Method of extracting nucleic acids using particulate carrier

Details Method of extracting nucleic acids using particulate carrier Method of extracting nucleic acids using particulate carrier

2002-02-07

2003-06-24

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091300, C536S025400

Reexamination Certificate

active

06582922

ABSTRACT:

The present invention relates to a method of extracting and isolating nucleic acids from a material containing nucleic acids, using a nucleic acid-binding particulate carrier. More specifically, the present invention relates to a nucleic acid extraction method using a porous particulate carrier having a particle diameter of 0.5 to 15.0 &mgr;m, a pore diameter of 50 to 500 nm and a pore volume of 200 to 5000 mm
3
/g.
Recent advances in the genetic engineering and molecular biology fields enable analyses of infections and genetic diseases at the DNA/RNA level. In particular, detection of a trace amount of nucleic acid, which has so far been extremely difficult, becomes readily achievable, and therefore gene analyses are remarkably facilitated, owing to the invention of nucleic acid amplification methods, such as polymerase chain reaction (PCR; Science 230:1350-1354, 1985) and nucleic acid sequence based amplification method (NASBA; Nature 350: 91-92, 1991 and Japanese Patents Nos. 2648802 and 2650159).
However, for detecting a nucleic acid in a biological sample, or amplifying a nucleic acid before detection as required, it is necessary to selectively withdraw nucleic acids from the sample, since biological samples usually contain large amount of constituents other than nucleic acids, such as proteins, lipids and saccharides, which are likely to adversely affect the amplification or detection. Accordingly, manipulations are required to remove contaminants from biological samples and to extract and isolate nucleic acids.
Various techniques have been employed for nucleic acid isolation. Typical examples include techniques in liquid phases, such as the phenol-chloroform extraction method (Biochimica et Biophysica Acta 72:619-629, 1963) and the alkaline SDS method (Nucleic Acid Research 7:1513-1523, 1979). These techniques are widely used on a laboratory scale, but require technological skills and are difficult to carry out with good reproducibility since they use organic solvents that are toxic and difficult to dispose of, such as phenol and chloroform, and use hazardous materials such as sodium hydroxide.
Nucleic acid isolation techniques utilizing nucleic acid-binding carriers include one utilizing glass particles and a sodium iodide solution (Proc. Natl. Acad. Sci. USA 76-2:615-619, 1979), and one utilizing hydroxyapatite (Japanese Unexamined Patent Publication No. 263093/1988). Although these techniques do not employ toxic organic solvents or other toxic substances, but have the problem that they are not suitable for processing many samples at a time and thus require a prolonged period of time, since they involve a number of centrifugation operations.
In short, conventional nucleic acid isolation techniques as mentioned above have the drawback that they use hazardous reagents such as organic solvents and alkalis, or involve centrifugation operations and are unsuitable for processing many samples at a time. Further, the techniques encounter large problems when the extraction and isolation steps are automatized, although automatization of the extraction and isolation of nucleic acids is indispensable for processing a number of samples with good reproducibility and for reducing labor cost.
For automatization of extraction and isolation of nucleic acids, a technique utilizing silica particles and chaotropic ions has been proposed (J. Clinical Microbiology 28-3:495-503, 1990, and Japanese Unexamined Patent Publication No. 289596/1990). The technique comprises mixing a sample with nucleic acid-binding silica particles and chaotropic ions capable of releasing nucleic acids contained in the sample to thereby bind the nucleic acids to silica particles, removing contaminants by washing, and collecting the nucleic acids bound to the silica particles. This technique is advantageous in that it is suitable not only for extraction of DNA but also for extraction of less stable RNA, and capable of giving high-purity nucleic acids. However, the step of washing the particles having bound nucleic acids involves centrifugation, filtration using a filter, etc., and therefore is liable to become complicated when automatized.
For facilitating mixing and washing of the silica particles, Japanese Unexamined Patent Publication No. 19292/1997 discloses a technique comprising magnetizing the silica particles and mixing a sample with the particles utilizing the magnetic field, followed by stirring. In this technique, nucleic acids are bound to the magnetic silica particles, the particles are magnetically separated from the liquid phase, and after washing the particles, the nucleic acids are collected. The steps of the technique become simpler and easier when automatized. However, the influence of contaminants in the sample cannot be excluded by simply employing magnetized particles, and as the result, this technique often shows reduced collection efficiency.
Namely, when a sample containing a large amount of contaminants is used, contaminants in the sample cover the particle surfaces, whereby nucleic acids adsorb to the particle surfaces at a reduced adsorption rate.
To overcome the problem, Japanese Unexamined Patent Publication No. 262387/1999 proposes a technique utilizing a particulate carrier with a larger particle surface area, and Japanese Unexamined Patent Publication No. 178571/1999 proposes a technique wherein a particulate carrier is suspended in a surfactant beforehand in order to preclude adsorption of contaminants. However, it is still difficult to extract a sufficient amount of nucleic acids from samples containing a large amount of contaminants, such as whole blood samples.
The object of the present invention is to solve the above problems by providing a method for efficiently extracting and isolating nucleic acids from a material containing nucleic acids, in particular from a clinical sample or like sample containing a large amount of contaminants.
The present inventors conducted extensive research to achieve the above object, and found that a porous particulate carrier having a pore diameter of at least 50 nm and a pore volume of at least 200 mm
3
/g is useful for efficiently extracting nucleic acids from a biological material, in particular from a clinical sample or like sample containing a large amount of impurities. The present invention has been accomplished based on this novel finding.
Porous particulate carriers have a pore diameter (i.e., diameter of the openings of the pores) and a pore volume (i.e., interior volume of the pores). The present invention has been accomplished based on the finding that the pore diameter and the pore volume affect the collection efficiency of nucleic acids from a sample.
The present invention provides a method of extracting nucleic acids from a material containing nucleic acids using a nucleic acid-binding particulate carrier, wherein the particulate carrier has a particle diameter of 0.5 to 15.0 &mgr;m, a pore diameter of 50 to 500 nm and a pore volume of 200 to 5000 mm
3
/g.
Specifically, the present invention provides a method of extracting nucleic acids from a material containing nucleic acids, the method comprising the following steps (a) to (c):
(a) mixing the material containing nucleic acids, a nucleic acid-binding particulate carrier having a particle diameter of 0.5 to 15.0 &mgr;m, a pore diameter of 50 to 500 nm and a pore volume of 200 to 5000 mm
3
/g, and a nucleic acid extraction solution for allowing the nucleic acids to adsorb to the particulate carrier, to thereby bind the nucleic acids to the particulate carrier;
(b) separating a composite of the nucleic acids and the particulate carrier from the mixture obtained in Step (a) to remove contaminants; and
(c) eluting and collecting the nucleic acids from the composite of the nucleic acids and the particulate carrier.
The present invention also provides a nucleic acid extraction kit comprising a nucleic acid-binding particulate carrier having a particle diameter of 0.5 to 15.0 &mgr;m, a pore diameter of 50 to 500 nm and a pore volume of 200 to 5000 mm
3
/g, a nucleic acid extraction solu

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