A new thermal cycling mechanism for effective polymerase chain reaction in microliter volumes

Abstract

This study presents a new thermal cycler using infrared (IR) heating and water impingement cooling for polymerase chain reaction (PCR) amplification of 10 ll samples in thin glass capillary tubes. The thermal cycling system can achieve a temperature ramping-up rate of 65°C/s and a ramping-down rate of 80°C/s. Two other cooling mechanisms, natural convection and forced air convection, can also be used in the present system to obtain a ramping-down rate of 2°C/s and 6°C/s, respectively. The amplification of the 439 fragment of hepatitis B virus (HBV) DNA was successful. The PCR amplified products were analyzed by agarose gel electrophoresis with ethidium bromide staining for visualization. A comparison of results of the amplification products at three different ramping-down rates was made and the rapid thermal cycling of the present system can run DNA required amplification in 29 min for 20 thermal cycles that is only 1/3 the time spent in the conventional PCR machine used in comparison. IntroductionAmplification of specific DNA fragment in vitro using the polymerase chain reaction (PCR) requires a well controlled thermal cycling [1, 2, 3]. There are three stages in the PCR. The first stage is denaturation at which the sample is heated up from room temperature to denaturation temperature at approximately 90-95°C. The high temperature results in a separation of the double strands of DNA into two single stranded DNAs. The second stage is called annealing at which the sample temperature drops from the denaturation temperature to annealing temperature at approximately 40-65°C. At the annealing stage, added primers in the sample are paired with single stranded DNA through a Brownian motion in the solution and subsequent hydrogen-bonding. The final step is elongation (or extension) at which the sample temperature is heated up from the annealing temperature to the elongation temperature at approximately 72°C. Since the primers already have a strong ionic bonding to the DNA template on the specific locations at the annealing stage, dNTPs are added complementarily to the DNA template. A complete temperature cycle consists of the temperature variations in the three stages in PCR. To obtain enough yield of products, the number of temperature cycles required depends on the type of DNA for amplification.In a conventional PCR machine, thermal cycling is performed on a 10 ll sample in a microcentrifuge tube using a temperature controlled metal block. The major setback of this conventional thermal cycler is a long temperature lag in the sample behind the metal block. Consequently, amplification time for DNA products may usually take a few hours. To reduce the thermal cycling time, Wittwer and his co-workers constructed a rapid cycling system by introducing hot air to heat up the sample in thin capillary tubes [4]. In their system, the thermal cycling time for 20 cycles was reduced from 1-4 h in a metal block thermal cycler to 20 min. In addition, their system can obtain better specificity in amplification products with less transi...

Design and simulation of leg exoskeleton cycling-actuated wheelchair

“…Firstly, cycling is widely used as an effective and efficient mode of transportation for short to moderate distances. 28,29 Secondly, cycling provides sustained physical exercise that incorporates the coordination of muscle movement for the entire body. In general, the cycling motion action is a repeated motion that can also be used for rehabilitation exercises in terms both of quality and quantity (for exerted motion).…”

Rapid microfluidic thermal cycler for polymerase chain reaction nucleic acid amplification

“…Slower or inaccurate temperature transition from the denaturation step to the annealing step increases the chance of primer-dimer formation which is the bonding of the primers (forward and reverse) to each other, or to other primers in the multiplexed reaction. It also increases the chance of nonspecific amplification which is bonding of the primers to sequences in the DNA template that are not exactly complimentary to the primer [1]. Quick cycles can be achieved by decreasing PCR dwell times.…”

“…They reported 65°C/s and 20°C/s for heating and cooling, respectively. Lee et al [1] used infrared (IR) heating and water impingement cooling technique for PCR in thin glass capillary tubes. They reported a temperature rate of 65°C/s and 80°C/s for heating and cooling processes.…”

“…In the first step, the DNA template is heated up to the denaturation temperature (approximately 96°C) and will be heated for 20-30 s to separate the DNA double strands. In the second step, annealing, the temperature of the DNA decreases to the annealing temperature (approximately 50-65°C, depending on the primer length and percent guanine and cytosine content) and will be kept at this temperature for 20-40 s so that the primers can anneal to the single stranded DNA template through Brownian motion in the solution and subsequent hydrogen-bonding [1]. In the third step, elongation or extension, the DNA temperature is increased to the elongation temperature (approximately 72°C) to synthesize new DNA strands complementary to the DNA template strands.…”