The invention here concerns a method for increasing the uniformity of nucleic acid amplification A method is described to add stopping periods during the ramp step(s), which minimizes the effect of temperature overshoot or undershoot Many cyclers' algorithms cause the target temperature to be passed during ramp and then returned to within a short penod of time This overshoot or undershoot can cause non-uniformity between reactions in a micro- titer plate The addition of stopping peπods reduces the effects of overshoot or undershoot and increases uniformity between different reactions wells on a plate

Background

Nucleic acid amplification is a routine, high-throughput method carried out by procedures such as polymerase chain reaction (PCR), rtPCR, cycle sequencing and hgase chain reaction These methods follow the same general principle of thermal cycling involving alternating steps of melting the double-stranded DNA to its single-stranded form and then annealing primers complementary to the smgle-stranded or template DNA to initiate the amplification phase In thermal cycling, the temperature of the sample/ reaction mixture is purposely transitioned during the so-called ramping step and maintained accurately to a configured series of temperature steps with the temperature cycle repeating for a desired number of times The higher temperatures are required for melting or denaturing the double-stranded DNA and lower temperatures are required for the hybridisation or annealing step

For obtaining high yield and high selectivity, the optimal temperatures must be determined and carefully regulated during the reaction The temperatures for denaturation and annealing differ according to several parameters including length and nucleotide composition of the nucleic acids, enzymes and other components in the reaction mixture For assays, which require the analysis of multiple reactions conducted simultaneously, precise temperature regulation of each individual reaction mixture is important for obtaining accurate, reproducible results

Instruments such as thermal cyclers may be set up to perform multiple reactions simultaneously Micro-titer plates or tubes are placed on a thermal block, which is cooled or heated according to the configured series of temperatures required for each reaction step Thermal cyclers use thermoelectric (Peltier) modules, among other mechanisms, to heat and cool a thermal block from which heat is transferred to the thin-walled vessels (i e microfuge tubes) or chambers (wells m microtiter plates) Thermal performance is the key for any thermal cycler to produce high quality amplicons

The problem encountered when large-scale amplification is conducted m a high-throughput system is the lack of thermal uniformity across all samples In particular, in rectangular arrays (i e micro-titer plates) containing multiple chambers such as those used in thermal cyclers

There are several reasons for the lack of thermal homogeneity Fluid temperatures withm the wells of a micro-titer plate are inferred by measuring the thermal block temperature and predicting the fluid temperature using an algorithm Reaction volume and plate loading will change the thermal mass of the system and limits the ability of the instrument to accurately predict fluid temperatures within any particular well

According to the art, temperature differences occur between chambers located at the outer edge of a rectangular array and chambers located internally when placed on such a thermal block Unlike wells located internally, wells at the outer edge are not surrounded by other wells This introduces thermal asymmetry that can challenge the ability of the thermal block to control fluid temperatures within the well This is referred to in the art sometimes as the "edge effect" (Caitlm Smith, Conventional Thermal Cyclers The Next Generation)

The difference in amplification efficiency between sample reactions is especially prevalent, as noted in the art, when micro-titer plates are only partially filled with reaction mixtures and the rest of the wells are left empty Filling these empty wells with fluids such as water reduced the variability of amplification somewhat, however, the differences were not eliminated Additionally, filling empty wells with water may be time consuming and burdensome

Also according to the art, lack of thermal homogeneity is attributed to a fast ramping rate found in the newer models of thermal cyclers (Frey et al , PCR-amphfication of GC-nch regions 'slowdown PCR', 2008, 1312-1317) The ramping rate is the speed at which the thermal cycler cools and heats the thermal block to the configured series of temperature steps, i e , denaturation and annealing temperatures The new generation of instruments achieve faster ramp rates primarily by reducing the mass of the thermal block, increasing the power of the thermoelectric (Peltier) modules and increasing the transient temperature overshoot of the block

