Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Chemical analysis
Reexamination Certificate
2000-03-31
2002-12-10
Brusca, John S. (Department: 1637)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Chemical analysis
C435S006120, C435S091100, C435S091200, C706S045000, C706S047000, C712S200000
Reexamination Certificate
active
06493640
ABSTRACT:
The present invention relates to the optimisation of cycling conditions used to control polymerase chain reactions.
Optimisation of the temperature control used for PCR amplification requires careful consideration of reaction conditions. The complex nature of the reaction and the interactions between essential reaction components means that traditional kinetic methods of analysis cannot be readily applied to predict optimum cycling conditions. The process described here overcomes these problems by predicting the level of amplification using a novel combination of “grey-box” modelling, genetic algorithms and neural networks, to model and predict the level of amplification for defined sets of cycling conditions. This can be used to determine which parts of the temperature profile have greatest effect on the reaction. Genetic algorithms are used to model the effects changes in the temperature profile have on amplification. These algorithms can then be used to define temperature cycles that give increased reaction performance. Linking this modelling process with on-line monitoring of the amplification process, real-time optimisation of reactions is possible. This is of particular importance to quality control sensitive procedures such as PCR-based diagnostics.
U.S. Pat. No. 4,683,195 (Mullis et al., Cetus Corporation) discloses a process for amplification of nucleic acid by the polymerase chain reaction (PCR). Short oligonucleotide sequences usually 10-40 base pairs long are designed to flanking regions either side of the target sequences to be amplified. These primers are added in excess to the target sequence DNA. A suitable buffer, magnesium chloride ions, a thermostable polymerase and free nucleotides are also added.
A process of thermal cycling is used to amplify the DNA typically several million-fold. Amplification is facilitated through cycling temperature. The target DNA is initially denatured at 95° C. and then cooled to generally between 40° C. to 60° C. to enable annealing of the primers to the separated strands. The temperature is raised to the optimal temperature of the polymerase, generally 72° C., which extend the primer to copy the target sequence. This series of events is repeated (usually 20 to 40 times). During the first few cycles, copies are made of the target sequence. During subsequent cycles, copies are made from copies, increasing target amplification exponentially.
Describing PCR mathematically may not be possible using traditional kinetic notation because of the complex interactions between reaction components. (see “A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methods” Cobb and Clarkson, (1994)Nucleic Acids Research. Vol.22, No.18, pp. 3801-3805). Mg
2+
and deoxynucleotide triphosphates have been shown to affect the efficiency of priming and extension by altering the kinetics of hybridisation and disassociation of primer-template duplexes at denaturing, annealing and extension temperatures. These components are also involved in altering the efficiency with which the polymerase recognises and extends such duplexes. Concentrations of Mg
2+
and deoxynucleotide triphosphates required for optimal amplification depends largely on the target and primer sequences, with the nucleotides at the 3′ end of the primer having a major effect on the efficiency of mismatch extension. Certain mismatch nucleotide combinations may be amplified more efficiently under certain reaction conditions that others. The presence of excess Mg
2+
in a reaction may result in the accumulation of non-specific amplification products, and insufficient concentrations reduce product yield. In addition, deoxynucleotide triphosphates quantitatively bind Mg
2+
ions, so that any modification in dNTP concentration requires a compensatory adjustment of MgCl
2
.
PCR optimisation conventionally requires repetitive trial-and-error adjustment of important reaction parameters. Reactions optimised in this way are generally not robust and are susceptible to small variations in the temperature profile and/or minor fluctuations in the composition of the reaction mixture. The complexity, and to a certain degree the uncertainty of the reaction, means that modelling is difficult. Where models have been proposed see: “Polymerase chain reaction engineering” Hsu et al., (1997). Biotechnology and Bioengineering, Vol 55, No.2. pp.359-366, important reaction elements have been ignored. Importantly, current models assume that denaturation, extension and annealing occur at fixed temperatures in the cycle, predominantly due to the way in which thermal cyclers are programmed with fixed temperatures for each of these principle events. However, this is an over simplification since the rate of these events is temperature dependent such that they occur over a wide temperature range.
Theoretically, amplification of specific template sequences should have an exponential function, i.e. under perfect conditions the amount of template amplified will double after each cycle of the reaction. However, the fidelity and rate of amplification is controlled by a complex interaction between the reaction components so that the theoretical optimum is never achieved. Under normal conditions, the accumulation of product becomes limited during the later cycles since the number of duplexes for extension exceeds the enzyme activity in the reaction. At this point, the accumulation of product becomes linear. This is compounded by thermal inactivation of the polymerase with prolonged exposure to temperatures in excess of 80° C. Amplification can be optimised by careful consideration of annealing temperatures, annealing times and annealing ramps. It is possible to increase the annealing temperature to avoid non-specific priming, by adjusting the ramp rate in order to compensate for the reduced rate of priming. This will increase the cycle range during which exponential accumulation of the target sequence occurs. The rate of priming and temperature range over which priming occurs will depend on the amount of free Mg
2+
.
Similar optimisation of denaturation times and ramps will have an impact on amplification since Taq polymerase becomes denatured with excessive exposure to the high denaturation temperatures (typically ≧94° C. for 1 min to 5 min) (see: “Kinetics of inactivation for thermostable DNA polymerase enzymes” Mohapatra and Hsu, (1996), Biotechnology Techniques, Vol.10, pp.569-572). Although polymerase is normally added in excess, successive denaturation steps in the PCR have significant impact on the amount of polymerase denaturation. Additionally, these temperature conditions cause depurination of the DNA template (typically every 2 Kb at 94° C. min
−1
). Since denaturation occurs before and after the set denaturation temperature has been achieved (typically DNA denatures with increasing velocity between 70° C. and 90° C.), modification of ramp times can be used to limit the times at 94° C. Polymerases such as Taq have been well characterised. They show classic temperature dependency, with a gradual increase in extension rate at high temperatures. Activity reaches an optimum (typically ≈70° C.), after which activity drops sharply (typically ≧80° C.). Extension will occur over an extended temperature range. It is possible to reduce extension times by consideration of the total amount of extension over this range. For example, a significant amount of extension will occur at ca. 60° C. Oligonucleotides that hybridise at this temperature will be extended immediately. Extension times can be reduced, or in some instances eliminate altogether.
The present invention seeks to provide optimisation of cycling conditions used to control polymerase chain reactions.
According to an aspect of the present invention there is provided a method of optimising the cycling conditions used to control a polymerase chain reaction as specified in claim
The preferred embodiment provides a process which allows intelligent control of the PCR. This is achieved by modelli
Clarkson John Michael
Cobb Benjamin David
Brusca John S.
Gitler Stewart L.
Kent Evan M.
KIm Young
Molecular Sensors Limited
LandOfFree
Control of nucleic acid amplification procedures including... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Control of nucleic acid amplification procedures including..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Control of nucleic acid amplification procedures including... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2970029