Labeled primer for use in and detection of target nucleic acids

Details Labeled primer for use in and detection of target nucleic acids Labeled primer for use in and detection of target nucleic acids

1998-12-14

2001-06-19

Jones, W. Gary (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S006120, C435S091500, C435S091520, C435S810000, C435S183000, C435S184000, C435S194000, C435S091200, C536S024310, C536S024330

Reexamination Certificate

active

06248526

ABSTRACT:

The invention relates to a labeled primer for nucleic acid amplification reactions (for example, the polymerase chain reaction) and to a process for detecting a nucleic acid sequence by means of a nucleic acid amplification process in which a labeled primer is used.
As is known, the polymerase chain reaction (PCR) is a very effective method for detecting small quantities of a known nucleic acid sequence in a sample (Erlich H. A., Gelfand, D. Sninsky JJ. (1991), Science, 252, pp. 1643-1651, which publication is incorporated herein by reference; PCR Protocols. Current methods and applications (1993) edited by B. A. White, Humana Press, Totowa, N.J., ISBN 0-89603-244-2, which publication is incorporated herein by reference). If the sequence of, for example, a viral DNA is already known, it is possible to synthesize a pair of primers which are complementary to regions on opposite single strands and which flank a target nucleic acid sequence of interest (e.g., a DNA sequence). Under PCR conditions, which are known per se, the primers can be annealed, or hybridized, to the target DNA, and a number of reaction cycles, normally more than 30, can then be used to produce large quantities of a specific DNA in vitro. The PCR cycles amplify a DNA fragment, which is of a specific size and which is composed of the lengths of the two primers plus the length of the DNA between them, when the target DNA is present in the sample. The PCR technique is so sensitive that it can be used to detect extraordinarily small quantities of a DNA with a high degree af reliability.
International Patent Application WO 92/02638, herein incorporated by reference, discloses a process for detecting a DNA sequence, in which process a sample, which contains or is suspected to contain the DNA to be detected (as a single strand), i.e., the target DNA, is hybridized with two different primers, i.e., the forward primer and the reverse primer, which flank the target DNA strand to be amplified. A labeled oligonucleotide probe, which is provided in a preferred embodiment of WO 92/02638 with a fluorescent dye system as label at both the 5′ end and at the 3′ end of the probe, is also employed in the reaction. This labeled probe is selected such that it hybridizes to the target DNA. In the fluorescent dye system, the fluorescence of one of the dyes, the reporter dye, is decreased (“quenched”) by the proximity of the second molecule, i.e. the quencher, by a process known as fluorescence resonance energy transfer (FRET) (Stryer, L. 1978; Fluorescence energy transfer as a spectroscopic ruler. Ann. Rev. Biochem. 47: 819-846, which is incorporated herein by reference). A labeled probe, has, as an example, the following sequence:
5′
FAM-TGG TGG TCT GGG ATG AAG GTA TTA TT-TAMRA
3′
wherein FAM represents the reporter dye, and TAMRA represents the quencher dye. A probe having the sequence and label can be ordered and obtained from a number of companies and is intended for use in a 5′-nuclease assay, i.e. the TaqMan® assay, which is described in detail by Livak K. J., Flood S. J. A., Marmaro, J., Giusti W., Deetz K., Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization, PCR Method and Appl, 1995; 4:357-362, which is incorporated herein by reference.
The special feature of this quenched probe system is that the fluorescence of the reporter dye (FAM), which is attached to the 5′ end of the probe, is reduced by proximity to the quencher dye (TAMRA), which is attached to the 3′ end of the probe, see above.
As the new DNA strand is formed under the influence of a suitable, preferably thermostable, DNA polymerase, e.g. the TaqDNA polymerase, the polymerase not only displaces the labeled probe from the single strand but also, by means of its 5′→3′ nuclease activity, degrades the probe and thereby releases the two fluorescent dyes. The fluorescence of the reporter dye is now no longer suppressed by the quencher dye and increases. A fluorescence spectrometer can then be used to measure the fluorescence at the wavelength of the reporter dye, which is proportional to the quantity of newly formed DNA.
The fact that a labeled probe is required, in addition to the forward primer and the reverse primer, in order to be able to observe or measure the amplification of the DNA segment to be detected has to be regarded as a disadvantage of this method. Therefore, a need arose to simplify this known process.
It has now been found that the use of an additional labeled probe in the polymerase chain reaction is unnecessary if at least one of the two primers is labeled, e.g., with an interactive label system in which care is taken to ensure that the labeled primer contains at least one nucleotide that is not complementary to the DNA strand to be amplified, i.e., a portion of the primer is deliberately mismatched.
The invention therefore provides a labeled primer and a process for the detection of a target nucleic acid. The labeled primer is deliberately mismatched in at least one nucleotide, and preferably two to five or more nucleotides, at the 3′ end of the primer. The labeled primer is incubated with a sample that contains or is suspected to contain the target nucleic acid or DNA under conditions sufficient to allow annealing or hybridization and said sample is subsequently exposed to nucleic acid polymerase having a 3′ to 5′ proofreading or functionally equivalent nuclease activity, or a mixture of enzymes having such proofreading activity, under conditions sufficient to permit said 3′ to 5′ proofreading activity to cleave said forward and/or reverse primer in said 3′ mismatched portion, thereby releasing said label or part of the label system. In a preferred embodiment, the labeled primer is used in a process of nucleic acid amplification to detect the target nucleic acid.
In another preferred embodiment, a label or label system is attached to the primer at or near its 3′ end and has an interactive label. For example, the interactive label system has a reporter dye molecule and a quencher molecule. At least one, and preferably at least the last two to five, or more, nucleotides at the 3′ end of the primer are deliberately mismatched to the DNA or nucleic acid sequence to be amplified. The label or part of a label system is attached to the 3′-terminal mismatched portion, preferably to the 3′ end nucleotide. The length of the unpaired region is selected and/or optimized by methods known to those skilled in the art for the particular label and particular polymerase used. Such a labeled primer is not able to undergo complete base pairing at its 3′-end with the DNA sequence to be amplified. Under the influence of the polymerase employed for the amplification, which possesses proof-reading or nuclease properties equivalent to such, the unpaired bases of the labeled primer, together with the label, e.g., the reporter dye molecule or quencher molecule, are released by the 3′→5′ nucleolytic activity of the polymerase before the actual elongation reaction takes place. In the process, the quencher is removed from spatial proximity to the reporter dye. The fluorescence of the reporter dye therefore increases, indicating presence of a target nucleic acid. The most preferred embodiment is illustrated in FIG.
1
.
The invention also relates to a process for detecting a target nucleic acid by means of nucleic acid amplification, in which process one of the primers possesses the abovementioned features. In the amplification, for which it is possible to use one or more thermostable DNA polymerases, at least one of which must also have proof-reading or functionally equivalent nuclease properties, the unpaired bases of the labeled primer, together with the label or part of a label system, e.g., a reporter dye which is attached to the labeled primer, are then released, resulting in a signal increase, e.g. fluorescence increasing at the wa

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