The consequences of the increased ramping rate are greater overshooting and/or the undershooting of the configured temperatures The term overshooting here describes temperatures, which reach above the denaturation temperature as configured in the thermal cycler prior to the start of a reaction Similarly, in the case of undershooting, the temperature of the reaction mixtures reaches those temperatures below the configured annealing temperature

A result of the reduced block mass, increased ramp rates and transient overshoot in fast cyclers is that the ability to control for reaction volume variation and partial filling of the plate is compromised further resulting in greater potential for overshoot or undershoot m some samples As the thermal cycler continues to heat or cool the block such that the entire block approaches the configured temperature, certain samples may become over-cooled or overheated It is also possible that certain samples with larger volumes require a longer period of time to reach a particular temperature

The consequences of undershooting and/or overshooting are inefficient amplification of the nucleic acids or perhaps even failure of any amplification to take place at all and reduced selectivity In the case of overshooting of the denaturation temperature, the enzyme is exposed to higher temperatures, which reduce its efficacy During undershooting of annealing temperatures, lower temperatures promote non-specific hybridisation of nucleic acids resulting in unwanted products and ultimately inaccurate results

Minimizing undershooting and/or overshooting may be particularly difficult when the thermal block is very large The thermal cycler is unable to transition and maintain temperatures for heating and cooling large numbers of samples As mentioned before, the thermal block may be exposed to elements of the instruments and the atmosphere, which influence the local temperature of the heating block Description of the Invention

Accordingly, the problem underlying the present invention was to provide a method for amplifying a nucleic acid with increased consistency This problem is solved by at least one of several possible steps Step a) can be used with steps b) through d) individually or in any combination

The invention comprises the following steps a) One of these possible steps for solving the problem is the introduction of "stopping periods" at various junctures during the rampmg step The term stopping period describes a period of time during which the temperature is not changed during the rampmg step In other words, the thermal cycler does not cool or heat the thermal block during the stopping period within the ramping step Stopping periods are introduced particularly when the sample temperature approaches the denaturing or "lowered" denaturation temperature and the annealing or "raised" annealing temperature, respectively (see below for exact definitions of "lowered" and "raised")

The advantage of having a stopping period is that it prevents the overshooting and/or undershooting of the denaturation or lowered denaturation temperature and annealing or raised annealing temperature and to keep the temperatures uniform in all sample wells The denaturation or lowered denaturation temperature and the annealing or raised annealing temperature are incrementally reached thereafter While the stopping period does not eliminate the overshooting and or undershooting, it can mitigate the effects thereof For instance if the denaturation temperature is 95°C an overshoot of 4°C can bnng the sample temperature to 99°C which can irreversibly reduce enzyme efficacy By introducing a stopping period at 90°C, the overshoot at the stopping period would be 94°C The smaller temperature change from 9O ⁰ C to the denaturation temperature of 95°C does not exhibit significant overshoot because a high ramp rate is not achieved with such a small temperature change b) In one embodiment, the method comprises the use of a "lowered denaturing temperature" In order to obtain this, first, the empirical denaturing temperature of the nucleic acid is determined Once determined, the actual denaturation step is carried out at a temperature lower than the empirical temperature (lowered denaturation temperature) The new "lowered" denaturation temperature diminishes the likelihood of the enzyme (polymerase) being repeatedly exposed to temperatures that reduce its activity Therefore, the lowered denaturation temperature indirectly increases amplification efficiency c) In one embodiment, the method comprises the use of a "raised annealing

temperature" To obtain a raised annealing temperature, the empirical annealing temperature of the primer and template DNA is determined first Once determined, the reaction is, however, carried out at temperatures higher than the empirical temperature (raised annealing temperature) to prevent non-specific annealing between pπmers and templates Consequently, highly specific reactions are selected for by raising the annealing temperature d) In one embodiment, the method comprises the use of a rampmg rate lower than the optimal rate for carrying out the amplification reaction In this case, the optimal rampmg rate of the reaction is determined through testing amplification efficiency and selectivity A rampmg rate is then selected m the thermal cyclers for a setting lower than the optimal rate e) In one embodiment, the method comprises the use of an initial incubation of the reaction mixture at a high temperature to activate the polymerase

