Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-05-25
2004-12-21
Sisson, Bradley L. (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C435S183000, C435S270000, C436S501000, C436S094000, C436S800000, C536S023100, C536S024300, C536S024310, C536S024330, C536S025300
Reexamination Certificate
active
06833257
ABSTRACT:
The present invention provides a method for detecting a target polynucleotide in a sample, for example by quantitatively monitoring an amplification reaction, as well as to probes and kits for use in these methods. The method is particularly suitable for the detection of polymorphisms or allelic variation and so may be used in diagnostic methods
Known fluorescence polymerase chain reaction (PCR) monitoring techniques include both strand specific and generic DNA intercalator techniques that can be used on a few second-generation PCR thermal cycling devices.
Generic methods utilise DNA intercalating dyes that exhibit increased fluorescence when bound to double stranded DNA species. Fluorescence increase due to a rise in the bulk concentration of DNA during amplifications can be used to measure reaction progress and to determine the target molecule copy number. Furthermore, by monitoring fluorescence with a controlled change of temperature, DNA melting curves can be generated, for example, at the end of PCR thermal cycling.
When generic DNA methods are used to monitor the rise in bulk concentration of nucleic acids, these processes can be monitored with a minimal time penalty (compared to some other known assays discussed below). A single fluorescent reading can be taken at the same point in every reaction. End point melting curve analysis can be used to discriminate artifacts from amplicon, and to discriminate amplicons. Melting peaks of products can be determined for concentrations that cannot be visualized by agarose gel electrophoresis.
In order to obtain high resolution melting data, for example for multiple samples, the melt experiment must be performed slowly on existing hardware taking up to five minutes. However, by continually monitoring fluorescence amplification, a 3D image of the hysteresis of melting and hybridization can be produced. This 3D image is amplicon dependent and may provide enough information for product discrimination.
It has been found that DNA melting curve analysis in general is a powerful tool in optimizing PCR thermal cycling. By determining the melting temperatures of the amplicons, it is possible to lower the denaturing temperature in later PCR cycles to this temperature. Optimization for amplification from first generation reaction products rather than the target DNA, reduces artifact formation occurring in later cycles. Melting temperatures of primer oligonucleotides and their complements can be used to determine their annealing temperatures, reducing the need for empirical optimization.
The generic intercalator methods however are only quasi-strand-specific and therefore is not very useful where strand specific detection is required.
Strand specific methods utilise additional nucleic acid reaction components to monitor the progress of amplification reactions. These methods often use fluorescence energy transfer (FET) as the basis of detection. One or more nucleic acid probes are labeled with fluorescent molecules, one of which is able to act as an energy donor and the other of which is an energy acceptor molecule. These are sometimes known as a reporter molecule and a quencher molecule respectively. The donor molecule is excited with a specific wavelength of light which falls within its excitation spectrum and subsequently it will emit light within its fluorescence emission wavelength. The acceptor molecule is also excited at this wavelength by accepting energy from the donor molecule by a variety of distance-dependent energy transfer mechanisms. A specific example of fluorescence energy transfer which can occur is Fluorescence Resonance Energy Transfer or “FRET”. Generally, the acceptor molecule accepts the emission energy of the donor molecule when they are in close proximity (e.g. on the same, or a neighboring molecule). The basis of fluorescence energy transfer detection is to monitor the changes at donor and acceptor emission wavelengths.
There are two commonly used types of FET or FRET probes, those using hydrolysis of nucleic acid probes to separate donor from acceptor, and those using hybridization to alter the spatial relationship of donor and acceptor molecules.
Hydrolysis probes are commercially available as Taqman™ probes. These consist of DNA oligonucleotides that are labeled with donor and acceptor molecules. The probes are designed to bind to a specific region on one strand of a PCR product. Following annealing of the PCR primer to this strand, Taq enzyme extends the DNA with 5′ to 3′ polymerase activity. Taq enzyme also exhibits 5′ to 3′ exonuclease activity. TaqMan™ probes are protected at the 3′ end by phosphorylation to prevent them from extension. If the TaqMan™ probe is hybridized to the product strand, an extending Taq molecule may also hydrolyze the probe, liberating the donor from acceptor as the basis of detection. The signal in this instance is cumulative, the concentration of free donor and acceptor molecules increasing with each cycle of the amplification reaction.
The fact that signal generation is dependent upon the occurrence of probe hydrolysis reactions means that there is a time penalty associated with this method. Furthermore, the presence of the probe may interrupt the smooth operation of the PCR process.
In addition, it has been found that hydrolysis can become non-specific, particularly where large numbers of amplification cycles, for instance more than 50 cycles, are required. In these cases, non-specific hydrolysis of the probe will result in an unduly elevated signal.
This means that such techniques are not very compatible with rapid PCR methods which are becoming more prominent with the development of rapid hot air thermal cyclers such as the RapidCycler™ and LightCycler™ from Idaho Technologies Inc. Other rapid PCR devices are described for example in co-pending British Patent Application Nos. 9625442.0 and 9716052.7. The merits of rapid cycling over conventional thermal cycling have been reported elsewhere. Such techniques are particularly useful for example in detection systems for biological warfare where speed of result is important if loss of life or serious injury is to be avoided.
Furthermore, hydrolysis probes do not provide significant information with regard to hysteresis of melting since signal generation is, by and large, dependent upon hydrolysis of the probe rather than the melt temperature of the amplicon.
U.S. Pat. No. 5,491,063 describes a method for in-solution quenching of fluorescently labeled probes which relies on modification of the signal from a labeled single stranded oligonucleotide by a DNA binding agent. The difference in this signal which occurs as a result of a reduced chain length of the probe following probe cleavage (hydrolysis) during a polymerase chain reaction is suggested for providing a means for detecting the presence of a target nucleic acid.
Hybridization probes are available in a number of forms. Molecular beacons are oligonucleotides that have complementary 5′ and 3′ sequences such that they form hairpin loops. Terminal fluorescent labels are in close proximity for FRET to occur when the hairpin structure is formed. Following hybridization of molecular beacons to a complementary sequence the fluorescent labels are separated, so FRET does not occur, and this forms the basis of detection.
Pairs of labeled oligonucleotides may also be used. These hybridize in close proximity on a PCR product strand bringing donor and acceptor molecules together so that FRET can occur. Enhanced FRET is the basis of detection. Variants of this type include using a labeled amplification primer with a single adjacent probe.
The use of two probes, or a molecular beacon type of probe which includes two labeling molecules increases the cost involved in the process. In addition, this method requires the presence of a reasonably long known sequence so that two probes which are long enough to bind specifically in close proximity to each other are known. This can be a problem in some diagnostic applications, where the length of conserved sequences
Fuerst Roderick
Lee Martin A
Sisson Bradley L.
The Secretary of State for Defence
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