Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-09-10
2003-11-18
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C436S094000
Reexamination Certificate
active
06649349
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is related to the field of probe-based nucleic acid sequence detection, analysis and quantitation. More specifically, this invention relates to novel methods, kits and compositions pertaining to Linear Beacons.
2. Description of the Related Art
Quenching of fluorescence signal can occur by either Fluorescence Resonance Energy Transfer “FRET” (also known as non-radiative energy transfer: See: Yaron et al.,
Analytical Biochemistry
95: 228-235 (1979) at p. 232, col. 1, lns. 32-39) or by non-FRET interactions (also known as radiationless energy transfer; See: Yaron et al.,
Analytical Biochemistry
95 at p. 229, col. 2, lns. 7-13). The critical distinguishing factor between FRET and non-FRET quenching is that non-FRET quenching requires short range interaction by “collision” or “contact” and therefore requires no spectral overlap between the moieties of the donor and acceptor pair (See: Yaron et al.,
Analytical Biochemistry
95 at p. 229, col. 1, lns. 22-42). Conversely, FRET quenching requires spectral overlap between the donor and acceptor moieties and the efficiency of quenching is directly proportional to the distance between the donor and acceptor moieties of the FRET pair (See: Yaron et al.,
Analytical Biochemistry
95 at p. 232, col. 1, ln. 46 to col. 2, ln. 29). Extensive reviews of the FRET phenomenon are described in Clegg, R. M.,
Methods Enzymol
., 221: 353-388 (1992), and Selvin, P. R.,
Methods Enzymol
., 246: 300-334 (1995). Yaron et al. also suggested that the principles described therein might be applied to the hydrolysis of oligonucleotides (See: Yaron et al.,
Analytical Biochemistry
95 at p. 234, col. 2, lns. 14-18).
The FRET phenomenon has been utilized for the direct detection of nucleic acid target sequences without the requirement that :labeled nucleic acid hybridization probes or primers be separated from the hybridization complex prior to detection (See: Livak et al. U.S. Pat. No. 5,538,848). One method utilizing FRET to analyze Polymerase Chain Reaction (PCR) amplified nucleic acid in a closed tube format is commercially available from Perkin Elmer. The TaqMan™ assay utilizes a nucleic acid hybridization probe which is labeled with a fluorescent reporter and a quencher moiety in a configuration which results in quenching of fluorescence in the intact probe. During the PCR amplification, the probe sequence specifically hybridizes to the amplified nucleic acid. When hybridized, the exonuclease activity of the Taq polymerase degrades the probe thereby eliminating the intramolecular quenching maintained by the intact probe. Because the probe is designed to hybridize specifically to the amplified nucleic acid, the increase in fluorescence intensity of the sample, caused by enzymatic degradation of the probe, can be correlated with the activity of the amplification process.
Nonetheless, this method preferably requires that each of the fluorophore and quencher moieties be located on the 3′ and 5′ termini of the probe so that the optimal signal to noise ratio is achieved (See: Nazarenko et al.,
Nucl. Acids Res
. 25: 2516-2521 (1997) at p.
2
516, col. 2, lns. 27-35). However, this orientation necessarily results in less than optimal fluorescence quenching because the fluorophore and quencher moieties are separated in space and the transfer of energy is most efficient when they are close. Consequently, the background emission from unhybridized probe can be quite high in the TaqMan™ assay (See: Nazarenko et al.,
Nucl. Acids Res
. 25: at p. 2516, col. 2, lns. 36-40).
The nucleic acid Molecular Beacon is another construct which utilizes the FRET phenomenon to detect target nucleic acid sequences (See: Tyagi et al.