Step a) can be earned out alone whereby one stopping period is introduced before the reaction reaches the denaturation or lowered denaturation temperature and another stopping period is introduced before the thermal block reaches the annealing or raised annealing temperature Preferably, steps a) and d) are carried out together More preferably, steps a) and c) are carried out together Most preferably, all four steps are carried out together

In one embodiment, the present invention provides a method for amplifying a nucleic acid with

• a denaturation temperature for dissociating the nucleic acid,

• an annealing temperature for nucleic acid hybridization, • an extension step for enzymatically extending the primers, and

• a ramping step during which the temperature of the reaction mixture cycles between the denaturing temperature and the annealing temperature, comprising introducing a stopping period during said ramping step(s) during which the temperature is not changed for a given time

It is understood that the method contemplates cycling protocols which have only two temperatures as well as cycling protocols that have more than two temperatures In one embodiment, the method contemplates distinctly different annealing and extension temperatures In another embodiment, the extension step is performed at the same temperature as the annealing temperature

In another embodiment, the nucleic acid is double stranded In another embodiment, the method further comprises an initial incubation performed to activate a polymerase

In another embodiment, the method comprises

a) determining an empirical/optimal rampmg rate at which the temperature of a reaction mixture cycles between a denaturing temperature and an annealing temperature, and

b) carrying out a ramping rate that is below the empirical/optimal rampmg rate

The inventor has surprisingly found a process for obtaining homogeneous reproducible amplification of multiple parallel reactions undergoing thermal cycling Significantly, reactions can be carried out in half-filled plates without having to fill the entire plate with water, which can be laborious Additionally, the "edge effect" as it is known in the art can be avoided

Another advantage of this method is that it may be used in any type of thermal cycler, but especially in older cyclers, which do not contain new additions, such as, sensors for small samples to maintain uniform temperatures or lids for reaction vessels that prevent evaporation Thus, this method is significantly more cost effective than buying new PCR equipment The invention may be adapted to any kind of amplification procedure for nucleic acids where thermal cycling is involved The method according to the invention may be conducted for only a few cycles or for all cycles of a PCR application

In one embodiment, the present invention relates to the use of method for maintaining a homogenous lowered denaturation temperature and a homogenous raised annealing temperature of the reaction mixture

Determination of Empirical Denaturation Temperature and Empirical Annealing Temperature The annealing and denaturation temperatures of a double-standed nucleic acid are determined empirically

Several formulas are used to predict the melting temperature (denaturation temperature) (T _n , ) of a DNA molecule in the art Factors influencing the temperature include the length of the molecule and most particularly the amount of guanine and cytosine present or as it is referred to the "GC" content of the nucleic acid duplex As the base-pairing between guanines and cytosmes are generally stronger than bonds formed by adenosines and thymines, a high GC content would mean a high T _m value to melt the double-stranded DNA The following three equations are preferred to predict the melting temperature of a given nucleic acid at Wallace Rule (2 + 4 Rule)

The formula below is used to determine the T _m value for very short sequences (up to about 15 base pairs) As part of the calculation, 2 ^C C for each AT-pair and of 4°C for each GC-pair is added to the melting temperature of a double stranded nucleic acid (R B Wallace, J Schaffer, R F Murphy, J Bonner, K Itakura, Nuc Acids Res 1979, 6, 3543)

T _m = 2°C x (A + T) + 4°C x (G + C) bi Calculation by GC-content

The GC content is taken into account in the following equation and is applicable for long sequences (P M Howley, M F Israel, M-F Law, M A Martm, M A J Biological Chemistry 1979, 254, 4876, R Teoule, H Bazm, B Fouque, A Roget, S Sauvaigo, Nucleosides & Nucleotides 1991, 10, 129)

