PCR method using irradiation of nanoparticles

Abstract

The invention relates to a method for the duplication of nucleic acids by means of a polymerase chain reaction, in the case of which a cycle consisting of the steps of denaturing, annealing and elongation is repeatedly performed. In one embodiment, in at least one passage of the cycle, the quotient of the duration of effect t.sub.A and the reaction volume V.sub.r irradiated by the energy source is less than 1 seconds per microliter. In another embodiment, in at least one passage of the cycle, the ratio of the duration of effect (t.sub.A) and the duration of the PCR cycle (t.sub.c) is less than 20%. In certain embodiments, the yield (g) of nucleic acids at the end of at least one of the passages of the cycle is less than 80% of the nucleic acids present at the start of the passage.

Claims

1. A method for amplifying nucleic acids in a reaction volume V.sub.r by means of a polymerase chain reaction, the method comprising performing a denaturing step, an annealing step and an elongation step, wherein a cycle consisting of the denaturing step, the annealing step and the elongation step is performed repeatedly on at least one nucleic acid, wherein, in at least one passage of the cycle, during a duration of effect t.sub.A the reaction volume V.sub.r is irradiated by an energy source suitable for denaturing by heat a point in the reaction volume on which the energy source acts, and wherein the quotient tA/Vr of the duration of effect t.sub.A and the reaction volume V.sub.r irradiated by the energy source is less than 1 seconds per microliter.

2. The method according to claim 1, wherein the yield (g) of nucleic acids at the end of at least one of the passages of the cycle is less than 80% of the nucleic acids present at the start of the passage.

3. The method according to claim 1, wherein in at least one of the passages of the cycle, the duration of effect (t.sub.A) is less than 10 seconds.

4. The method according to claim 1, wherein the number of the passages of the cycle of the polymerase chain reaction is greater than 45.

5. The method according to claim 1, wherein the concentration of the nucleic acid or portion thereof to be amplified in the method is less than 1 nM at the start of the method.

6. The method according to claim 1, wherein the cycle duration (t.sub.c) is shorter than 20 seconds in at least one of the passages of the cycle.

7. The method according to claim 1, wherein the method includes a global heating step.

8. The method according to claim 1, wherein the temperature at which the annealing step occurs is equal to the temperature at which the elongation step occurs.

9. The method according to claim 1, further comprising the use of a DNA polymerase that is thermolabile.

10. The method according to claim 1, wherein the concentration of the products of the amplification reaction is determined by test probes.

11. The method according to claim 1, wherein in at least one passage of the cycle, the ratio of the duration of effect (t.sub.A) and the duration of the PCR cycle (t.sub.c) is less than 20%.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows in a schematic illustration nanoparticles that are conjugated with filling molecules, spacer sequences, abasic modifications and primer sequences;

(2) FIG. 2 shows in a schematic illustration a structure for carrying out the method according to the invention with a laser, a two-dimensional mirror scanner and a sample;

(3) FIG. 3 shows in a schematic illustration a further structure for carrying out the method according to the invention with a laser, a two-dimensional mirror scanner and sample tubes in a water bath;

(4) FIG. 4 shows the idealized temperature profile of a conventional PCR (dotted line);

(5) FIG. 5 shows the amplification factor N.sub.k/N.sub.o as a function of the time for different parameters;

(6) and

(7) FIG. 6 shows in four diagrams the results of amplification reactions with different cycle times and numbers of cycles.

DETAILED DESCRIPTION OF THE INVENTION BY REFERENCE TO A PLURALITY OF EXEMPLARY EMBODIMENTS

(8) In known PCR methods for the amplification of a short nucleic acid (e.g. with fewer than 300 base pairs), which work with tempering by means of thermocyclers, the process duration is generally limited by the tempering time, which accounts for a large part of the cycle duration. In order to achieve a process duration that is as short as possible, it is endeavoured in such cases, with respect to the yield g.sub.h (the index “h” points towards this conventional case) per passage of the cycle, to arrive close to the theoretical threshold of 100%, in order to achieve the desired result with as few cycles as possible. According to the findings of the inventors, however, in methods with shorter tempering times, e.g. in methods that work with local heating by means of nanoparticles, a maximization of the yield g is no longer necessarily the best strategy. Instead, here, taking into account a reduced yield, a shortening of the cycle duration that outweighs the disadvantages of the lower yield can be achieved, such that overall, despite lower yield, shortening of the process duration results.

(9) Without adhering to a certain theory, it is firstly necessary to observe the characteristic time constants required by different processes during the PCR and a simple mathematical model is to be formulated.