Nature Biotechnology
, 14: 303-308 (1996). A nucleic acid Molecular Beacon comprises a probing sequence embedded within two complementary arm sequences (See: Tyagi et al,
Nature Biotechnology
, 14: at p. 303, col. 1, lns. 22-30). To each termini of the probing sequence is attached one of either a fluorophore or quencher moiety. In the absence of the nucleic acid target, the arm sequences anneal to each other to thereby form a loop and hairpin stem structure which brings the fluorophore and quencher together (See: Tyagi et al.,
Nature Biotechnology
, 14: at p. 304, col. 2, lns. 14-25). When contacted with target nucleic acid, the complementary probing sequence and target sequence will hybridize. Because the hairpin stem cannot coexist with the rigid double helix that is formed upon hybridization, the resulting conformational change forces the arm sequences apart and causes the fluorophore and quencher to be separated (See: Tyagi et al.
Nature Biotechnology
, 14: at p. 303, col. 2, lns. 1-17). When the fluorophore and quencher are separated, energy of the donor fluorophore does not transfer to the acceptor moiety and the fluorescent signal is then detectable. Since unhybridized “Molecular Beacons” are non-fluorescent, it is not necessary that any excess probe be removed from an assay. Consequently, Tyagi et al. state that Molecular Beacons can be used for the detection of target nucleic acids in a homogeneous assay and in living cells. (See: Tyagi et al.,
Nature Biotechnology
, 14: at p. 303, col. 2; lns. 15-77).
The arm sequences of the disclosed nucleic acid Molecular Beacon constructs are unrelated to the probing sequence (See: Tyagi et al.,
Nature Biotechnology
, 14: at p. 303, col. 1; ln. 30). Because the Tyagi et al. Molecular Beacons comprise nucleic acid molecules, proper stem formation and stability is dependent upon the length of the stem, the G:C content of the arm sequences, the concentration of salt in which it is dissolved and the presence or absence of magnesium in which the probe is dissolved (See: Tyagi et al.,
Nature Biotechnology
, 14: at p. 305, col. 1; lns. 1-16). Furthermore, the Tyagi et al. nucleic acid Molecular Beacons are susceptible to degradation by endonucleases and exonucleases.
Upon probe degradation, background fluorescent signal will increase since the donor and acceptor moieties are no longer held in close proximity. Therefore, assays utilizing enzymes known to have nuclease activity, will exhibit a continuous increase in background fluorescence as the nucleic acid Molecular Beacon is degraded (See:
FIG. 7
in Tyagi et al: the data associated with (∘) and (▭) demonstrates that the fluorescent background, presumably caused by probe degradation, increases with each amplification cycle.) Additionally, Molecular Beacons will also, at least partially, be degraded in living cells because cells contain active nuclease activity.
The constructs described by Tyagi et al. are more broadly described in WO95/13399 (hereinafter referred to as “Tyagi2 et al.” except that Tyagi2 et al. also discloses that the nucleic acid Molecular Beacon may also be bimolecular wherein they define bimolecular as being unitary probes of the invention comprising two molecules (e.g. oligonucleotides) wherein half, or roughly half, of the target complement sequence, one member of the affinity pair and one member of the label pair are present in each molecule (See: Tyagi2 et al., p. 8, ln. 25 to p. 9, ln. 3). However, Tyagi2 et al. specifically states that in desig unitary probe for use in a PCR reaction, one would naturally choose a target complement sequence that is not complementary to one of the PCR primers (See: Tyagi2 et al., p. 41, ln. 27). Assays of the invention include real-time and end point detection of specific single-stranded or double stranded products of nucleic acid synthesis reactions, provided however that if unitary probes will be subjected to melting or other denaturation, the probes must be unimolecular (See: Tyagi2 et al., p. 37, lns. 1-9). Furthermore, Tyagi2 et al. stipulate that although the unitary probes of the invention may be used with amplification or other nucleic acid synthesis reactions, bimolecular probes (as defined in Tyagi2 et al.) are not suitable for use in any reaction (e.g. PCR) wherein the affinity pair would b
Coull James M.
Gildea Brian D.
Hyldig-Nielsen Jens J.
Boston Probes, Inc.
Gildea Brian D.
Horlick Kenneth R.
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