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In this experiment, DNA amplification was done with different concentration of MgCl2 and range of annealing temperature. From previous literatures, the optimum concentration of MgCl2 that has been used is 1.5 mM and the annealing temperature was 55.0Â°C. Therefore, we plan our experiment with different concentration of MgCl2 such as 1.5 mM, 2.0 mM, 2.5 mM and 3.0 mM. Besides, the range of annealing temperature were 50.0Â°C to 60.0Â°C and some were 55.0Â°C to 65.0Â°C.
Marker 3'-SAG2 is the first markers that were optimized. This experiment carried out with annealing temperature from 45.0Â°C to 68.0Â°C for each concentration of MgCl2. The best band at ~222 bp obtained when the temperature is 63.0Â°C with 1.5 mM MgCl2. For marker 5'-SAG2, experiment done with the range of annealing temperature 58.0Â°C to 68.0Â°C and 1.5 mM and 2.0 mM MgCl2. The best band obtained at ~242 bp when the temperature is 59.9Â°C with 2.0 mM MgCl2. Marker BTUB was optimized with annealing temperature 55.0Â°C to 65.0Â°C and 1.5 mM and 2.0 mM MgCl2. However the result shows that at temperature 58.7Â°C with 1.5 mM MgCl2, this marker has the ~ 411 bp band. The next marker was cB21-4. It was optimized with annealing temperature 50.0Â°C to 60.0Â°C and 1.5 mM, 2.0 mM and 3.0 mM MgCl2. The 53.7Â°C annealing temperature and 1.5 mM MgCl2 was the optimal conditions to get the ~502 bp band of marker cB21-4. Then, for optimization of marker GRA6, experiment carried out with annealing temperature 55.0Â°C to 65.0Â°C and 1.5 mM MgCl2. The gel electrophoresis result shows that the ~351 bp band obtained at optimum condition of temperature is 57.8Â°C with 1.5 mM MgCl2. After that, marker SAG3 was optimized with annealing temperature 66.0Â°C to 68.0Â°C and 2.0 mM MgCl2. However, the best band (~311bp) obtained at temperature 66.0 Â°C with 2.0 mM MgCl2. For marker SRS1, optimization was done with annealing temperature 55.0Â°C to 65.0Â°C and 1.5 mM, 2.0 mM, 2.5 mM and 3.0 mM MgCl2. When the condition is 64.2Â°C and 3.0 mM MgCl2, the best band at ~1386 bp was obtained. Finally, marker GRA1 was optimized with annealing temperature 55.0Â°C to 65.0Â°C and 1.5 mM MgCl2. The best band (~827 bp) achieved at temperature 61.1Â°C and 1.5 mM MgCl2.
All the optimization was performed using Taq DNA polymerase and confirmation done with Pfu DNA polymerase.
The purpose of this study was to optimize the PCR condition, especially concentration of MgCl2 and annealing temperature for all the eight markers. DNA isolated from the RH strains (Type I) of T.gondii has been used as a template. All PCR primers were designed based on Ferreira et al., (2008) for 3'-SAG2 and 5'-SAG2, Su et al., (2006) for BTUB and SAG3, de Melo Ferreira et al., (2006) for cB21-4 and GRA6, and Khan et al., (2005) for SRS1 and GRA1 as reference. However, all the sequence of PCR primer will be reconfirmed again based on Toxoplasma Genome Map Database.
PCR is a effective technique to amplify a single or few copies of a piece of DNA. Theoretically, basis of PCR has three nucleic acid segments: the segment of double-stranded DNA to be amplified and two single-stranded oligonucleotide primers flanking it. Besides, there is a DNA polymerase (protein component), deoxyribonucleoside triphosphates (dNTP), a buffer and salts. PCR is regularly used because it is the most sensitive assay for rare sequences and not only detects rare DNAs but quantitates them as well. The first step of PCR is mixing the template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerase, dNTP and a buffer. The mixture is cycled through specific temperatures that allow denaturation, annealing and synthesis to amplify a product of specific size and sequence. The PCR products are then displayed on an appropriate gel and examined for yield and specificity.
Many variables can influence the result of PCR products. The accurate concentration of the MgCl2 is critical. Magnesium ions required for polymerase stability and processivity and also increase hybridization of DNA polymerase with DNA template. A magnesium ion also helps to reduce nonspecific primer-template interactions and improve amplification efficiency (Kramer and Coen, 1999). Sufficient magnesium ions are required to enhance the binding activity of DNA polymerase with DNA template.
Denaturation, annealing and extension are each quite rapid at the optimal temperatures. Time to achieve the desire temperature inside the reaction tube that is the ramp time is usually longer than either denaturation or primer annealing (Kramer and Coen, 1999). For marker 3'-SAG2, optimization done with different set of annealing temperature which start with 45.0Â°C to 55.0Â°C , 55.0Â°C to 65.0Â°C and 63.0Â°C to 68.0Â°C. Almost for each temperature multiple bands were obtained but only at 63.0Â°C single band able to see. This result shows that primers need a specific annealing temperature for a successful amplification. The number of cycles depends on both the efficiency of the reaction and the amount of template DNA in the reaction. Greater cycle numbers (>40) can reduce the polymerase specific activity and increase nonspecific amplification (Kramer and Coen, 1999). In this experiment, amplification was done with 35 cycles.
Contamination such as detergent SDS and sodium acetate during DNA preparation also can decrease the efficiency of PCR. One of the ways to minimize the contamination is by repeatedly washing the pellet with 70% ethanol. The amount of DNA template that used in PCR reaction also must be sufficient in order to be visualizing the PCR products using ethidium bromide. However using too much of DNA template is not suitable when optimizing for MgCl2 or other parameters because it may decrease amplification efficiency (Kramer and Coen, 1999).
Highly thermostable DNA polymerase is used in PCR reactions as it can withstand the repeated heating and cooling inherent in PCR and synthesize DNA at high temperatures. Besides, consideration of error rate also important in selecting the efficient DNA polymerase. The error rate of Pfu DNA polymerase in PCR is 2.6 x 10-6 nucleotides/cycle and lower than Taq DNA polymerase which is 2.0 x 10-4 nucleotides/cycle. Pfu DNA polymerase is the enzyme that catalyzes the template dependent polymerization of nucleotides into duplex DNA in the 5' to 3' direction. Pfu DNA polymerase also exhibits 3' to 5' exonuclease (proofreading) activity that enables the polymerase to correct nucleotide incorporation errors. However, Pfu DNA polymerase is more expensive compare to Taq DNA polymerase. Therefore, in this experiment we used Taq DNA polymerase for optimization and reconfirmation of results is done with Pfu DNA polymerase.
Kramer, M.F. and Coen, D.M. (1999). Current Protocols in Molecular Biology. Chapter 15: 15.1.1-15.1.15. John Wiley & Sons, Inc.