T _m = 81 5 + 0 41 (%GC) + 16 6 log c(M+) - 500/n - 0 61 (%F) - 1 2 D where %GC Percentage of GC-pairs

c(M+) = Concentration of mono-valent cations

n = Number of nucleotides

%F = Percentage of formarmde in the buffer

D - Percentage of mismatches

c) Nearest-Neighbor Method

Interaction between adjacent bases along the backbone of the helix also significantly influences the melting temperature for a DNA molecule The meltmg temperature calculation is based on the thermodynamic relationship between adjacent bases and is applicable for nucleic acid sequences 20 to 60 bases long (W Rychhk, W J Spencer, R E Rhoads, Nuc Acids Res 1990, 18, 6409-6413)

Tm = [(1000 x dH/(A + dS + R x In (C/4))] - 273 15 + 16 6 x log c(K+)

where H = Sum of the enthalpies of all pairs

dS = Sum of the entropies of all pairs

A = -10 8 cal, Entropy of helix formation

R = I 984 cal/grad x mol, Gas constant

C = Oligonucleotide concentration (250 pmol/1)

c(K+) - Concentration of potassic ions in the ohgo solution (50 mmol/1)

The preferred formulas give only a theoretical value of the denaturation temperature, which is particularly useful for PCR methods carried out on long stretches of DNA For real-time PCR, however, the denaturation temperature need not be determined using these formulas It is know in the art that 95°C, for example, is more than sufficient for denaturing short pieces of DNA (< 1000 bp) The starting value for realtime PCR for short pieces of DNA may, therefore, be 95 ⁰ C This value is further optimised through testing a range of denaturation temperatures (usually + 2°C) and determining via the highest amplification efficiency the optimal temperature, which is referred to as the empirical denaturation temperature The adjusted value derived experimentally is used to determine the lowered denaturing temperature, which is below the empirically determined denaturation temperature

Too high an annealing temperature results in no hybridisation between the primer and template and too low a temperature results in non-specific products The annealing temperature is determined using the following preferred formula ("Optimization of the annealing temperature for DNA amplification in vitro" by W Rychhk, W J Spencer and R E Rhoads Nucleic Acids Res 1990 Nov 11 , 18(21 ) 6409- 12)

Ta - 0 3 x Tm (primer) + 0 7 Tm (product) - 14 9

where, Tm(pπmer) = Melting Temperature of the primers

Tm(product) ^~ Melting temperature of the product

As in the case with the denaturation temperature, the value obtained is a theoretical value and is further optimised by carrying out several amplifications over a range of annealing temperatures (usually ± 2°C) The optimized value, which is referred to as the empirical annealing temperature, is then used to determine the raised annealing temperature The raised annealing temperature is above the empirically determined annealing temperature

Determination of Optimal Ramping Rate

The best approach to determine the optimal ramping rate is to test the amplification efficiency and specificity using different rampmg rates as known m the art Accordingly, specificity and efficiency are measured starting at the highest technically achievable rampmg rate, which depends on the particular model of the themal cycler being employed, and stepwise reducing the rate The variety of models produced by different firms are each programmed for a different "highest setting" of the rampmg rate

Additionally, other parameters, such as the empirical denaturation and empirical annealing temperatures, are maintained for maximal amplification efficiency and specificity The actual ramping rate used according to this invention may be lower then the optimal ramping rate to reduce overshooting and/or undershooting, thus, assuring equivalent amplification across all sample reactions The thermal cycler is then configuied for the new slower ramping rate, and consequently, the instrument requires a longer amount of time to reach the configured temperatures

Preferred embodiments of the invention are described with reference to the dependent claims

