Microcontroller based Low cost PCR Thermal Cycler for DNA Amplification

Microcontroller based Low cost PCR Thermal Cycler for DNA Amplification

Sidhant Sanjay Kulkarni Asst.Prof., Dept. of E& Tc SRES, COE, Kopargaon

Abstract

Polymerase Chain Reaction is a common molecular biology technique for amplifying a specific sequence De-oxyrhibose nuclic acid molecule for sequencing or detection purposes. PCR is a cyclical reaction in which the number of DNA molecules of interest is doubled with each repeat of the reaction.

Keywords: DNA, Polymerase, PCR, temparature Cycles

1. Introduction

   PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA. Previously, amplification of DNA involved cloning the segments of interest into vectors for expression in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient so that untold numbers of copies can be made of the DNA.

   Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the

   use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions [1].

   There are three major steps involved in a PCR as shown in Fig.1 for amplification of DNA. These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler [2].

   Fig. 1. Steps involved in amplification of DNA by PCR .

   Thermal Cycler is a device which rapidly heats and cools the test tubes containing the reaction mixture. As shown in Table I each step like initial denaturation (alteration of structure) for 1 to 4 min, annealing (joining) for 1 to 1.5 min, and extension takes place at a different temperature depending on DNA. And the process is repeated for number of cycles to produce multiple copies of same DNA structure. Once the cycles are over the reaction mixture undergoes a last step of final elongation for 5 to 6 min.

   The procedure for performing PCR consists of first preparing the reagents and then adding them to a DNA

   sample in a sample block of usually 60 to 96 vials (In our case 8 vials) of 0.2mL to 0.5mL volume each as The thermal cycler cycles the block temperature using heating and cooling air-flow as per the requirement as indicated in Table 1[3][4].

   Table 1. PCR Thermal Cycling Program

   Cycle Description	Temperature	Time (min)
   Initial Denaturation	95C	1-4
   Denaturation	95C	0.5 to 1
   Annealing	50 to 65C	1 to 1.5
   Extension	72C	0.75 to 3
   Final elongation	72 C	5

2. System Design

   1. Block Diagram

      Fig. 2. Block Diagram of Low cost PCR Thermal Cycler

      In our system, the process will have a closed loop intelligent system that will continuously monitor the temperature of copper block which contains the reaction mixture. The current parameter readings are shown on a display module. The moment any parameter crosses the set point opto-coupler is energized to take control of the process.

      A block diagram of the various parts of the cycler is shown in Fig. 2. It consists of Micro controller 89C51 to control entire process of cycler. The heater coil for heating the reaction mixture and a AC fan for cooling the reaction tubes its driver circuit composed of optocoupler IC MOC3011, A temperature sensor LM 35A is used to sense the temperature of copper block and the output of the sensor is given to ADC and The output from ADC is directly fed to the micro-controller to constitute the onoff electronic control. The cycling parameters are entered into the micro-controller through the computer through Visual Basics and can be monitored on the display during cycling. When the temperature of the tube exceeds the set-point, micro-controller switches off the heating and turns

      ON the Fan circuit and vice versa..A micro- controller with LCD display is implemented to display sensing parameter continuously.For programming the cycling parameters A Graphical user interface (GUI) has also been developed on PC[5].

   2. Hardware Design

      1. Copper Block Design

         Fig.3. Design of copper block with PCR tubes.

         The copper block is designed to carry 8 vials which carries the reaction mixture or sample and this sample is heated by thermal conduction by the heater. Block is made of copper to ensure good thermal conduction between copper block and vials (PCR tubes)[6].

      2. Fan Design (CFM Calculations)

         Cooling was carried out by blowing air from AC powered from a 220 V ac source, and controlled by a Optocoupler [6].

