Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-13
2001-04-10
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C530S350000
Reexamination Certificate
active
06214555
ABSTRACT:
BACKGROUND OF THE INVENTION
This application relates to a method for detection and identification of microorganisms, including in particular pathogenic microorganisms, and to compositions and kits useful in practicing the method. The invention can be applied to detection of viruses, including HIV and hepatitis, bacteria, including Chlamydia, fungi, including
Cryptococcus neoformans
and protozoa, including
Trypanosoma cruzi
. This application also relates to DNA sequencing reactions, and in particular to improved bi-directional sequencing reaction protocols making use of thermally stable polymerase enzymes.
Detection of the presence of pathogenic microorganisms through DNA-based technology is emerging as an important tool in the diagnosis of many diseases. For example, diagnosis of
Chlamydia trachomatis
infections, the most common bacterial sexually transmitted disease in North America, is shifting from traditional methods such as culture, enzyme immunoassay (EIA) and direct fluorescent antibodies (DFA) to DNA-hybridization diagnostics. Roche Diagnostic Systems, Inc. (Nutley, N.J.) manufactures Amplicor™, a test which detects
C. trachomatis
and
Neisseria gonorrohoeae
by the hybridization of a pathogen specific probe to PCR amplified products, detectable by a color change/optical density technique. Abbott Laboratories (Abbott Park, Ill.) makes UriProbe, also a test for
C. trachomatis
and
N. gonorrohoeae
, which relies on the ligase chain reaction (LCR). The LCR method, described in Patent Applications WO 9320227, WO 9300447, WO 9408047, WO 9403636, EP 477 972 uses thermostable ligase enzyme to ligate two DNA probes which hybridize in ligatable juxtaposition on a template DNA strand, thus generating a detectable ligated DNA fragment only if the template DNA is present. A multiplex PCR assay for
C. trachomatis
has also been described in Mahony et al.,
J. Clin. Microbiol.
33: 3049-3053 (1995).
The ideal DNA sequencing procedure for use in a diagnostic environment would have the following characteristics: (1) it would be able to utilize a DNA-containing sample which had been subjected to only minimal pretreatment to make the DNA accessible for sequencing; (2) it would require combining this sample with only a single reaction mixture, thus reducing risk of error and contamination, and increasing the ease with which the procedure can be automated; and (3) it would require a short amount of time to perform the sequence determination, thus decreasing the marginal costs in terms of equipment and labor for performing the test.
A wide variety if infectious pathogens that can be detected by DNA-based methods are listed in
Diagnostic Molecular Microbiology
, Persing et al., eds. American Society for Microbiology, Washington D.C. (1993). This text details diagnostic tests for bacteria, virus, fungi, and protozoa. Diagnostic tests are also proposed for identifying the presence of drug resistance genes or toxin genes.
Although these tests are generally effective for identifying an infectious disease-causing organism if present, they do not routinely provide information concerning the specific serotype, variant or form of the infecting organism. Depending on the organism in question, this information can be significant in determining the likely course of the infection, for determining the most appropriate therapeutic approach and for epidemiological purposes. Furthermore, the previously known assays involve several steps and are therefore more susceptible to systematic error than would be a test with fewer steps. Thus, there remains a need for a simple test format which is generally applicable to the detection of microorganisms, including infectious disease-causing microorganisms, and particularly for a simple test which provides an indication of the specific nature, e.g., the serotype, of the organism. It is an object of the present invention to provide such a test.
It is an object of the present invention to provide reagent combinations useful in performing tests for infectious disease-causing microorganisms, including Chlamydia human papilloma virus(HPV) and HIV.
It is a further object of the present invention to provide kits useful in performing tests for infectious disease-causing microorganisms, including Chlamydia, HPV and HIV.
It is an additional object of the present invention to provide an improved method for bi-directional sequencing of DNA samples which is well-suited for use in the diagnostic environment and for automation.
It is an additional object of the invention to provide a method for bi-directional sequencing of DNA which utilizes a DNA-containing sample which has been subjected to only minimal pretreatment to make the DNA accessible for sequencing.
It is still a further object of the invention to provide a method for bi-directional sequencing of DNA which requires combining a complex DNA-containing sample with only a single reaction mixture, thus reducing risk of error and contamination, and increasing the ease with which the procedure can be automated.
SUMMARY OF THE INVENTION
The present invention provides a method for the evaluation of a sample for the presence of a target microorganism which can be performed directly on a natural abundance DNA preparation obtained from the sample in a single reaction vessel. The method of the invention comprises the steps of:
(a) combining the natural abundance DNA preparation with first and second primers, a nucleotide triphosphate feedstock mixture, a chain-terminating nucleotide triphosphate and a thermally stable polymerase enzyme which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than 0.4 times the rate of incorporation of deoxynucleotides to form a reaction mixture, said first and second primers binding to the sense and antisense strands of the DNA of the target microorganism, respectively, and flanking a selected region within the genome of the target microorganism;
(b) exposing the reaction mixture to a plurality of temperature cycles each of which includes at least a high temperature denaturation phase and a lower temperature extension phase, thereby producing a plurality of species of terminated fragments if DNA from the target microorganism is present in the sample, each species of terminated fragment corresponding to a different incorporation position for the chain-terminating nucleotide triphosphate in the DNA of the target microorganism; and
(c) evaluating the terminated fragments produced to determine the incorporation positions of the chain-terminating nucleotide triphosphate. Based on the incorporation positions, not only the presence but also the specific nature, e.g. the serotype, of any target microorganism present can be determined.
The present invention also provides a composition comprising a mixture of four deoxynucleotide triphosphates and at least one dideoxynucleotide triphosphate corresponding to one of the four deoxynucleotide triphosphates, wherein the dideoxynucleotide triphosphate is present in a mole ratio to the corresponding deoxynucleotide triphosphate of from 1:50 to 1:500, said composition further comprising a thermally stable polymerase enzyme which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than 0.4 times the rate of incorporation of deoxynucleotides.
Also provided is a kit for detection of a target microorganism comprising, in packaged combination, the above composition and a pair of primers which bind to the sense and antisense strands, respectively, and flank a selected region within the genome target microorganism.
The present invention also provides a method for bi-directional sequencing a region of interest in a DNA sample. The method can be carried out using a single set of reagents which is added to a minimally-treated sample to produce useful sequencing results.
DETAILED DESCRIPTION OF THE INVENTION
The compositions and kits of the present invention can be employed in a bi-directional DNA sequencing method in many contexts including: (1) detection of mutations, part
Dunn James M.
Hui May
Lacroix Jean-Michel
Leushner James
Campbell Eggerton A.
Oppedahl & Larson LLP
Visible Genetics Inc.
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