1 In one embodiment, the stopping period during the rampmg step is introduced when the reaction mixture temperature is within 1°C to 10 ⁰ C of the denaturing or lowered denaturing temperature For example, if the denaturing or lowered denaturing temperature is determined to be 93 ⁰ C, then a stopping period may be introduced at any point between 92°C and 83°C During the stopping period, the ramping step is completely halted It is preferred that a stopping period is introduced 2 ⁰ C to 7 ⁰ C below that of the denaturing or lowered denatuπng temperature Most preferably, a stopping period is introduced withm 3 ⁰ C to 5 ⁰ C of the denaturing or lowered denaturing temperature In another embodiment, the stopping period during the ramping step is introduced when the reaction mixture temperature is below 1°C to 10 ⁰ C of the lowered denatuπng temperature In another embodiment, the stopping period during the rampmg step is introduced when the reaction mixture temperature is below 2°C to 7°C of the lowered denaturing temperature In another embodiment, the stopping period during the rampmg step is introduced when the reaction mixture temperature is below 3°C to 5°C of the lowered denaturing temperature In another embodiment, the stopping period during the ramping up step is introduced wherein the annealing temperature is increased by I ⁰ C to 1O ⁰ C from the empirical annealing temperature In another embodiment, the method stopping period during the ramping up step is introduced wherein the annealing temperature is increased by 2°C to 7°C from the empirical annealing temperature In another embodiment, the method stopping period during the ramping up step is introduced wherein the annealing temperature is increased by 3°C to 5°C from the empirical annealing temperature

Similarly, during the annealing phase, the stopping period during said ramping step is introduced when the reaction mixture temperature is within I ⁰ C to 10 ⁰ C of the annealing or raised annealing temperature For instance, if the annealing or raised annealing temperature is 58 ⁰ C, then a stopping period may be introduced at any point between 59°C and 68 ⁰ C Again, as in the case with the denaturation step, during the stopping period, the ramping step is completely halted A preferred embodiment is when a stopping period is introduced 2°C to 7°C above that of the annealing or raised annealing temperature Most preferably, a stopping period is introduced 3 ⁰ C to 5°C above of the annealing or raised annealing temperature

The duration of said stopping period is between two to ten seconds In other words, the temperature of the thermal block is neither increased nor decreased for the given amount of time, which may be anywhere between two to ten seconds Preferably, the duration of the stopping peπod is between three to seven seconds More preferred, the duration of the stopping period is between four to six seconds, and most preferred, the duration is five seconds

Similarly, as a result of the insertion of the stopping periods and the reduction of the ramping rate, the duration of the ramping step is increased by 1 to 60 seconds as compared ramping step known in the art Preferably, the ramping step is increased by 1 to 20 seconds, and most preferably, it is increased by 2 to 10 seconds

As discussed above, the basis of the "lowered" denaturation temperature is the empirical temperature The preferred equations above give an approximate temperature, which is further optimised through experimentation The lowered denaturing temperature is decreased by 1°C to 10 ⁰ C from the empirical temperature Thus, if the empirical temperature is given as 95°C, the lowered denaturation temperature would be between 94°C and 84°C It is preferred that the lowered denaturation temperature is decreased by 2°C to 7°C, and most preferred, the temperature is decreased by 3°C to 5 ⁰ C from the empirical denaturing temperature

Similarly, the "raised" annealing temperature is based on the empirical annealing temperature as determined initially by the preferred equation given above and optimised through experimentation Preferably, the raised annealing temperature is increased by 1°C to 10 ⁰ C above the empirical annealing temperature More preferably by 2°C to 7°C and most preferably by 3 ⁰ C to 5°C from the empirical annealing temperature Preferably, the initial incubation as described in step e) above is performed at a temperature between 90 ⁰ C and 98 ⁰ C to activate the polymerase More preferably, the incubation is conducted between 93 ⁰ C and 95 ⁰ C

With regard to reaction vessels, any vessel may be used to contain the reaction mixture including gas capillaries, plastic capillaries, optical tubes and micro-titer plates, m particular partially loaded micro-titer plates and partially loaded capillary systems