(10) Firstly, a conventional PCR method is assumed, which is carried out in a customary thermocycler, e.g. with a Peltier element or an air stream in order to temper the reaction volume (usually 5 to 50 microlitres) from externally. Such customary thermocyclers typically achieve heating and cooling rates of approximately 5 K/s, even if the average tempering speed may be significantly lower. This means that the process of heating a sample from an annealing temperature of 60° C. to a denaturing temperature of 95° C. and then cooling it again to the annealing temperature can require at least approximately: 2.Math.35° C./(5° C./s)=14 s.

(11) Added to this is the fact that, with methods used thus far, the thermalization of the sample volume can additionally take a few seconds until approximately the same temperature prevails everywhere in the sample, such as on the heated or cooled vessel walls. Consequently, the total tempering time t.sub.t per cycle in conventional protocols is typically more than 14 s.

(12) For a PCR, the annealing and elongation times are also important. The annealing time in the case of sufficiently high primer concentrations (e.g. more than 300 nM) under suitable conditions in the prior art is frequently a few seconds until a primer is hybridized to the majority (>90%) of the targets. For example an annealing time of 1 s can also be realized.

(13) The time required by the polymerase for elongation of the primers depends upon the length of the amplicon and the write speed of the polymerase used. In order to elongate, for example, 80 base pairs, the elongation under suitable conditions, in the case of a polymerase with effective write speed of 100 BP/s, takes approximately 0.8 s.

(14) The hybridization time and the elongation time together are referred to below as the required productive time t.sub.ph. In the above example, the required productive time for a short amplicon is for example t.sub.ph≈2 s, if one second is assumed for annealing and a further second for elongation.

(15) The duration of a PCR cycle t.sub.ch in the abovementioned example is the sum of the tempering time t.sub.th and the required productive time t.sub.ph, i.e.:
t.sub.ch=t.sub.th+t.sub.ph. (3)

(16) If any dwell time at the denaturing temperature is disregarded, as it can be selected to be very short, e.g. it can be shorter than one second.

(17) If the tempering time of approximately 14 s is compared with the required productive time of approximately 2 s in the above example, it can be seen that, in a conventional thermocycler for short nucleic acids, the following inequalities typically apply: t.sub.th»t.sub.ph and t.sub.ch≈t.sub.th. This means that the tempering time, i.e. the heating and cooling times, thus the time taken to bring the sample from the annealing temperature to the denaturing temperature and cool it back down steadily to the annealing temperature, generally determines the duration of each cycle and thus also the total duration of the PCR.

(18) If the number of copies N.sub.0 of the template is to be increased to N.sub.k copies with the PCR, this can—as already explained above—be achieved with a number k of temperature cycles, wherein k=log.sub.(1+gh)(N.sub.k/N.sub.0) if the average yield in each cycle is g.sub.h. It is assumed once again for simplification purposes that the yield per cycle remains constant during the PCR. If f.sub.co cycles are carried out per time unit (with f.sub.ch=1/t.sub.ch), the number of the copies N.sub.k after a time t can be given by:
N.sub.k=N.sub.0(1+g.sub.h).sup.fc.sup.h.sup..Math.t (4)
wherein g.sub.h=0 . . . 100%. The process duration T of the whole PCR can be given by the cycle duration t.sub.ch multiplied by the necessary number of cycles k:

(19) $\begin{matrix} T = t_{c_{h}} .Math. k = t_{c_{h}} .Math. \log_{(1 + g_{h})} (\frac{N}{N_{0}}) . & (5) \end{matrix}$

(20) For example, an amplification by the factor N.sub.k/N.sub.0=10.sup.12, depending on the value of g.sub.h, can require the times shown in Table 1.

(21) TABLE-US-00001 TABLE 1 Preferred PCR durations T in units of the cycle duration t.sub.ch to the 10.sup.12 times amplification of a target. g.sub.h T[t.sub.ch] 0.4 82 0.6 59 0.80 47 1.00 40

(22) It follows from this that in the case of a conventional PCR, wherein t.sub.th»t.sub.ph and t.sub.ch≈t.sub.th, a short process duration can be achieved by the yield per cycle being maximized, g.sub.h thus being close to 100%. In this case, the number of copies N.sub.k with the time can be given by:
N.sub.k=N.sub.0(1+1).sup.fc.sup.h.sup..Math.t=N.sub.0.Math.2.sup.fc.sup.h.sup..Math.t, (6)
i.e.: for each cycle, the number of copies thus far can have added to it the same number (i.e. being doubled for each cycle). The shortest possible PCR duration T.sub.min for an amplification by the amplification factor N.sub.k/N.sub.0 can then be given by:

(23) $\begin{matrix} T_{m i n} = t_{c} .Math. k = t_{c} .Math. \log_{2} (\frac{N_{k}}{N_{0}}) . & (7) \end{matrix}$

(24) A different situation can emerge if the tempering times no longer determine the cycle duration. This case includes in particular also the sub-case that heating and cooling steps, including the thermalization, are negligibly short, i.e. if the following inequalities apply: t.sub.t«t.sub.p and t.sub.c≈t.sub.p.