         The science of heat transfer provides the basis for cooling system design. The basic formula of heat transfer is written as:

         Q= m×T×Cp (1) Where:

         q = heat transferred m = mass

         T = difference in temperature Cp = specific heat

         A fundamental implication of the above formula is that the amount of heat that can be transferred from one thing to another is directly proportional to the difference in temperature between the two things(T). The heat transfer formula from above can be re-written to apply directly to a cooling fan as,

         cfmr = Q/T..(2)

         Where:

         cfmr = the required airflow generated by the cooling fan

         Q = the required amount of heat rejected into the air to maintain proper temperature.

         T= temperature difference

         Fig. 4 Relationship between CFM and Temp. difference

         In PCR it consist of various temperature cycles in which either energy is needed to supplied when we want to increase the temperature and particular amount of energy needed to be removed when temperature need to be reduced.

         In our case,

         Table 2. Energy and CFM Calculations for Fan

         Temperature difference	energy to be released	CFM needed
         95-72 ºC	2849Joules	77
         72 to RT	4004Joules	77

         Therefore we require AC fan with CFM rating equal to 77

         we have selected the AC fan with standard CFM rating of 115 (Model FP1087).

      3. Heater Design (Power calculations for Heater) The amount of heat actually transferred from copper block to polypropylene by conduction when there is temperature difference between two bodies is calculated by the formula,

         Heat conducted trough several walls in good thermal contact can be expressed as

         q =( T1 – Tn) /((s1/k1A) + (s2/k2A) + … (sn/knA))..(3)

         where

         q=conductive heat transfer T1=temperature of copper block Tn=temperature of polypropylene S1=thickness of copper S2=thickness of polypropylene K1=thermal conductivity of copper

         K2=thermal conductivity of polypropylene A1=heat transfer area of copper

         A2=heat transfer area of polypropylene.

         Surface Area in contact of polypropylene tube =

         Total surface area of cone + total surface area of upper cylinder

         Fig. 5. Dimensional structure of Polypropylene tube

         Surface area of cone= r2 + r l(4)

         Where, r is radius of cone l is slant height

         = r (r+l)

         = × 3mm (13.44mm)

         =126.66 mm2

         Therefore, total surface area of cone in contact = 126.66mm2

         Surface area of cylinder = 2 r2 + 2rh.(5)

         Where, h =height of cylinder r =radius of cylinder

         =2r (r+h)

         =2× 3mm(3mm+5mm)

         =150.79mm

         Therefore, total surface area of cylinder in contact

         = 150.79mm2

         Therefore,

         Total surface area in contact with copper block

         = 126.66+150.79

         =277.45mm2

         Therefore,

         The conductive heat transfer through the copper wall can be calculated by equation (1)

         by experiments it is observed that maximum temperature difference between copper and polypropylene is 15C. so taking T1-Tn=15C

         q=15/(1 X10-3)/(0.22 X 277.45 X 10-6)+ (2 X 10-

         3)/(393 X 277.45 X 10-6)

         q=0.9146 watt/m2

         This is the amount of heat actually transferred from copper block to polypropylene by conduction when the temperature difference is 15C, which is very negligible as compared with enrgy consumed by copper block. Therefore, we assume that energy supplied by the heater is entirely used for raising the temperature of reaction mixture and no

         heat loss takes place in copper block and polypropylene tubes.

         According to the formulas of heat transfer,

         Energy = Mass X Specific Heat X Temp. Difference

         (4)

         And,

         Power = Energy / Time .(5)

         Putting specific heat of copper=0.385J/gC mass of copper block carrying vials=200gm

         temperature difference =(95C-RT) and assuming required heating rate = 1C we get

         Wattage of heater required to raise the temperature of DNA sample from RT to 95C is 77 watts.

   3. Software design

      1. Flowchart

         Fig.6 Flowchart of main program Fig. 6 shows the flowchart of microcontroller

         program which will start to execute after user enters cycling details. In our system on-off control is used for carrying temperature cycles[8].

