Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-29
2001-09-11
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S287200, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06287772
ABSTRACT:
BACKGROUND OF THE INVENTION
Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids. Nucleic acid probes have long been used to analyze samples for the presence of nucleic acid from bacteria, fungi, virus or other organisms and are also useful in examining genetically-based disease states or clinical conditions of interest. Nonetheless, probe-based assays have been slow to achieve commercial success. This lack of commercial success is, at least partially, the result of difficulties associated with specificity, sensitivity and reliability.
Nucleic acid hybridization is a fundamental process in molecular biology. Sequence differences as subtle as a single base (point mutation) in very short oligomers (<10 base pairs “bp”) can be sufficient to enable the discrimination of the hybridization to complementary nucleic acid target sequences as compared with non-target sequences. However, nucleic acid probes of greater than 10 bp in length are generally required to obtain the sequence diversity necessary to correctly identify a unique organism, disease state or clinical condition of interest. The ability to discriminate between closely related sequences is inversely proportional to the length of the hybridization probe because the difference in thermal stability decreases between wild type and mutant complexes as the probe length increases. Consequently, the power of probe-based hybridization to correctly identify the target sequence of interest from closely related (e.g., point mutations) non-target sequences can be very limited.
Hybridization assays hold promise as a means to screen large numbers of patient samples for a large number of mutations. In practice, however, it is often difficult to multiplex an assay given the requirement that each of the many very different probes in the assay must exhibit a very high degree of specificity for a specific target nucleic acid under the same or similar conditions of stringency.
Peptide Nucleic Acid (PNA) is a non-naturally occurring polyamide (pseudopeptide) which can hybridize to nucleic acid (DNA and RNA) with sequence specificity (See U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, 5,786,461 and Egholm et al.,
Nature
365: 566-568 (1993)). Being non-naturally occurring molecules, unmodified PNAs are not known to be substrates for the enzymes which are known to degrade peptides or nucleic acids. Therefore, unmodified PNAs should be stable in biological samples, as well as have a long shelf-life. Likewise, when complexed to a nucleic acid, PNAs shield the nucleic acid from degradation (See: WIPO patent application: Stanley et al., WO95/15974). Unlike nucleic acid hybridization which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength, conditions which strongly disfavor the hybridization of nucleic acid to nucleic acid (Egholm et al.,
Nature,
at p. 567). The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated (Tomac et al.,
J. Am. Chem. Soc.,
118: 5544-5552 (1996)). Sequence discrimination is more efficient for PNA recognizing DNA than for DNA recognizing DNA (Egholm et al.,
Nature,
at p. 566). However, the advantages in point mutation discrimination with PNA probes, as compared with DNA probes, in a hybridization assay appears to be somewhat sequence dependent Nielsen et al.,
Anti
-
Cancer Drug Design
8: 53-65, (1993) and Weiler et al.,
Nucl. Acids Res.
25: 2792-2799 (1997)). As an additional advantage, PNAs hybridize to nucleic acid in both a parallel and antiparallel orientation, though the antiparallel orientation is preferred (See: Egholm et al.,
Nature
at p. 566).
Because PNAs hybridize to nucleic acids with sequence specificity, PNAs are useful candidates for developing probe-based assays. However, PNA probes are not the equivalent of nucleic acid probes. Even under the most stringent conditions both the exact target sequence and a closely related sequence (e.g., a non-target sequence having a single point mutation (a.k.a. single base pair mismatch)) will often exhibit detectable interaction with a labeled nucleic acid or labeled PNA probe (Nielsen et al.,
Anti
-
Cancer Drug Design at p.
56-57 and Weiler et al.,
Nucl., Acids Res.,
25: 2792-2799 (1997)). Any hybridization to a closely related non-target sequence will result in the generation of undesired background signal. Because the sequences are so closely related, point mutations are some of the most difficult of all nucleic acid modifications to detect using a probe-based assay. Numerous diseases, such as sickle cell anemia and cystic fibrosis, are caused by a single point mutation of genomic nucleic acid. Consequently, any method, kits or compositions which could improve the specificity, sensitivity and reliability of probe-based assays would be useful in the detection, analysis and quantitation of nucleic acid containing samples and particularly useful for nucleic acid point mutation analysis.
SUMMARY OF THE INVENTION
This invention relates to methods, kits and compositions suitable for the improved detection, quantitation and analysis of nucleic acid target sequences using probe-based hybridization assays. The invention is more specifically directed to methods, kits and compositions suitable for specifically detecting, quantitating and optionally isolating target nucleic acid in a sample, even in the presence of non-target sequences. The present invention directly improves the specificity and sensitivity of the assay thereby improving the signal to noise ratio of the assay. The present invention will also result in improvements in reliability since the incidence of false positives and false negatives should also be reduced. Because the methods, kits and compositions of this invention are directed to the specific detection of target nucleic acid, even in the presence of non-target sequences, they are particularly well suited for the development of sensitive and reliable probe-based hybridization assays designed to analyze for point mutations. The methods, kits and compositions of this invention should also find utility for the detection, quantitation or analysis of organisms (micro-organisms), viruses, fungi and genetically based clinical conditions of interest.
The present invention is drawn to a method of forming a PNA probe triplex, comprising contacting a sample containing target nucleic acid under hybridization conditions to at least one probe set comprising three probes wherein the first and second probes comprise a probing segment and a PNA arm segment and wherein the third probe comprises a PNA arm segment. The probing segment of the first probe hybridizes to a first site of hybridization on the target nucleic acid which is in close proximity to a second site of hybridization of the probing segment of the second probe such that the PNA arm segments of the first, second and third probes form a PNA probe triplex when the first and second probes hybridize to the first and second sites of hybridization on a target nucleic acid, respectively. In a preferred embodiment, the PNA arm segments of the first and second probes do not hybridize to each other but interact only in the presence of the PNA arm segment of the third probe.
The present invention further comprises a method of detecting the presence or quantity of target nucleic acid present in a sample. A sample containing target nucleic acid is contacted, under hybridization conditions, to at least one probe set comprising three probes wherein the first and second probes comprise a probing segment and a PNA arm segment and wherein the third probe comprises a PNA arm segment. The probing segment of the first PNA probe hybridizes to a first site of hybridization on the target nucleic acid which is in close proximity to a second site of hybridization of the probing segment of the second probe such that the PNA arm segments of the first, second and third probes form a PNA pro
Coull James M.
Hyldig-Nielsen Jens J.
Peterson Kenneth H.
Stefano Kyriaki
Stender Henrik
Boston Probes, Inc.
Hamilton Brook Smith & Reynolds P.C.
Sisson Bradley L.
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