recovery with the prior art Boom- method

101-120 106, 110, 112, 113, 114, 117 121-140 121, 129, 130, 132, 135, 136, 139 141-160 161-180

In order to prove that the impaired recovery of ss-RNA was not due to RNAse activity in the elution buffer, 200 ng of MS2RNA were spiked to 20 μL of the final eluate after extraction of 20 feces samples and incubated this mixture for 10 minutes at 56 °C. We found no RNAse activity in the final eluate and these results are shown in the lower panel of figure 1. The feces samples are flanked by marker lambda x Hindlll and 100% recovery (200 ng) MS2 RNA marker. Examnle 2

Mechanism of low or no recovery of ss-RNA

The mechanism of low or no recovery is for two feces specimens is demonstrated in fig. 2. MS2 RNA was not recovered from these specimens (lane 1 and 2). This was due to the absence of binding to silica particles, since MS2 RNA could be recovered from the remaining supernatant by adsorption to fresh particles (lane 3 and 4). The silica particles remaining after the initial binding step were tested for their potency to bind MS 2 RNA by adding fresh binding buffer and MS2 RNA. Lanes 5 and 6 show that no MS2 RNA was recovered. This was due to the absence of binding to silica particles since MS2 RNA could be recovered from the remaining supernatant by adsorption to fresh silica particles (lanes 7 and 8). Therefore, it can be concluded that silica particles bind a component of feces, which interferes with ss-RNA binding. In fig. 2, the feces samples are flanked by marker lambda x Hindlll and 100% recovery (200 ng) MS2 RNA marker.

Example 3 pH-effect on low or no recovery of ss-RNA

The recovery of MS2 RNA was restored with the Boom-method when the pH of the silica particles was decreased. This pH-effect was shown in Figure 3 using four feces specimens A, B, C and D which showed no or low recovery of MS2 RNA when using the standard silica particles with a pH of 5.5. Lanes 1 to 4, recovery of MS2 RNA from feces sample A after extraction with the standard silica particles (pH of 5.5), lanes 5 to 8 after extraction with silica particles with a pH of 5.3, lanes 9 to 12 after extraction with silica particles with a pH of 5.0, and lanes 13 to 16 after extraction with silica particles with a pH of 4.7, respectively. In fig. 3, the feces samples are flanked by marker lambda x Hindlll and 100% recovery (200 ng) MS2 RNA marker

As it has been determined by means of using commercially available high resolution pH indicator papers, the latter value corresponds to final pH of the lysis buffer/silica particle suspension of pH 4.3, whereas the final pH of the lysis buffer/silica particle suspension according to the Boom method is about 6.4. The results were confirmed by recovery of MS2 RNA from 10 other randomly selected specimens either in a suspension according to the Boom method, or in a buffer/silica particle suspension of pH 4.3, prepared by with a suspension of fractionated silica particles having a pH of 4.7. The results of which are shown in fig. 4, upper panel (Boom method) and lower panel (pH according to the invention). In the fig. 4, the feces samples are flanked by marker lambda x Hindlll and 100% recovery (200 ng) MS2 RNA marker.

Example 4

Inhibition of reverse transcriptase activity

The isolated RNA was subsequently monitored by means of a reverse transcription and subsequent gel electrophoresis using an MS-2 RNA specific primer and Superscript II reverse transcriptase (Invitrogen), which resulted in generation of a 940 bp cDNA fragment. After reverse transcription the cDNA products were incubated with 1 μg/mL RNAse A.

80 randomly selected feces samples were tested for inhibition of reverse transcription. Inhibition of cDNA synthesis was found in 15% of the 80 randomly chosen feces samples (Table 3).

Table 3:

Feces sample Inhibition of RT 101-120 105, 113 121-140 129, 132, 139 141-160 145, 146 161-180 163, 166, 172, 173, 180

Combination of these results with the results of impaired recovery according to example 1 showed an additional effect of both independent phenomena with a total of 31% inconclusive results (Table 4). Table 4:

Feces sample Impaired recovery with the Inhibition of RT standard Boom-method

101-120 106, 110, 112, 113, 114, 117 105, 113 . 121-140 121, 129, 130, 132, 135, 136, 129, 132, 139 139 141-160 - 145, 146 161-180 - 163, 166, 172, 173, 180

Example 5

Example of RT activity with and without alpha- Casein

A reverse transcriptase reaction according to example 4 was repeated on RNA spiked into 10 randomly chosen different feces specimen in the presence or absence of 0,4mg/ml alpha-Casein. Results are shown in fig. 5, demonstrating that in contrast to samples that have been reverse transcribed in the absence of this additive (upper panel), MS2 cDNA was obtained from all specimens in case of addition of alpha-Casein. Thus it can be concluded that inhibition of reverse transcriptase activity can effectively be compensated by addition of alpha-Casein.

List of References

Boom, R., et al., J. Clin. Microbiol. 28 (1990) 495-505 Boom, R., et al., J. Clin. Microbiol. 37 (1999) 615-619 EP 0 389063 WO 99/61454

Claims

Patent Claims
1. Method for the isolation of nucleic acid from a sample containing cells or complex biological material, said method, comprising the steps a) contacting said sample with a suspension comprising a buffer and a solid support, said suspension having a pH value below pH 5.0 characterized in that said buffer causes lysis of said cells or said biological material contained in said sample and makes said nucleic acid to be isolated accessible for direct binding onto said solid support, b) binding said nucleic acid to said, solid support, c) washing the generated nucleic acid/solid support complex with an appropriate washing solution, and d) eluting the nucleic acid from said solid support with an appropriate elution solution.
2. Method according to claim 1, wherein said isolated nucleic acid is single stranded RNA.
3. Method according to claims 1 to 2, wherein said sample is selected from a group consisting of feces, sputum, nasal swab, throat washes and bronchoalveolar lavage (BAL), spinal fluid or soil.
4. Method according to claim 3, wherein said sample is feces.
5. Method according to claims 1 to 4, wherein said solid support is selected from a group consisting of silica particles, diatoms, glass particles and magnetic glass particles.
6. Method according to claims 1 to 5, wherein said buffer comprises a chaotropic salt.
7. Method according to claim 6, wherein said washing buffer comprises a chaotropic salt.
8. Method for synthesizing a cDNA comprising a) isolation of single stranded RNA according to claim 2, b) reverse transcription of at least a part of said single stranded RNA.
9. Method according to claim 8, wherein step b) is performed in the presence of alpha-Casein.
10. A suspension comprising a buffer and a solid support, said suspension having a pH value below pH 5.0, characterized in that said buffer causes lysis of cells contained in said sample, further characterized in that said solid support is capable of binding nucleic acid.
11. A suspension according to claim 10, characterized in that said buffer below pH 5.0 comprises a chaotropic salt.
12. A suspension according to claims 10 to 11, characterized in that said solid support is selected from a group consisting of silica particles, diatoms, glass particles and magnetic glass particles.
13. A kit comprising a buffer and a solid support for preparing a suspension according to claims 10 to 12.
PCT/EP2005/002667 2004-03-16 2005-03-12 Improved method of isolating nucleic acids WO2005093065A1 (en)

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