Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1992-07-27
2001-03-06
Elliott, George C. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C536S024300
Reexamination Certificate
active
06197499
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the detection of a DNA sequence in real time using fluorescence.
2. Background Information
The polymerase chain reaction (PCR) developed by Saiki et al. [Science 230, 1350-1354 (1985)] provided a method for rapidly amplifying small specific segments of DNA. The PCR technique has greatly simplified the analysis of DNA sequences at the genomic level.
Detection or identification of a segment of DNA involves a two step procedure. The first step is the PCR amplification reaction using carefully selected primers. The second step identifies whether the desired DNA segment was amplified.
Prior to the present invention, the second step has required the use of a radioactively labeled DNA probe to precisely identify a PCR amplified DNA segment. There are several disadvantages associated with the use of a radioactively labeled probe. For example, radioactive probes are dangerous to handle and when labeled with
32
P, which is the standard protocol, the probe has a short useful life (a few weeks at most). In addition, the use of a radioactive probe prevents detection of the results in real time, that is, a delay for an autoradiographic exposure time is required.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide a method for detecting an amplified DNA sequence without the use of a radioactive probe.
It is another object of the present invention to provide a method for detecting an amplified DNA sequence in real time.
In one embodiment the present invention relates to a method of detecting a DNA sequence by means of a fluorescent DNA:DNA hybrid comprising the steps of:
i) asymmetrically amplifying said DNA sequence;
ii) hybridizing a fluorescently labeled probe complementary to a segment of the amplified DNA sequence to the amplified DNA wherein the hybridization is effected in solution;
iii) separating the hybridized probe from the unhybridized probe by electrophoresis; and
iv) detecting during electrophoresis the presence or absence of a DNA:probe hybrid by fluorescence detection.
In another embodiment the present invention relates to a method of screening a sample for a pathogen comprising the steps of:
i) asymmetrically amplifying a DNA sequence unique to said pathogen;
ii) hybridizing a fluorescently labeled probe complementary to a segment of the amplified DNA sequence to the amplified DNA sequence wherein the hybridization is effected in solution;
iii) separating the hybridized probe from the unhybridized probe by electrophoresis; and
iv) detecting during electrophoresis the presence or absence of a DNA:probe hybrid by detection of the fluorescent label.
In a further embodiment the present invention relates to a method of simultaneously screening a sample for the presence of several pathogens comprising the steps of:
i) asymmetrically amplifying a DNA sequence unique to each pathogen whose presence or absence is being determined;
ii) hybridizing a fluorescently labeled probe for each pathogen being detected complementary to a segment of the pathogen's amplified DNA sequence to the amplified DNA wherein the hybridization is effected in solution;
iii) separating the sample by electrophoresis; and
iv) detecting during electrophoresis by a laser scanner-imaging device the presence or absence of a DNA: probe hybrid for each pathogen by detection of the fluorescent label.
Various other objects and advantages of the present invention will become obvious from the drawings and the following description of the invention.
REFERENCES:
patent: 4683202 (1987-07-01), Mullis
Kumar et al., A Method For The Rapid Screening Of Human Blood Samples For The Presence Of HIV-1 Sequences: The Probe-Shift Assay, Aids Research and Human Retroviruses, 1989, vol. 5, No. 3, pp. 345-354.
Siegler, N., DNA-Based Testing: A Progress Report, ASM News, 1989, vol. 55, No. 6, pp. 308-312.
Wu et al. Allele-Specific Enzymatic Amplification of B-Globin Genomic DNA For Diagnosis Of Sickle Cell Anemia, Proceedings of the National Academy of Sciences, Apr. 1989, vol. 86, pp. 2757-2760.
Brumbaugh et al., Continuous, On-Line DNA Sequencing Using Oligodeoxynucleotide Primers With Multiple Fluorophores, Proceedings of the National Academy of Sciences, Aug. 1988, vol. 85, pp. 5610-5614.
Gebeyechu et al. Novel Biotinylated Nucleotide—Analogs For Labeling and Colorimetric Detection Of DNA, Nucleic Acids Research, 1987, vol. 15, Issue 11, pp. 4513-4534.
Saiki et al., Enzymatic Amplification of B-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickel Cell Anemia, Science, 1985, vol. 230, pp. 1350-1354.
Landegren, Ulf et al. “A Ligase-Mediated Gene Detection Technique” Science (1988) 241:1077-1080.
Gyllensten et al., PNAS, vol. 85, pp. 7652-7656, Oct. 1988.
Brumbaugh John
Hughes Stephen H.
Kumar Ramesh
Auth Dorothy R.
Elliott George C.
Feiler William S.
Morgan & Finnegan L.L.P.
The United States of America as represented by the Department of
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