Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-03-28
2004-07-27
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C435S267000, C435S269000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06767723
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for synthesis of nucleic acids, especially to a method for synthesis of nucleic acids by means of a polymerase chain reaction (hereinafter abbreviated as a PCR).
2. Description of the Related Art
A PCR method is a procedure capable of amplifying an intended DNA fragment as much as several hundred thousand-fold by repeating a process comprised of dissociation of a DNA strand into single strands, binding of primers with sandwiching a particular region of the DNA strand, and a DNA synthesis reaction by the action of a DNA polymerase. The PCR method is described in Japanese Laid-open Patent Publication No.S61-274697 which is an invention by Mullis et al.
A PCR procedure can be used as a highly sensitive method for analyzing nucleic acids in various samples, and particularly it can be used in analysis of nucleic acids in a sample derived from an animal body fluid. The PCR procedure is therefore used for such a purpose of diagnosis or monitoring of an infection, a hereditary disease, and a cancer. The PCR procedure is also suited to DNA typing tests for a transplantation, a paternity test, medical treatments based on an individual genetic information, and the like. For these purposes, a peripheral blood is often selected as a test object.
One drawback of the PCR procedure is that the reaction is inhibited by pigments, proteins, saccharides, or unknown contaminants. Namely, many DNA polymerases including TaqDNA polymerase derived from Thermus aquaticus, a typical thermostable DNA polymerase, are widely known to allow the PCR to be inhibited potently by even a trace amount of living body-derived contaminants existing in the PCR reaction solution. Therefore, the PCR procedure requires a process in which a cell(s), a protozoan (protozoa), a fungus (fungi), a bacterium (bacteria), a virus(es) and the like (hereinafter referred to as a gene inclusion body) are isolated from a subject and then nucleic acids are extracted from the gene inclusion body prior to a DNA amplification. Such process has conventionally been a procedure in which the gene inclusion body is decomposed using an enzyme, a surfactant, a chaotropic agent, or the like, and then nucleic acids are extracted from the decomposed product of the gene inclusion body using, for example, phenol or phenol/chloroform. Recently, an ion-exchange resin, a glass filter, or a reagent having an effect of agglutinating proteins is used in the step of the nucleic acid extraction.
It is difficult, however, to completely remove impurities by purifying nucleic acids in a sample using these procedures, and furthermore, an amount of nucleic acids in a sample recovered by these purification procedures often varies among experiments. For these reasons, a subsequent nucleic acid synthesis may sometimes be unsuccessful, especially when a content of the intended nucleic acid in the sample is low. In addition, these purification procedures involve complicated manipulations and are time-consuming, and there is a high opportunity for contamination during the procedures. Therefore, a simpler, more convenient and effective method of a sample pretreatment is desired in order to solve these problems.
When a body fluid such as blood and a liquid excretion sample such as urine are left to stand, sedimentation of solid components such as cells, bacterial body components and the like occurs to cause heterogeneity in the distribution of cells or bacterial bodies containing an intended nucleic acid. It was therefore necessary to uniformly distribute the solid components in these samples by agitating the samples in advance before the sample addition, when these samples are used for a direct amplification of a nucleic acid.
A peripheral blood is often used, as a test material for a genetic testing. We have hitherto devised methods that provide a nucleic acid synthesis procedure capable of directly amplifying an intended nucleic acid in blood. However, when a whole blood sample is left to stand, sedimentation of blood cells, bacterial body components, or other components occurs to cause heterogeneity in the distribution of blood cells, bacterial bodies, and other components containing the intended nucleic acid. It was therefore necessary to achieve uniform distribution by agitating the sample in advance before its addition, when the blood sample is directly subjected to the PCR. Likewise, the same step was also required when a body fluid and a liquid excretion sample are directly used in the PCR.
SUMMARY OF THE INVENTION
The present inventor invented a process in which a sample is treated with a surfactant, then stored and used without further treatments, as a template for nucleic acid synthesis. The present invention is particularly useful in a method of nucleic acid synthesis in which a body fluid such as blood and a liquid excretion itself such as urine are mixed with a nucleic acid amplification reaction solution and allowed to react, for treating a sample with a surfactant before the reaction and, for example, thereby destructing solid components containing nucleic acids to disperse them uniformly in the sample liquid. In addition, such treatment is expected to have bactericidal, virucidal, and the like effects and thereby reduce the possible risk of infection of a worker caused by a biological sample that is inherent in handling biological samples.
Thus, the present invention is a method for synthesis of nucleic acids to amplify an intended nucleic acid from a sample which comprises homogenizing a living body-derived sample and then directly adding the homogenized sample to a reaction solution to amplify the nucleic acid.
The present invention is the method for synthesis of nucleic acids wherein the sample is homogenized using a surfactant.
The present invention is the method for synthesis of nucleic acids wherein the surfactant is an ionic surfactant.
The present invention is the method for synthesis of nucleic acids wherein the ionic surfactant is an anionic surfactant.
The present invention is the method for synthesis of nucleic acids wherein the anionic surfactant is at least one selected from the group consisting of salts of N-lauroylsarcosine and dodecyl sulfates (e.g. SDS).
The present invention is the method for synthesis of nucleic acids wherein the homogenized sample is subjected to nucleic acid synthesis in a reaction solution containing a nonionic surfactant.
The present invention is the method for synthesis of nucleic acids wherein Tween 20 and/or Nonident P40 is used as the nonionic surfactant.
Further, the present invention is a method of sample storage, which comprises homogenizing a living body-derived sample and storing the homogenized sample.
According to the present invention, by treating the sample with the surfactant before the reaction, solid components such as cells or bacterial bodies containing nucleic acids can be destructed and uniformly dispersed in the sample liquid, and therefore it is not necessary to agitate the sample in advance for uniformly distributing the solid components in the sample. In addition, the present invention enables long-term sample storage.
REFERENCES:
patent: 5501963 (1996-03-01), Burckhardt
Steiner et al., “A rapid one-tube genomic DNA extraction process for PCR and RAPD analyses,” Nucleic Acids Research, 1995, vol. 23, No. 13, pp. 2569-2570.*
Liu et al., “Effects of three sample preservation methods on total DNA preparation of porcine whole blood,” Di-San Junyi Daxue Xuebao, 1999, vol. 21, No. 1, Abstract only.
Horlick Kenneth R.
Kim Young J.
Rader & Fishman & Grauer, PLLC
Shimadzu Corporation
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