In one embodiment, the present method can be used for analytical purposes, which use thermal cycling An example of this could be in vitro diagnostic testing for viral pathogens GMO (Genetically Modified Organisms) testing of food products may also be included Another example could be the testing of oncogenes such as K-ras in tumor cells where only a small subset of tumor cells displays a mutation Depending on the presence of the mutation in the cells, the course of therapy is determined

Preferably, the nucleic acid is amplified through a polymerase chain reaction (PCR), which comprises the following variations on the basic technology but is not limited to only these variations AFLP (Amplified fragment length polymorphism) AFLP PCR, Allele-specific PCR, AIu PCR (for AIu repeats), Assembly PCR, Asymmetric PCR, Colony PCR, Heavy Methyl Assay, Hehcase-dependent Amplification PCR, Hot Start PCR, Immuno PCR, Inverse PCR, In Situ PCR, ISSR (Intersequence-specific) PCR, Late PCR, Long PCR, MethyLight assay, Methylation-specific PCR (MSP), Nested PCR, Reverse Transcriptase-PCR, real-time PCR, Random Amplification of Polymorphic DNA (RAPD-PCR), Quantitative-PCR and multiplex applications thereof

Alternatively, the nucleic acid is amplified through cycle sequencing Cycle sequencing is a procedure through which DNA fragments are generated Fluorescent dyes are employed to analyse the samples of DNA fragments using an automated DNA sequencing machine PCR methods are used to produce the DNA fragments Thus, this application also utilizes thermal cycling to denature the double-stranded DNA and anneal a primer to the single-stranded template Here, also uniform concentrations of DNA fragments must be obtained from multiple samples for accurate results Alternatively, the nucleic acid is amplified through an alternative method, such as the ligase chain reaction In the ligase chain reaction, the nucleic acid is amplified only when there is no mismatch in the junction between the primers allowing a thermostable ligase to join the primers Thereafter, PCR amplification may be carried out as usual using the conjoined primers

As in a typical PCR application, in a ligase chain reaction, there is thermal cycling to allow for the denaturation of the DNA, annealing of the primers, and ligation All of these steps can be carried out in a high-throughput manner Similar to PCR applications, the need for homogenous temperatures in all samples is critical for accurate results.

In a further aspect, the invention refers to a computer program, which is designed to carry out the method on a computer-driven thermal cycler The computer program may be installed on a typical thermal cycler and the cycler carries out the invention On the other hand, a computer program may be installed such that the program in the older thermal cyclers can be circumvented if the computer program according to the invention is launched and implemented at the behest of the user

A further aspect of the invention refers to a thermal cycler, which is programmed to amplify a double stranded nucleic acid More specifically, the thermal cycler is programmed such that stopping periods, slower ramping rates, and adjusted denaturing and annealing temperatures are automatically calculated and implemented once the appropriate data has been entered into the instrument In doing so, the operator of the thermal cycler does not have to manually program the instrument

Figures

Figures 1 to 10 show data described under examples below The figures shows the graphic results of a duplex real-time PCR assay, which amplifies methylated Septm 9 and an internal control, beta-actm (HB 14) The x-axis of the graph indicates the number of cycles (Cycle) completed in the assay whereas the y-axis shows the fluorescent output minus the background (dRn)

Figure 1

In this experiment, all the wells in the micro-titer plate were filled with PCR mastermix The PCR reaction present in well Al is depicted as the bold line in the graph in this figure and in the other figures The conditions of the PCR are the following fast ramping rate (1 5°C/sec for cooling and 1 5°C/sec for heating) activation at 93°C for 30 mm , denaturation temperature at 95°C, annealing temperature at 57°C The curves show a generally similar Ct (threshold cycle) for all samples, but a somewhat varying curve shape for all samples