Example 1

(25) The effect of shortening the cycle duration is to be examined below. The cycle duration in this example is described as t.sub.ci (the optional index “i” is used below to emphasize the solution according to the invention for the parameters, which describes a PCR with shortened cycle durations), wherein the example cycle duration t.sub.ci has been shortened, with respect to the cycle duration t.sub.ch in the conventional case, by a shortening factor x with x∈ custom character .sup.>1

(26) so that the following applies:

(27) $t_{c_{i}} = \frac{t_{c_{h}}}{x} .$
i.e.: the new cycle frequency f.sub.ci=x.Math.f.sub.ch can be calculated from the cycle frequency f.sub.c of the conventional case. The yield in this example is described with g.sub.i. The example yield can be equal to the yield in the conventional case (g.sub.i=g.sub.h), but it can also be smaller than this (g.sub.i<g.sub.h). If there is a reduction in the yield per cycle, this can be described by an efficiency loss factor y, wherein:

(28) $g_{i} = \frac{g_{h}}{y} .$

(29) It follows that in this example:

(30) 0 $\begin{matrix} N_{i} = {N_{0} (1 + \frac{g_{h}}{y})}^{f_{c_{h}} .Math. x .Math. t} = {N_{0} (1 + g_{i})}^{f_{c_{i}} .Math. t} = {N_{0} (1 + g_{i})}^{f_{c_{h}} .Math. x .Math. t} . & (8) \end{matrix}$

(31) The process duration T.sub.i of the whole PCR in this example is therefore:

(32) $\begin{matrix} T_{i} = \frac{t_{c_{h}}}{x} .Math. \log_{(1 + g_{i})} (\frac{N_{i}}{N_{0}}), & (9) \end{matrix}$

(33) for which the fact that preferably

(34) $f_{c_{h}} = \frac{1}{t_{c_{h}}} .$
has one again been utilized.

(35) A shortening of the process duration in comparison with the conventional case can be achieved both if g.sub.i=g.sub.h and also if gi<g.sub.h, provided that the disadvantage of the lower yield is outweighed by the advantage of shortening of the cycle duration.

(36) If for example amplification by the factor N.sub.1/N.sub.0=10.sup.12 is assumed, according to Equation (9) the following values can be given for the process duration in units of t.sub.ch as a function of the selection of the values for g.sub.i and x.

(37) TABLE-US-00002 TABLE 2 PCR durations T of a conventional PCR and preferred PCR durations T.sub.i in units of the conventional cycle duration t.sub.ch to 10.sup.12 times amplification of a target. Conventional PCR duration χ T with x = 1 1.11 1.25 1.33 1.67 2.00 4.00 10.00 g.sub.i 0.05 566 * 510 * 453 * 425 * 340 * 283 * 142 * 57 * 0.10 290 * 261 * 232 * 217 * 174 * 145 * 72 * 29 0.15 198 * 178 * 158 * 148 * 119 * 99 * 49 * 20 0.20 152 * 136 * 121 * 114 * 91 * 76 * 38+ 15 0.25 124 * 111 * 99 * 93 * 74 * 62 * 31 12 0.30 105 * 95 * 84 * 79 * 63 * 53 * 26 11 0.35 92 * 83 * 74 * 69 * 55 * 46 * 23 9 0.40 82 * 74 * 66 * 62 * 49 * 41 * 21 8 0.45 74 * 67 * 59 * 56 * 45 * 37+ 19 7 0.50 68 * 61 * 55 * 51 * 41 * 34 17 7 0.55 63 * 57 * 50 * 47 * 38+ 32 16 6 0.60 59 * 53 * 47 * 44 * 35 29 15 6 0.65 55 * 50 * 44 * 41 * 33 28 14 6 0.70 52 * 47 * 42 * 39+ 31 26 13 5 0.75 49 * 44 * 39+ 37 30 25 12 5 0.80 47 * 42 * 38 35 28 24 12 5 0.85 45 * 40 * 36 34 27 22 11 4 0.90 43 * 39 34 32 26 22 11 4 0.95 41 * 37 33 31 25 21 10 4 1.00 40 * 36 32 30 24 20 10 4

(38) The values marked with * illustrate the range in a theoretical observation of the combinations of g.sub.i and x, for which no acceleration arises with respect to a conventional PCR with g.sub.h≈100%.

