Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-04-23
2002-09-10
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S022100, C536S023100, C536S024300, C536S024330, C536S025300, C536S025320
Reexamination Certificate
active
06448015
ABSTRACT:
This invention relates to the identification of genetic mutations and, more particularly, to a method of detecting point mutations in DNA utilizing fluorescence energy transfer. This invention was supported by National Institutes for Health grant DK36288 to the Center for Biotechnology, University of Nebraska-Lincoln.
BACKGROUND
The detection of mutations in sequences of DNA is becoming increasingly important in medical science. The detection of such a mutation in a DNA sequence typically involves the use of an oligodeoxyribonucleotide probe that is complementary to the target DNA sequence. The probe is designed to present some moiety, such as a radioactive element, that signals the occurrence of hybridization in a filter assay or an electrophoretic gel. The identification of hybridization has been used diagnostically for specific bacterial infections by detection of
Mycobacterium tuberculosis
genomic DNA, gonorrhea rRNA, Chiamydia genomic and plasmid DNA and
Escherichia coli
and
Bacillus subtilis
rRNA. Hybridization assays have also been developed for viral detection, including cytomegalovirus (CMV), human papilloma virus (HPV), and HIV-1.
By combining target amplification with allele specific oligonucleotides, small samples of human DNA can be analyzed for purposes of genetic screening, including the study of genetic changes associated with well-known inherited diseases. For instance, cancers typically display familial site-specific clustering. The identification of this kind of clustering can aid in the determination of enhanced risk for the development of the particular cancer. In addition, hereditary metabolic variations in DNA have been identified that affect the metabolism of known carcinogens. A variation that would increase the metabolism of a carcinogen may impact the likelihood of the development of cancer and, if developed, the speed of the cancer's growth.
Traditional hybridization methods have been developed which employ radioactive probes with separation on filters. While radioactive probes have performed suitably well, growing concern over the use of radioactive materials has stimulated a search for alternative probes that achieve similar levels of sensitivity and performance without the risks and dangers associated with radioactive materials. For instance, biotin has been incorporated into an oligodeoxyribonucleotide for use in biotinavidin-linked analyses. In addition, numerous modifications of DNA have been used in the development of other alternative probes, including links to antibodies, gold-antibodies, mercury for double antibody reactions, eupsoralen, and fluorescent dye links for fluorescence detection of hybridization. These alternative methods typically allow approximately 10
5
to 10
6
copies of the DNA to be detected.
These and related advancements in the art have given rise to several methods of DNA mutation detection. These methods include denaturing gradient gel electrophoresis (DGGE), single-strand conformational polymorphisms (SSCP), temperature gradient gel electrophoresis (TGGE), the heteroduplex method (HET), ribonuclease cleavage, chemical cleavage of mismatch (CCU), ligase assay, allele-specific amplification (ASA) dideoxy fingerprinting (ddF), and allele-specific oligonucleotides (ASO). DGGE, SSCP, TGGE, HET, and ddF are frequently used to locate which exons of a gene contain mutations.
The currently available non-radioactive methods for detecting mutations in DNA have been somewhat problematic. For example, these methods have been generally unable to consistently provide accurate results in detecting point mutations in DNA. These detection methods have also proven to be time-consuming and quite costly to use. In addition, these non-radioactive mechanisms require a significant amount of DNA to perform their detecting function, though many times only a small quantity of DNA is available for analysis. Moreover, these methods are difficult to use, often requiring complex instruments and highly trained technicians not available in many laboratories. Finally, the materials utilized in these methods are generally either fragile or prone to degradation during the testing procedure.
SUMMARY OF THE INVENTION
It is, therefore, a primary object of the present invention to provide a method for quickly and accurately detecting point mutations in DNA.
It is also an object of the present invention to provide a method for quickly and accurately detecting point mutations in DNA that does not utilize radioactive material.
It is a further object of the present invention to provide a method for quickly and accurately detecting point mutations in DNA that requires only a relatively small amount of DNA to perform its detection function.
It is another object of the present invention to provide a method for quickly and accurately detecting point mutations in DNA that is inexpensive, non-complex and easy to use.
It is yet another object of the present invention to provide a method for quickly and detecting point mutations in DNA that is durable and less likely to experience degradation of its constituent components during the testing procedure.
It is yet another object of the present invention to provide a method for deteting point mutations in DNA which requires minimal sample preparation.
To accomplish these and related objectives, the present invention relates to a method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and fluorescence resonance energy transfer (FRET). The selected probe is labeled at each end with a fluorescent dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from a probe/target duplex stage, wherein the probe is hybridized to the complementary strand of target DNA, to the single strand stage, when the probe is melted to become detached from the target DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the target DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T
m
. Where there is a base mismatch between the probe and the target DNA strand, indicating a point mutation in the target DNA strand, the T
m
has been found to be significantly lower than the T
m
for a perfectly match probe/target duplex. The present invention allows for the detection of the T
m
, which allows for the quick and accurate detection of a point mutation in the target DNA strand.
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A.H.N. Hopman, J. Wiegant, G.I. Tesser, and P. Van Dujin, “A Non-Radioactive In Situ Hybridization Method Based on Mercurated Nucleic Acid Probes and Sulfhydryl-Hapten Ligands,” Nucleic Acids Res. 14, 6471-6488 (1986).
A. Oser, W.K. Roth, and G. Valet, “Sensitive Non-Radioactive Dot-Blot Hybridizat
Middendorf Lyle
Parkhurst Kay M.
Parkhurst Lawrence J.
Board of Regents of University of Nebraska
Riley Jezia
Shook Hardy & Bacon LLP
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