Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-30
2001-09-11
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06287769
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of amplifying DNA fragments, an apparatus for amplifying DNA fragments, a method of assaying microorganisms, a method of analyzing microorganisms and a method of assaying a contaminant.
2. Description of the Prior Art
In recent years, a garbage disposal for composting organic waste (the so-called kitchen garbage) discharged from a household kitchen or the like is now actively researched and developed. In the garbage disposal, microorganisms such as bacteria and protozoa degrade organic matter to form compost.
During the composting process (organic degradation process) in such a garbage disposal, the degree of composting is evaluated by monitoring the temperature or the like. The state of the garbage disposal is adjusted to prepare high-quality compost on the basis of the evaluation.
In order to prepare high-quality compost, it is necessary to obtain information of the microorganisms (at least the types of the microorganisms) functioning in the garbage disposal. The information of the microorganisms is also necessary for excellently controlling degradation of the kitchen garbage with the microorganisms. In order to improve soil by adding the prepared compost, it is also important to obtain information of microorganisms contained in the soil.
In general, information of microorganisms such as bacteria, for example, is obtained by a method of isolating each bacterium included in the bacteria and biochemically examining the same. However, this method requires much time, and it is difficult to analyze a bacterium which is hard to isolate by this method.
On the other hand, a PCR (polymerase chain reaction) method is employed for amplifying DNA (U.S. Pat. Nos. 4,683,195, 4,683,202, 4,965,188, 5,038,852 and 5,333,675). In the PCR method, a primer having a base sequence complementary to that at both ends of DNA (template DNA) to be amplified and heat-resistant DNA polymerase are employed for repeating a cycle formed by three stages of a thermal denaturation step, an annealing (heat treatment) step and an extension reaction step thereby enabling amplification of DNA fragments substantially identical to the template DNA. Employing this PCR method, a prescribed fragment in DNA of one of a small amount of bacteria can be amplified to hundred thousand to million times, for example.
In order to employ the PCR method, however, the base sequence of at least at both ends of a part of the template DNA must be known. If the types and base sequences of the microorganisms functioning in the garbage disposal or existing in the soil are unknown, therefore, DNA fragments of the microorganisms cannot be amplified in the conventional PCR method.
In this regard, there has been proposed a RAPD (random amplified polymorphic DNA) method or AP-PCR (arbitrarily primed-polymerase chain reaction) method of simultaneously amplifying many types of DNA fragments from a single type of DNA with a single primer, with no information of the base sequence. According to this method, the annealing temperature for the primer is reduced while the magnesium ion concentration in a reaction solution is increased during PCR, thereby reducing sequence specificity of the primer in bonding. Thus, the primer is bonded to chromosome DNA of a microorganism with mismatching, to duplicate DNA fragments.
According to the RAPD method or AP-PCR method, some DNA fragments are amplified in a large amount with a single primer, with no information on the base sequence of the DNA to be amplified. A DNA fingerprint is obtained by separating the amplified DNA fragments by gel electrophoresis. The state of the microorganism can be elucidated by analyzing the DNA fingerprint.
When applying the conventional RAPD method or AP-PCR method to a group of microorganisms formed by a plurality of microorganisms, however, the number of types of amplified DNA fragments is so large that it is difficult to associate a microorganism which is a template with amplified DNA fragments, and hence it is difficult to discriminate an ecosystem formed by the group of microorganisms.
In order to examine presence/absence of contaminants in soil, food or the like and the degree thereof, the soil or food must be analyzed by suitable methods varying with the types of the contaminants. Particularly when examining the contaminated state of organic matter, it is necessary to predict the types of contaminants for analyzing the same since the analytic methods vary with the elements contained in the organic matter. Thus, awaited is a method of effectively predicting the types of the contaminants.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method of amplifying DNA fragments capable of correctly discriminating a plurality of different DNA, an apparatus for amplifying DNA fragments, and a highly reliable method of assaying microorganisms and a method of analyzing microorganisms employing the same.
Another object of the present invention is to provide a method of assaying a contaminant capable of readily assaying presence/absence of a contaminant in soil, food or the like and the degree thereof.
A method of amplifying DNA fragments according to an aspect of the present invention comprises steps of preparing a plurality of primers having different amplification probabilities and simultaneously applying a polymerase chain reaction method of repeating a thermal denaturation step, a primer annealing step and an extension reaction step with polymerase in this order to a plurality of different DNA with each of the plurality of primers, thereby amplifying DNA fragments of the plurality of different DNA. The DNA includes not only that of organisms but also DNA fragments.
In the method of amplifying DNA fragments according to this aspect of the present invention, DNA fragments of a plurality of DNA having different base lengths can be amplified by simultaneously applying the polymerase chain reaction method to the plurality of different DNA with each of the plurality of primers having different amplification probabilities. Therefore, a plurality of microorganisms included in a group of microorganisms can be correctly discriminated.
Further, a plurality of types of DNA fragments are obtained from single DNA by employing the plurality of primers having different amplification probabilities. Thus, a plurality of information can be obtained from each microorganism included in the group of microorganisms, for improving the precision of assay.
Preferably, the method further comprises a step of employing a reference primer having a known base sequence and applying the polymerase chain reaction method to reference DNA having a base sequence complementary to that of the reference primer thereby amplifying a DNA fragment of the reference DNA, simultaneously with amplifying the DNA fragments of the plurality of different DNA.
The reference DNA has the base sequence complementary to the reference primer. Therefore, the DNA fragment is reliably amplified from the reference DNA by the polymerase chain reaction method. The amplification efficiency for the DNA fragment of the reference DNA in polymerase chain reaction can be obtained by quantitatively analyzing the amplified DNA fragment of the reference DNA. In this case, the polymerase chain reaction method is simultaneously applied under the same conditions to the reference DNA and the plurality of different DNA, and hence the amplification efficiency obtained from the DNA fragment of the reference DNA is also applicable to the DNA fragments of the plurality of different DNA. Therefore, the quantity of the DNA fragments of the plurality of different DNA can be corrected on the basis of the obtained amplification efficiency.
The method may further comprise a step of classifying the DNA fragments amplified from the plurality of different DNA by a discrimination method. Thus, a plurality of microorganisms included in a group of microorganisms can be correctly discriminated.
Further, the discrimination method may be electrophoresis.
Akin Gump Strauss Hauer & Feld L.L.P.
Jones W. Gary
Sanyo Electric Co,. Ltd.
Taylor Janell E.
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