Figure 2

The landscape plot shows the distribution of fractional cycle number (FCN) and maximum ratio (MR) of a full micro-titer plate The center of the plate shows an MR value of approximately O i l and an FCN of 41 6 The edges of the plate differ from the values in the middle FCN and MR values are characteristic numbers, which describe the curve shape of the amplification curve (Sham, E , Nucleic Acids research, 2008) Efficient PCR amplifications show early FCN values and higher relative MR values

Figure 3

The figure shows the amplification efficiency in the wells of columns 2 and 10 In the first column and between columns 2 and 10, the wells have been filled with water whereas the columns to the right of column 10 have been left empty The conditions of the PCR are the following fast ramping rate, activation at 93 ⁰ C for 30 mm , denaturation temperature at 95°C, annealing temperature at 57°C As can be seen from the graph, the wells located in column 10 have highly variable curve shapes and Ct values while the curve shapes and Ct values in column 2 are more uniform Figure 4

The figure shows the curve shapes in the wells of columns 2 and 10 In the first column and between columns 2 and 10, the wells have been filled with water whereas the columns to the right of column 10 have been left empty The conditions of the PCR are the following fast ramping rate, activation at 93°C for 30 mm , denaturation temperature at 93°C, annealing temperature at 57°C The graph shows less variability in curve shape as compared to the results of Figure 3

Figure 5

The figure shows the curve shape in the wells of columns 2 and 10 In the first column and between columns 2 and 10, the wells have been filled with water whereas the columns to the right of column 10 have been left empty The conditions of the PCR are the following fast ramping rate, activation at 93°C for 30 min , denaturation temperature at 95°C, annealing temperature at 58°C The amplification curves are significantly more homogeneous than the curves obtained in Figure 3 Additionally, the curves have shifted to the left and moved up indicating increased uniformity

Figure 6

The figure is a preferred embodiment and shows the curve shapes in the wells of columns 2 and 10 In the first column and between columns 2 and 10, the wells have been filled with water whereas the columns to the right of column 10 have been left empty The conditions of the PCR are the following slow ramping rate (1 5°C/sec for cooling and 0 8°C/sec for heating), activation at 93°C for 30 mm , denaturation temperature at 95°C, annealing temperature at 57°C, and a stopping period at 62°C for 5 seconds The amplification curves are significantly more uniform here as compared to those in Figure 3

Figure 7

The figure is a preferred embodiment and the curve shapes in the wells of columns 2 and 10 In the first column and between columns 2 and 10, the wells have been filled with water whereas the columns to the right of column 10 have been left empty The conditions of the PCR are the following slow ramping rate, activation at 93°C for 30 mm , denaturation temperature at 93 ⁰ C, annealing temperature at 58°C, and a stopping period at 62°C for 5 seconds Here, the amplification curves are more homogeneous in comparison to the amplification curves shown in Figure 3

Figure 8

The figure shows the amplification in the wells of columns 2 and 10 with the addition of a brake at both the annealing and denaturing temperatures In the first column and between columns 2 and 10, the wells have been filled with water whereas the columns to the right of column 10 have been left empty The conditions of the PCR are the following fast ramping rate, activation at 93°C for 30 mm , denaturation temperature at 95°C, annealing temperature at 58°C, and the addition of two stopping periods at 90 ⁰ C for 5 seconds and at 62°C for 5 seconds The amplification curves have significantly improved as compared to the amplification curves found in Figure 3

Figure 9

The figure depicts the amplification of all the wells in a micro-titer plate fully loaded with sample reactions The conditions of the real-time PCR assay is given as the following slow ramping rate, activation at 93°C for 30 mm , denaturation temperature at 93°C, annealing temperature at 58 ⁰ C, and a stopping period at 62°C for 5 seconds The amplification curves in this figure are much more homogeneous in curve shape as compared to the ones obtained in Figure 1

Figure 10

The figure shows the landscape plot of a PCR plate with the improved conditions of PCR as given in Figure 9 As can be seen, the distribution of MR and FCN across the plate is more uniform as compared to the plot in Figure 2