Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-10-15
2004-11-23
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C435S007100, C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06821771
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns the field of genetics.
More precisely, the present invention relates to a device for amplifying target nucleic acid sequences, to reaction cartridges for use in the device, and to methods of application of this device.
The aim of the present invention is the detection and, if required, real-time quantification of target nucleic acid sequences in one or more samples.
BACKGROUND AND PRIOR ART
Detecting target nucleic acid sequences is a technique that is being used to a greater and greater extent in many fields, and the range of applications of that technique is predicted to widen as it becomes more reliable, cheaper and faster. In the human health field, detecting certain nucleic acid sequences can in some cases provide a reliable and rapid diagnosis of viral or bacterial infections. Similarly, detecting certain genetic peculiarities can allow susceptibilities to certain diseases to be identified, or provide an early diagnosis of genetic or neoplastic diseases. The detection of target nucleic acid sequences is also used in the agroalimentary industry, in particular to provide product traceability, to detect the presence of genetically modified organisms and to identify them, or to carry out food checks.
Detection procedures based on nucleic acids almost systematically involve a molecular hybridisation reaction between a target nucleic acid sequence and one or more nucleic acid sequences complementary to that target sequence. Such processes have a number of variations, such as techniques known to the skilled person as “transfer techniques” (blot, dot blot, Souther blot, Restriction Fragment Length Polymorphism, etc.), or such as miniaturised systems on which the complementary sequences of the target sequences are previously fixed (microarrays). Within the context of such techniques, complementary nucleic acid sequences are generally termed probes. A further variation, which can in itself constitute the basis of a diagnostic procedure or may simply be a supplementary step in one of the techniques mentioned above (in particular to increase the concentration of the target sequence and thus, the sensitivity of the diagnosis), consists of amplifying the targeted nucleic acid sequence. A number of techniques that can specifically amplify a nucleic acid sequence have been described, the most popular technique being the Polymerase Chain Reaction (PCR). Within the context of that technique, complementary nucleic acid sequences of target sequences, termed primers, are used to amplify those target sequences.
PCR reactions involve repeated cycles, generally 20 to 50 in number, and each is composed of three successive phases, namely: denaturation, primer annealing, strand elongation. The first phase corresponds to transforming double-stranded nucleic acids into single-stranded nucleic acids; the second phase is molecular hybridisation between the target sequence and the complementary primers for said sequence, and the third phase corresponds to elongation of the complementary primers hybridised to the target sequence, using a DNA polymerase. Those phases are carried out at specific temperatures: generally, 95° C. for denaturation, 72° C. for elongation, and between 30° C. and 65° C. for annealing, depending on the melting temperature (Tm) of the primers used. It is also possible to carry out the annealing and elongation steps at the same temperature (generally 60° C.).
Thus, a PCR reaction consists of a sequence of repetitive thermal cycles during which the number of target DNA molecules acting as the template is theoretically doubled for each cycle. In practice, the PCR yield is less than 100%, so the quantity of product X
n
obtained after n cycles is:
X
n
=X
n-1
(1+
r
n
),
where
X
n-1
is the quantity of product obtained in the preceding cycle, and r
n
is the PCR yield in cycle n (0<r
n
≦1).
Assuming the yield to be a constant, i.e., identical for each cycle, the quantity of product X
n
obtained after n cycles from an initial quantity X
0
is:
X
n
=X
0
(1+
r
)
n
(A)
In practice, the yield r reduces during the PCR reaction, due to a number of factors such as a limiting quantity of at least one of the reagents necessary for amplification, deactivation of the polymerase by its repeated passes at 95° C., or its inhibition by pyrophosphates produced by the reaction.
Because of this reduction in yield, the PCR reaction kinetics firstly exhibit an exponential phase (where r is a constant), which then changes into a plateau phase when r reduces.
During the exponential phase, equation (A) above applies, and can also be written as:
log(
X
n
)=log(
X
g
)+
n
log(1+
r
)
Thus, in the exponential phase of the PCR, the curve showing the quantity of product on a logarithmic scale as a function of the number of cycles is a straight line with slope (1+r) which intersects the ordinate at a value equal to the logarithm of the initial concentration.
Real-time measurement of the quantity of product obtained can thus provide the initial concentration of the template, which is of particular importance in a large number of applications, for example when measuring the viral change in a patient, or to determine the variability of a transcriptome.
Generally, the PCR employs reaction volumes of 2 &mgr;l to 50 &mgr;l and is carried out in tubes, microtubes, capillaries or systems known in the art as “microplates” (integral assemblies of microtubes). Each batch of tubes or equivalent containers must thus be successively heated to the three temperatures, corresponding to the different phases of the PCR, for the desired number of cycles.
Using tubes or similar systems obliges the operator to carry out many manipulations to prepare as many tubes and solutions (known in the art as mix PCR) as there are target sequences to be amplified, even when using a single sample of nucleic acids, with the exception of multiplex amplification procedures, which amplify a plurality of target sequences simultaneously in the same container, either using low specificity primers that can hybridise with a plurality of target sequences, such as RAPD—random amplified polymorphism DNA, or using specific primers in larger numbers, where each pair of primers used amplifies a single target sequence. Multiplex amplifications correspond to particular cases and are not in routine use. Further, they do not guarantee freedom from interactions of one amplification reaction with another, and because of possible hybridisations between primers, can only be very limited in the number of target sequences amplified per container.
Those different manipulations cause a number of disadvantages.
Firstly, they are time consuming. Secondly, they are not risk-free as regards possible contamination from one tube to another or from the external environment (dust, bacteria, aerosols or other contaminants that may contain nucleic acid molecules or molecules that may influence the efficacy of the amplification reaction). Further, homogeneity of volume and reagent concentration from one tube to another is not guaranteed. Finally, the volumes are necessarily manipulated manually and are generally greater than 1 &mgr;l, which affects the costs of carrying out PCR as the reagents employed are expensive.
The use of devices designed for at least partial automation of such manipulations can overcome some of those disadvantages. However, those instruments are relatively expensive and their use is, therefore, only economically justified when carrying out many PCR amplifications, for example for genome sequencing.
Some instruments also exist that can carry out kinetic PCR amplifications. As seen above, kinetic PCR necessitates real-time, specific quantification of the amplified target sequence. The use of a fluorescent reporter in the reaction mixture allows the increase in the total quantity of double-stranded DNA to be measured in that mixture. However, that method cannot discriminate amplification of the target sequence from background noise or from possib
Genesystems
Merchant & Gould P.C.
Siew Jeffrey
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