Example 2

(39) This example is based on Example 1 and includes the case in which the cycle duration t.sub.ci according to the invention is preferably selected to be shorter than the conventional cycle duration t.sub.ch, but furthermore in such a way that the yield per cycle can remain approximately the same as in the case of the selection of the cycle duration to date t.sub.ch, i.e. g.sub.i≈g.sub.h (this can, e.g. make it necessary for the annealing and the elongation to continue in each cycle to run with approximately the same efficiency as when the cycle duration thus far t.sub.ch is selected). In other words, here the efficiency loss factor y=1, as according to definition in this example no efficiency loss arises.

(40) In this case the number of cycles necessary for a desired amplification can then remain constant, and the duration of each cycle can be shortened by the factor x and the process duration according to the invention can be shortened corresponding to T.sub.i=T/x. In other words: with the example values indicated in Table 2 the PCR duration of a hypothetical conventional PCR T can be read within the scope of this theoretical observation in the second column. The process duration for a PCR according to the invention with g.sub.0i=g.sub.0 can then be read in the same line as the conventional comparative value.

(41) In an example realization of these examples, the cycle duration is selected so that it continues to be greater than the required productive time t.sub.ci−t.sub.ti»t.sub.pi, so that, e.g. approximately g.sub.i=g.sub.h≈100% is reached.

Example 3

(42) This example is also based on Example 1. However, it is now assumed that the shortening of the example cycle duration t.sub.ci in comparison with the conventional cycle duration t.sub.ch leads to a reduction in the average yield per cycle g.sub.i in comparison with the average yield thus far g.sub.h

(43) $(i . e . the efficiency loss factor =^{y = \frac{g_{h}}{g_{i}}} > 1) .$
As further assumed, this decrease in the yield per cycle, which can result from the shortening of the cycle duration by factor x, but can be more than compensated through more temperature cycles (which can be carried out more quickly by the factor

(44) $x = \frac{t_{c_{h}}}{t_{c_{i}}}),$
i.e. the increase in the amplicon concentration per time unit is nevertheless higher (wherein the time unit under observation is preferably to be selected to be very much longer than t.sub.ch).

(45) A decrease in the average yield per cycle can be realised, e.g., by the cycle duration becoming even shorter than the required productive time, i.e. t.sub.ci<t.sub.pi, wherein t.sub.pi=t.sub.ph remains, so that the yield per cycle is g.sub.h«100% (e.g. because only few copies of the template can hybridize in the time with a primer and/or the polymerase cannot, in the time, elongate all the primers or the denaturing does not take place completely, since, e.g., the duration of effect is so short that the DNA double strand cannot sufficiently unravel.

Example 3a

(46) It is assumed in this example that the following relationship applies for the yield:

(47) $\begin{matrix} g_{h} \geq g_{i} \geq \frac{g_{h}}{x} . & (10) \end{matrix}$

(48) In other words, the average yield per cycle increases in this embodiment preferably maximum linearly with the shortening x of the cycle duration, whereby this can arise for example if the cycle duration no longer suffices for a large part of the template DNA to be able to hybridize with a primer (i.e. the efficiency loss factor is here 1<y≤x). The decrease in the yield per cycle, which is maximum factor x, can thereby be more than compensated by x times more cycles per time unit. In this case it can be written as follows:

(49) $\begin{matrix} N_{i} \geq {N_{0} (1 + \frac{g_{h}}{x})}^{f_{c_{h}} .Math. x .Math. t} = {N_{0} (\underset{\underset{:= α}{︸}}{{(1 + \frac{g_{h}}{x})}^{x}})}^{f_{c_{h}} .Math. t} = N_{0} .Math. α^{f_{c_{h}} .Math. t} & (11) \end{matrix}$

(50) In this case the basis of the exponential function a can be greater than in a conventional PCR, where the basis of the exponential function can be according to Equation (4) (1+g.sub.h) and, in the best case scenario, is equal to two. This is summarized in the following table, which contains values for (1+g.sub.h) and also for α:

(51) TABLE-US-00003 TABLE 3 Values for α in comparison with a hypothetical basis, thus far, of the exponential function (1 + g.sub.h). Conven- tional (1 + g.sub.h) 1.11 1.25 1.33 1.67 2.00 4.00 10.00 g.sub.h 0.40 1.40 1.41 1.41 1.42 1.43 1.44 1.46 1.48 0.60 1.60 1.62 1.63 1.64 1.67 1.69 1.75 1.79 0.80 1.80 1.83 1.86 1.87 1.92 1.96 2.07 2.16 1.00 2.00 2.04 2.08 2.11 2.19 2.25 2.44 2.59

(52) This means that the amplification taking place per time unit can be greater than conventionally, provided that g.sub.0>0 and Equation 10 is fulfilled. The inventors therefore refer to the process according to the invention also as “super-amplification”.