      2. GRAPHICS USER INTERFACE (GUI)

         Fig. 7 Data Entry Form of GUI

         Graphical user interface has also been developed on PC. The PC provides a user-friendly interface giving a graphical picture of the process details. It also shows the set point values. A facility has also been provided for the request of the process status from the system as shown in Fig 6. Microsoft Visual Basic provides a powerful event-driven programming platform, which is very useful to carry out the rest of the functionality of the system has to possess. The GUI is also provided with the following features:

         1. Graphical and numerical display of the current reading of the temperature.

         2. Temperature graph

         The microcontroller has been programmed to just read the data from the ADC, display it on the LCD, simultaneously sending the data to the PC via the RS232 channel. Further, depending on the data it receives from the PC entered by the user it takes a control action by turning ON and OFF to heater and fan to control the process.

         Fig. 8 Temprature Graph

3. Observations and Results

   The experiment was carried out and readings were taken for 10 subsequent trial runs and it was observed that approximate heating and cooling rates achieved after the experiments were 1 ºC/s and 0.25 ºC/s respectively [9] [10]. The heating and cooling rates for different steps in amplification process is as shown in Table 3.

   Table 3. Output for 10 subsequent trial runs

   Step Name	Temperatur	Time	Heating
   (DNA)	e	required for	/
   	Rise /fall	heating	Cooling
   		/cooling	rate
   Annelaing	RT to 95 ºC	65 to 70 sec	1 ºC/s
   denaturatio n	95 to 54 ºC	150 to 160sec	0.25 ºC/s
   Extension	54 to 72 ºC	25 to 28 sec	1 ºC/s

4. Conclusion

   In order to carry out DNA amplification by using the prototype module of PCR thermal cycler and to carry out different heating and cooling cycles we have developed a low cost model using simple nichrome coil heater and air forced convection technique by blowing the air at the specimen by using a AC fan with required CFM and air blowing capability. We have Developed user friendly GUI by using Visual Basics to enter different temperature profiles of DNA to be amplified and to observe the current temperature of the system.

5. References

1. P. Denton and H. Reisner, in PCR Protocols: A Guide to Method and Applications, edited by M. Innis et al. (Academic, New York, 1990), Chap. 52.

2. C. T. Wittver, G. B. Reed, K. M. Ririe, The Polymerase Chain Reaction, pp. 174-181, Birkhauser, Boston, MA, 1994.

3. D.S. Yoon, Y.S. Lee, Y. Lee, H.J. Cho, S.W. Sung, K.W. Oh, J. Cha, G. Lim, Precise temperature control and rapid thermal cycling in a micromachined DNA polymerase chain reaction chip, J. Micromech. Microeng. 12 (2002) 813823.

4. D.S. Lee, C.Y. Tsai, W.H. Yuan, P.J. Chen, P.H. Chen, A new thermal cycling mechanism for eective polymerase chain reaction in microliter volumes, Microsyst. Technol. 10 (2004) 579584.

5. P. K. Upadhyay, D. V. Gadre, A personal-computer- based thermocycler for polymerase chain reaction, Meas. Sci. Technol., 6, pp. 588-592, 1995.

6. Debjani Pala and V. Venkataraman,K. Naga Mohan and

   H. Sharat Chandra, Vasant Natarajan, A power-efcient thermocycler based on induction heating for DNA amplication by polymerase chain reaction.

7. C. T. Wittver, G. C. Fillmore, D. R. Hillyard, Automated Polymerase Chain Reaction in Capillary Tubes with Hot Air, Nucleic Acids Research, Vol. 17, No. 11, pp. 4353-4357, 1989.

8. D.S. Lee, C.Y. Tsai, W.H. Yuan, P.J. Chen, P.H. Chen, A new thermal cycling mechanism for eective polymerase chain reaction in microliter volumes, Microsyst. Technol. 10 (2004) 579584..

9. The PTC-0150 minicycler operations manual, version 4.0, MJ Research, USA.

10. C. F. Chou, R. Changrani, P. Roberts, Daniel James Sadler, J. Burdon, F. Zenhausern, S. Lin, A. Mulholland, N. Swami, R. Terbrueggen, A minitiarized cyclic PCR device

modeling and experiments, Microelectronic Engineering, 61-62, pp. 921-925, 2002.