Amplification of deoxyribonucleic acid (DNA) fragment using two-step polymerase chain reaction (PCR)

Polymerase chain reaction (PCR), an essential tool in many fields such as molecular biology, normally comprises three steps: denaturation at a high temperature, annealing at a low temperature and elongation at a moderate temperature. Here, we report a two-step PCR method which incorporates annealing and elongation step for significant time-saving and reduction in reagent use. To investigate whether the two-step method is as useful as the common three-step method, different lengths of DNA fragments were amplified by two-step PCR and three-step PCR and the influence of incorporated temperature and the amount of Taq polymerases were performed in the present study. The results showed that, all the DNA fragments of 300 to 1000 bp could be amplified by two-step PCR method and the temperature from 50 to 60.8°C could provide a viable range for annealing/extension steps and the fidelity of the PCR has not been affected by the two step method presented. Taken together, the twostep PCR could be used to amplify DNA fragment, which is time-saving, reliable and inexpensive.


INTRODUCTION
Polymerase chain reaction (PCR) has been widely used in many fields such as molecular biology, illness diagnosis, food detection, etc. (Saiki et al.,1985;Xiong et al., 2004;Umeda et al., 2010;Kawasaki et al.,2009;Yabuki et al., 2007;Sambrook and Russell, 2001).Generally, each PCR cycle comprises three steps, a 95°C step to denature the duplex DNA, an annealing step of around 55°C to allow the primers to bind and a 72°C elongation step.In practice, about 30 to 35 cycles are commonly used.So a complete PCR often takes more than two hours by this normal procedure (Sambrook and Russell, 2001).The annealing step and elongation step could be incorporated into one step which, along with the *Corresponding author.E-mail: wtianyuncn@126.com.denaturation step is named the two-step PCR method in many labs.The two-step protocol is designed for significant time-savings and a reduction in reagent use, relative to the standard PCR protocol.However, the knowledge of this method is limited now.In the present study, we investigated the influence of temperature and Taq polymerases on this method, whether different lengths of DNA fragments could be amplified by two-step PCR and the difference of DNA product quality produced by the two methods.

PCR template and reagents
Enterobacteria phage lambda DNA (GenBank no: V00636) served as template and PCR regents were all purchased from TaKaRa Biotechnology Company Limited (Dalian, China).Primers were designed according to the template gene sequence and synthesized by Shanghai Sangon Biological Engineering Technology and Co., Ltd (Shanghai, China) and their oligonucleotides sequences are shown in Table 1.

DNA fragments amplified by PCR
Seven different lengths of DNA fragments were amplified by twostep PCR in comparison with that by three-step PCR.Reaction mixture (100 µl) for each quantitative PCR contained 100 ng template DNA, 0.2 mM dNTP, 0.1 mM of each primer, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-Cl (pH 8.8), 2.5 U Taq polymerase.
The two-step PCR method contained the following procedure: Predenaturation for 3 min at 95°C and subjected to 30 cycles of 30 s denaturation at 94°C, 50 s step at 65°C, and 1 min extension at 65°C.The three-step PCR procedure was denaturation for 3 min at 95°C and subjected to 30 cycles of 30 s denaturation at 94°C, 30 s annealing at 55°C, 30 s elongation at 72°C and 1 min extension at 72°C.

Gradient denaturation temperature and Taq polymerases
The DNA fragment of 600 bp was amplified at a gradient of temperatures by two-step PCR method.The PCR system was prepared as previously described and the PCR procedure included denaturation for 3 min at 95°C and subjected to 30 cycles of: 30 s denaturation at 94°C, 50 s incorporated step at the gradient of 25 to 75°C.The DNA fragment of 600 bp was amplified at a gradient of amount of Taq polymerases by two-step PCR method.The PCR procedure was as same as two-step PCR method described previously, while the PCR system included a gradient of Taq polymerases of 0.5, 0.4, 0.3, 0.2, 0.1, 0.08, 0.072, 0.064, 0.056 and 0.048 units, respectively.

Purified and sequence of amplified DNA fragments
After PCR, the results were detected by 2.0% agarose gel electrophoresis, then they were purified and sequenced using T7

DNA fragments amplified by two-step PCR procedure
As shown in Figure 1, the seven different DNA fragments from 300 to 1000 bp in length could be effectively amplified, although, the quantity of DNA fragments was less in

Gradient denaturation temperature and Taq polymerases
We amplified the 600 bp DNA fragment at gradient denaturation temperature and Taq polymerases.The results demonstrated that, 600 bp DNA fragment could be obtained by two-step PCR at temperatures as high as 70°C and as low as 35°C (Figure 2).The PCR was effective when the temperatures were from 50 to 65°C (Lane 3 to 6, Figure 2).Although, DNA fragments could be amplified at 30 and 25°C, the PCR amount diminished greatly.In two-step PCR, annealing temperature and elongation temperature were quite adjacent, and the lower temperature was in favor of annealing, whereas, the higher temperature was fit for elongation, so the proper temperature will benefit both processes.In the present study, the large quantity of DNA could be produced at temperature from 65 to 35°C.The amount of DNA fragments will be less greatly and were not amplified when the temperature was beyond this range.
Optimum usage of Taq polymerase is necessary for PCR, too less could lead to the failure of PCR, but too much also could not benefit the results (Giovannelli et al., 2004;Al-Shanti et al., 2009;Abe et al., 2008;Wang et al., 2007).DNA fragments of 600 bp were amplified at a gradient of the amount of Taq polymerases by two-step PCR method (Figure 3).In 20 µl PCR system, the amount of

Table 1 .
Primers used in the experiment.