(53) The time required by a PCR according to the invention in this embodiment is given if Equation 9 for the PCR duration is re-written to:

(54) $\begin{matrix} T_{i} \leq \frac{t_{c_{h}}}{x} .Math. \log_{(1 + g_{0} / x)} (\frac{N}{N_{0}}) . & (12) \end{matrix}$

(55) In other words: In the example of Table 2 the PCR duration of a hypothetical conventional PCR can be read in the second column. In comparison with a conventional comparative value, the process duration according to the invention can then be read in an entry with values without * or + in the same line or above, depending on which value combination of g.sub.i and x is realised.

(56) A particularly interesting variant of this embodiment results for conventional PCRs, wherein the yield per cycle g.sub.0≈100% (lowermost line in Table 3).

(57) In this case, a in Equation 11 can be approximately re-written as

(58) $a = {(1 + \frac{1}{x})}^{x} .$
PIC. It can also be preferably achieved that x becomes very high, so that approximately the threshold formation

(59) $\lim_{x .fwdarw. \infty} {(1 + \frac{1}{x})}^{x} = e$
is admissible (e≈2.71828 . . . ), so that the value for N; can be approximated from Equation 11 as:
N.sub.i≈N.sub.0.Math.e.sup.fc.sup.h.sup..Math.t (13)

(60) It is shown here, in comparison with Equation 6, that the time-based amplification can no longer take place with 2.sup.fc.sup.h.sup..Math.t, but instead with e.sup.fc.sup.h.sup..Math.t i.e. the basis of the exponential function can be greater.

(61) From Equation 13, the process duration for the case in which x is very high, can be approximately estimated as

(62) 0 $\begin{matrix} T_{i} \approx \ln (\frac{N_{i}}{N_{0}}) t_{c_{h}}, & (14) \end{matrix}$

(63) for which the fact that f.sub.ch=1/t.sub.ch has again been utilized. In other words, in this case the process duration can go hand in hand with the natural logarithm of the amplification factor.

Example 3b

(64) This example is also based on Example 1. Shorter temperature cycles can also be used in this embodiment. However, in this embodiment the shortening according to the invention of the cycle duration t.sub.ci, with respect to the cycle duration t.sub.ch thus far, can lead to a reduction in the yield per cycle g.sub.i with respect to the yield g.sub.h thus far, so that the following can apply:

(65) $\begin{matrix} \frac{g_{h}}{x} > g_{i}, & (15) \end{matrix}$

(66) This means that the yield per cycle in this embodiment can decrease more than linearly with the shortening x of the cycle duration (i.e. the efficiency loss factor is here y>x). Also in this case, the decrease in the yield per cycle can be preferably overcompensated by more cycles, which are carried out more quickly than conventionally by the shortening factor x under suitable conditions.

(67) In the example of Table 2 the hypothetical process duration of a conventional PCR can be read in the lowermost entry of the second column for g.sub.h≈100% (in this case therefore: the value 40). The process duration in this embodiment of the invention can then be read in the entries, of which the values are marked with a +, provided that this value combination of g.sub.i and x can be realized.

(68) FIG. 1 shows an exemplary embodiment of the method according to the invention for the amplification of nucleic acids 1, which is carried out as a PCR. First nanoparticles 3 are contained in a reaction volume 2. The first nanoparticles 3 have oligonucleotides 4 at their surface, as shown in FIG. 1a. One class of oligonucleotides 4 contain, in each case as a sub-sequence, a primer sequence 5 with the sequence A and, as a further, optional sub-sequence, a spacer sequence 6 S and an optional abasic modification 7 between the primer sequence 5 A and spacer sequence 6 S. The primer sequence 5 thereby serves as a forward primer 8. The spacer sequence 6 S is used to keep the primer sequence 5 far enough away from the surface of the nanoparticles 9 so that a nucleic acid 1 to be amplified can bind with better efficiency to the primer sequence 5 and a DNA polymerase 11 can find better access to the primer sequence 5. The abasic modification 7 prevents the spacer sequence being overwritten by the polymerase 11. The oligonucleotides 4 with the primer sequence 5 A are, e.g., fixed with a thiol bound to the surface of the first nanoparticles 3, so that the 3′-end faces away from the first nanoparticle 3. Optionally, a further class of oligonucleotides 4 can be located on the surface of the first nanoparticles 3, these are the filling molecules 10 F. With the filling molecules 10 the charge of the nanoparticles 9 can be modulated so that undesired aggregations of the nanoparticles 9 do not arise. In addition the filling molecules 10 can increase the distance of the primer sequences 5 from each other on the surface of the nanoparticles 9, so that the nucleic acids 1 to be amplified and the DNA polymerase 11 have better access to the primer sequences 5. This can increase the efficiency of the method. The spacer sequence 6 is thereby preferably at least as long as the filling molecules 10, so that the primer sequences 5 advantageously project out of the filling molecules 10.

