Detecting nucleic acids

Details Detecting nucleic acids Detecting nucleic acids

1999-12-20

2002-11-05

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S022100, C536S025300, C548S100000, C548S120000, C548S146000, C548S148000, C548S181000, C548S215000

Reexamination Certificate

active

06475730

ABSTRACT:

The present invention relates to a method of detecting nucleic acids and particularly to a method of analysing for the presence and/or amount of a nucleic acid.
There are many known methods of detecting nucleic acids.
For example, in the case of analysing for the presence of a particular nucleic acid in a sample containing only a very small amount of the acid it is known to use the Polymerase Chain Reaction (PCR) to amplify the amount of the acid which may then be detected, e.g. by using a labelled oligonucleotide probe. It is however known that the PCR reaction has the disadvantage that it can give rise to artefacts resulting in “false positives”. Furthermore, PCR is difficult to apply in situ in cells and is time consuming as excess of probing reagents have to be removed.
A further technique for analysis of nucleic acids is Fluorescence Resonance Energy Transfer (FRET). Two oligonucleotide probes are used in this method, one being associated with a photosensitiser (A) and the other with a photoemitter (B). In a FRET assay, the probes are such that when hybridized to a target nucleic acid strand to be detected the distance between the photosensitiser (A) and the photoemitter (B) is 5-10 nm in any dimension. To perform the assay, photosensitiser (A) is excited with electromagnetic radiation of the appropriate wavelength. If the two probes have hybridized to the nucleic acid strand adjacent to each other then photosensitiser (A) is able to excite photoemitter (B) by resonance energy transfer. Radiation emitted by (B) (which is of different wavelength from the exciting radiation) may then be detected to confirm presence of the target strand. The FRET technique does however have the disadvantage that because the resonance energy transfer occurs over a distance of 5-10 nm (equivalent to a distance of 4 bp) it is possible for the FRET signal to arise between non-specifically hybridized probes or between probes and an interacting nucleic acid chain. If the FRET partners are also not optimally aligned the efficiency of energy transfer can be low thus limiting the magnitude of the detectable signal.
A further proposal for the assay of nucleic acids is disclosed in U.S. Pat. No. 5,332,659 (Kidwell). This technique relies on establishing a sufficiently high, localised concentration of excimer-forming components to result in formation of an excimer which may be detected to assay the nucleic acid. In the preferred embodiment of this technique a sensing nucleic acid strand is labelled with a plurality of the excimer forming components (e.g. pyrene) attached to the nucleic acid chain by flexible linkers whereby said components are able to move randomly relative to each other and interact to participate in excimer formation.
To perform the assay, a sample potentially containing the target nucleic acid is probed with the sensing strand whilst the system is irradiated (e.g. by a laser) with electromagnetic radiation capable of effecting formation of an excimer if two excimer-forming components are within sufficiently close proximity. In the non-hybridized sensing strand, there is a relatively high probability of excimer formation resulting from random movement of the excimer forming components. However when the sensing strand is hybridized to the target, the former (i.e. the sensing strand) adopts a configuration in which interaction of the excimer-forming components is considerably less likely so that there is a reduced signal compared to a control in which no target is present.
This proposal has the disadvantage of relying on a decrease in signal which may be difficult to detect and of achieving excimer formation by random movement (allowed by virtue of the linkers) of the component parts thereof. Further a decrease in signal would also result from non-specific hybridisation of probe DNA with other structurally similar regions.
In an alternative embodiment disclosed in U.S. Pat. No. 5,332,659, it is proposed that the sensing strand be more heavily labelled. In this embodiment, the length of the linker is selected to reduce the freedom of movement of the excimer-forming components thus minimising (but not eliminating) their interaction in solution. The sensing strand is capable of binding to the target strand such that the sensing strand undergoes structural rearrangement to bring the excimer-forming components sufficiently close to each other for excimer formation. However once again the formation of the excimer relies on a sufficiently high effective concentration of the excimer-forming components being provided, and being able to interact, by the presence of the linkers.
It is acknowledged in U.S. Pat. No. 5,332,659 that all of the disclosed embodiments relating to detection of polynucleic acids result in some complex formation whether the target polynucleic acid strand is present or absent. This could give rise to a false reading.
A similar technique is disclosed in U.S. Pat. No. 5,466,578 (which is Continuation-in-Part of U.S. Pat. No. 5,332,659) but with the modification of using a quaternary ammonium surfactant to enhance light or emission or absorbance. However the same disadvantages as discussed for U.S. Pat. No. 5,332,659 still apply.
EP-A-0 810 291 discloses a method for detection of nucleic acid (NA) sequences using hybridisation of nonradioactive probes with target NA sequences. The method is based on the hybridization of the target NA molecule with two (or more) oligonucleotides, ON1 and ON2, bearing chromophoric labelling groups at their 5′-terminus and 3′-terminus, respectively, and being complementary to the neighbouring base-pairing sites of the target NA. Thus, under hybridizing conditions, the two oligonucleotides are capable of hybridizing to the target sequence (if present) such that the 5′ terminal nucleotide of the first probe and the 3′ terminal nucleotide of the second probe are hybridized to adjacent bases of the target sequence.
The 5′ individual chromophoric labelling groups R1 and R2 are each capable of forming with the other chromophoric group a complex which fluoresces at a longer wavelength than either of the individual chromophoric groups. The complex is only formed on photo-irradiation if the two chromophoric groups are brought into complex forming relationship and this only occurs if both oligonucleotides probes are hybridized to the target sequence. Thus by monitoring for fluorescence at the emission wavelength for the complex it is possible to determine whether or not the particular polynucleotide target is present (since fluorescence of the emission wavelength of the complex will only be observed of the two probes are hybridized to the target).
To ensure that the complex can be formed, it is stated in EP-A-0 810 291 that intercalation of the chromophoric groups into the double stranded nucleic acid formed by hybridisation of the probes must be avoided.
It is possible in the technique of EP-A-0 810 291 to employ a plurality of pairs of probes as described each of which, if hybridized to the appropriate target sequence, will result in production of a complex having a different fluorescence emission so that by detecting for these different wavelengths the analysis procedure is capable of detecting a plurality of different target sequences.
The preferred complex for use in the technique of EP-A-0 810 291 is a excimer (i.e. a fluorescent complex) formed when two identical complex forming partners (e.g. pyrene) are brought into the correct positional relationship to each other and photo-irradiated and the invention is exemplified by excimers. Similar subject matter to EP-A-0 810 291 is disclosed in Ebata et al. Photochem. Photobiol. (1995) v 62, pp836-839 and Ebata et al. Nucl Acids Symp (1995) Series No 34, pp187-188) and in P. L. Paris et al. Nucl. Acids Res.1 (1998), v26, pp 3789-373.
There is also a reference in EP-A-0 810 291 to the possibility of the complex being an exciplex but no specific details with regard to such a complex are given. An exciplex is a heterocomplex analogue of an excimer and is formed, on photo-irradia

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