(69) In the reaction volume 2 there is a liquid sample 12, which contains the first nanoparticles 3 of FIG. 1a with the primer sequences 5, spacer sequences 6, abasic modification 7 and filling molecules 10, and which also has dNTPs and DNA polymerase 11. A nucleic acid 1 to be detected can be present in the sample 12. In this exemplary embodiment the nucleic acid 1 to be detected is a DNA single strand, which is also described as an original 13 or amplicon, and has a sub-sequence A′ and also a sub-sequence B′. The original 13 can also have further sub-sequences, e.g. as overhangs at the 5′-end or 3′-end or between the two sub-sequences A′ and B′. In FIG. 1b, the original 13 with its sub-sequence A′ binds to the primer sequence 5 A on the surface of the first nanoparticles 3. It is shown in FIG. 1c that a DNA polymerase 11 binds to the original 13 and the primer sequence 5 A hybridized with the original 13. Then, the DNA polymerase 11 synthesizes, in an elongation step shown in FIG. 1d, starting from the 3′-end of the primer sequence 5 A, a nucleic acid 1 that is complementary to the original 13 and is referred to as a complement 14 and is combined with the spacer sequence 6 on the surface of the first nanoparticle 3. In FIG. 1e, the first nanoparticle 3 is then irradiated with light, which is absorbed by the first nanoparticle 3 due to its plasmonic or material properties and is converted into heat. The heat is emitted to the environment of the first nanoparticle 3 and, in the area of the original 13 and the newly synthesized complement 14 hybridized with it, the heat is sufficient for the original 13 to denature from the complement 14. The original 13 is now free again, as shown in FIG. 1f, so that it can bind to a further primer sequence 5 and further nanoparticle-bound complements 14 can be synthesized in further cycles of the method. This produces a linear increase in the concentration of the complements 14 with an increasing number of cycles.

(70) In one embodiment of the method, after the extension of the primer sequence 5 on the surface 4 of the first nanoparticles 3, wherein a nanoparticle-bound complement 14 is produced, a free reverse primer 15 is used, which binds to the 3′-end of the complement. It is shown in FIG. 1g that the already synthesized complement 14 with the sub-sequences A and B, which is combined via a spacer sequence 6 and an abasic modification 7 on the surface of the first nanoparticle 3, hybridizes with a reverse primer 15 B′ that was previously free in the sample 12. The primer 8 has the sequence B′ and is combined with the sub-sequence B of the complement 14. Starting from the primer 8 with the sequence B′, the DNA polymerase synthesizes a copy of the original 13. The synthesis takes place only up to the abasic modification 7, as this cannot be overwritten by the polymerase 11. It is also shown in FIG. 1g that the original 13 has bound to a further primer sequence 5 A on the surface of the first nanoparticle 3 and a DNA polymerase 11 starting from the primer sequence 5 A synthesizes a further complement 14. The original 13, the copy of the original 13 and the two complements 14 combined with the first nanoparticle are shown in FIG. 1h. A subsequent denaturing through excitation of the first nanoparticles 3 leads to the original 13 and its copy becoming free. Both the original 13 and also its copy can thereby serve in subsequent steps of the method as a template for amplification. After a waiting period, which is possibly necessary for the hybridization of the original 13 and copies of the original 13 with primer sequences 5 A on the first nanoparticles 3 and free primers 8 B′ with primer sequences 5 already elongated on the first nanoparticles 3, the next cycle of the method can be carried out with a further excitation of the first nanoparticles 3. The cycle is preferably repeated until a sufficient number of extended primer sequences 5 are located on the first nanoparticles 3 and/or a sufficient number of copies of the original 13 are located in the sample 12, in order to be able to carry out a detection of the completed amplification or the presence of the original 13 in the sample 12. Through a free primer 8 B′, as shown in FIGS. 1g and 1h, an exponential amplification of the original 13 is possible. In FIGS. 1a to 1f, without this free primer 8, however, only a linear amplification of the nanoparticle-bound complement 14 can be achieved.

(71) FIG. 2 shows a structure that is suited for carrying out the method according to the invention. The structure contains a light source, which is implemented in this example as a laser 16, and a two-dimensional mirror scanner 17, which can guide light from the laser 16 to the sample 12. The two-dimensional mirror scanner 17 can thereby deflect the laser beam in two dimensions. The denaturing in the sample 12 takes place in this structure in that a laser beam is focussed on a part of the sample 12. In the course of the method the laser beam is deflected so that it impinges on different parts of the sample 12. In the example shown in FIG. 2, the laser beam is deflected by the mirror scanner 19 in such a way that the laser beam travels linearly over the reaction volume 2, in which the sample 12 is located. The path covered by the laser beam is shown in dotted lines in FIG. 3 in the sample 12. Due to the fact that at each time point of the method only parts of the sample 12 are excited, lasers 16 with a lower power can be used. As excitations of less than a microsecond suffice in order to denature DNA with the aid of optothermally heated nanoparticles 9, in the case of typical focus diameters of a laser 16 from approximately 10 to 100 μm, a laser beam with a speed of approximately 10 to 100 m/s can scan the sample 12 and thereby lead to a denaturing of the DNA at each point over which the laser beam travels. This facilitates a very rapid scanning also of large sample volumes. The complete scanning of a surface area of 1 cm.sup.2 takes only 128 ms, e.g. with a focus diameter of 78 μm and 128 lines at a line distance of 78 μm and a line length of 1 cm, with a speed of the scanning laser beam of 10 m/s. If the volume has e.g. a depth of 10 mm, a volume of 1 ml can be processed (for this it must of course be ensured, inter alia, that the intensity of the excitation is sufficiently high over the whole depth). This is advantageously substantially shorter than would generally be required by a denaturing step through global heating. With optical elements such as e.g. a mirror scanner 17 shown in FIG. 2, and so-called F theta lenses, a good homogeneity of the focus quality and size can be achieved over the whole scanned sample 12. Alternatively to a continuously emitting laser 16, a pulsed laser 16 or a thermal radiator can also be used.

(72) In the embodiment of the method shown in FIG. 1, first nanoparticles 3 of gold with a diameter of 60 nm are functionalized with oligonucleotides 4 ID1 (according to J. Hurst et al., Anal. Chem., 78(24), 8313-8318, 2006, the related content of which is part of the present disclosure by virtue of reference thereto). After functionalization and 6 washing steps, the first nanoparticles 3 are present in a concentration of 200 pM in a PBS buffer (5 mM PBS, 10 mM NaCl, 0.01% Tween 20, pH 7.5). The amplification reaction is carried out in a total volume of 10 μl in 100 μl sample tubes 18 (2 μl Apta Taq Mastermix 5× with MgCl2 (obtained from Roche), 1 μl NaCl 450 mM, 1 μl MgCl.sub.2 90 mM, 1 μl Tween 20 1%, 2 μl water, 1 μl of the functionalized first nanoparticles 200 pM, 1 μl oligonucleotide 4 ID2 5 μM as a dissolved reverse primer and 1 μl oligonucleotide ID3 as original 13 to be amplified). The concentration, to be determined, of the original 13 in the total volume of 10 μl, e.g. 0.1 fM of the oligonucleotide ID3 dissolved in water with 100 nM oligonucleotide 4 ID4 (oligonucleotide ID4 hereby serves for the saturation of surfaces, e.g. during the maintenance of the original 13 before the reaction.) As shown in FIG. 3, the sample tubes 18 are brought in a glass cuvette 19 in a water bath 20 to a temperature of 64° C., which constitutes both the annealing temperature and the elongation temperature. The water bath 20 serves, besides tempering, also for improved introduction of the laser 16 into the non-planar surface of the sample tubes 18. The water in the water bath 20 allows the refractive index difference between the outside and the inside of the sample tubes 18, filled with PCR reaction mix, to be reduced and to therefore prevent a refraction of the laser beam and hence a negative influence on the focus quality and sharpness. The coupling of the laser 16 is thereby advantageously improved. The laser 16 which is used to excite the nanoparticles is a frequency-doubled diode-pumped Nd:YAg-Laser (CNI Lasers Inc.), which is focused, with an output power of 2.5 W with a F-Theta lens (Jenoptik, focal length 100 mm) behind a mirror scanner 17 (Cambridge Technologies, Pro Series 1) into the sample tubes 18 in the water bath 20 (focus diameter approximately 20 μm). The mirror scanner 17 allows the focus to move line by line through the sample tubes 18, as also already shown in FIG. 3, and thus allows the whole PCR reaction volume to participate in the optothermal amplification. For each sample tube 18, 680 lines with a distance of approximately 12 μm, with a line speed in the sample tubes 18 of approximately 10 m/s, are covered with the focus. This corresponds to a cycle in the first sample tube 18. Subsequently all other sample tubes 18 are travelled over one after the other, so that each sample tube 18 has undergone a cycle. After a waiting period, which can be selected differently in each sample tube 18, the next cycle is started. This is repeated as often as needed with differences for each sample tube 18.

(73) FIG. 6 shows data for five different sample tubes, which contain as a starting concentration of the original ID3 in each case 0.1 fM. In the first sample tube a total of 200 cycles are carried out with a waiting time of 3 s between the individual cycles, in the second sample tube 120 cycles at 5 s, in the third sample tube 90 cycles at 6.6 s, in the fourth sample tube 60 cycles at 10 s and in the fifth sample tube 45 cycles at 13.3 s. This is shown in FIG. 6b. The script below the diagrams indicates in each case the waiting time. The total duration from the first to the last cycle is 10 minutes in each of the five sample tubes. This is shown in FIG. 6a. In order to determine the total amplification through the optothermal amplification reaction, after the end of the amplification reaction 1 μl of the sample is removed from each sample tube and diluted in 99 μl water. From this dilution or thinning, 1 μl is introduced into an amplification reaction to be quantified (real-time PCR) in order to determine the concentration of the copies of the original there that were produced in the different sample tubes by the optothermal amplification reaction. This dilution serves for possibly inhibiting or interfering content substances from the optothermal amplification reaction being diluted too greatly, so that they can no longer interfere in the subsequent quantifying amplification reaction. The quantifying amplification reaction is performed in a LightCycle II (Roche). Here, there is a cycle of 10 s denaturing at 94° C., 10 s annealing at 62° C. and 10 s elongation at 72° C. At the end of the 72° C. step, the measurement of the fluorescence is also carried out. Prior to the start of the first cycle, a once-only denaturing step takes place at 94° C. for 30 s. Besides 1 μl of the diluted copies of the original from the optothermal amplification reaction, 10 μl reaction volume for the quantifying amplification reaction contains 2 μl Apta Taq Mastermix 5× with MgCl.sub.2 (obtained from Roche), 2.8 μl water, 2 μl oligonucleotide 4 ID5 1 μM as dissolved forward primer, 2 μl oligonucleotide 4 ID6 1 μM as a dissolved reverse primer and 0.2 μl SYBRGreen 100× as intercalating colour dye in order to make the PCR product detectable during the real-time PCR. An additional standard curve, which is determined with a diluting or thinning series of known concentrations of oligonucleotide ID3 as original for the quantifying amplification reaction, allows the subsequent quantification of the copies used into the quantifying amplification reaction. The total amplification is thereby determined that was produced during the optothermal amplification reaction in the different sample tubes. This is shown in FIG. 6d. It can clearly be seen here that the total amplification, despite equal process time (in each case 10 minutes, see FIG. 6a), with increasing cycle duration (and thereby decreasing number of cycles), greatly decreases. Assuming that over the whole amplification reaction the amplification factor per cycle remains constant, the yield per cycle g can be calculated from Equation (2). The thus determined g is shown in FIG. 6c. It can clearly be seen here that g increases with increasing cycle duration. Despite the decreasing g with decreasing cycle duration, the total amplification with the same process duration increases with decreasing cycle duration.

(74) FIG. 4 shows the idealized temperature profile of a conventional PCR (dotted line) with a cycle duration of t.sub.ch=15 s. A constant slope of 5 K/s was assumed for the temperature flanks. In contrast, one embodiment of the PCR method according to the invention, with a cycle duration t.sub.ch=2 s, is shown with a constant slope of the temperature flanks of 3000K/s, as can be achieved for example through optical excitation of nanoparticles according to the invention (solid line; for better legibility, the temperature profile was displaced by 1° C. downwards).

(75) FIG. 5 shows the amplification factor N.sub.k/N.sub.0 as a function of the time for different parameters. The pointed line shows the amplification of a typical conventional PCR with a cycle duration of t.sub.ch=25 s and a yield per cycle of g.sub.h=100%. The dotted line shows the amplification in a preferred conversion according to the invention with

(76) $g_{i} = 2 5 %, x = 4$ $t_{c_{i}} = \frac{t_{c_{h}}}{x} = 6.25 s$

(77) The solid line shows another preferred conversion according to the invention with g.sub.i=100%, x=2 and

(78) $t_{c_{i}} = \frac{t_{c_{h}}}{x} = 12.5 s$

(79) The features disclosed in the above description, the claims and the drawings can be significant both individually as well as in any combination for the realisation of the invention in its different embodiments.

REFERENCE SYMBOL LIST

(80) 1 Nucleic acid 2 Reaction volume 3 First nanoparticles 4 Oligonucleotide 5 Primer sequence 6 Spacer sequence 7 Abasic modification 8 Forward primer 9 Nanoparticle 10 Filling molecule 11 DNA polymerase 12 Sample 13 Original; amplicon 14 Complement 15 Reverse primer 16 Laser 17 Mirror scanner 18 Sample tube 19 Glass cuvette 20 Water bath
Sequences
(from 5′ to 3)
/iSp9/=abasic modification “Spacer9”
[sequences as per the original German text]

PCR method using irradiation of nanoparticles

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