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The objection of this practical is to amplified a segment of actin gene from cauliflower genome DNA (gDNA). Practical conducted consisted of extraction of cauliflower gDNA and amplification of the partial Î²-actin gene from extracted gDNA using two cauliflower-specific primers, BRAS-6F and BRAS-6R. Chicken liver DNA is used as target DNA in this prac to see if the specific designed primers can amplified the same region of the chicken Î²-actin gene. Finally, the PCR samples were run through gel electrophoresis.
Polymerase Chain Reaction(PCR) is a powerful method developed by Kary Mullis in 1983. PCR has been widely applied in molecular biology, genetics and forensic biology (Bartlett and Stirling, 2003) Utilizing the ability of DNA polymerase to synthesize new strand of DNA complementary to the template, it enable researchers to amplify specific genetic sequences from a low concentration of sample.
The PCR reaction is based on thermal cycling. One typical PCR cycle involves with three steps(McPherson and Møller, 2006):
(1) Denaturation. The two strands of the parent DNA molecule are separated into single-stranded DNA by heating the solution to 94-96° C.
(2) Annealing. The solution is quickly cooled to 50-65°C to let the primers anneal to a DNA strand. One primer anneals to the 3' end of the target strand while the other primer anneals to the 3' end of the complementary target strand. Then each copy will be the template in the next cycle. This primer annealing depends on the melting temp (Tm) of the primer.
(3) Extension. The solution is then heated to 72°C which is the optimal temperature for Taq DNA polymerase, a kind of DNA polymerase working at high temperature. This polymerase elongates both primers at 3' ends, in the direction of the target. DNA synthesis continues until the temperature rises and the double-stranded DNA is denatured again.
In this practical, we amplified a fragment (193bp) of the ß-actin gene from the cauliflower genome which had been extracted in earlier practice. Actin is a constitutively expressed protein which is quite conserved across organisms. The primers BRAS-6F and BRAS-6R were specifically designed based on the sequence of the cauliflower actin gene. We also tested the primers on chicken liver DNA to see if they can amplify the same region of the chicken ß-actin gene.
Materials and Methods
Target DNA Preparation
Cauliflower DNA was extracted from cauliflower inflorescences. Tissues were homogenized in CTAB buffer and heated in 65°C water bath for 10min to make DNA dissolved completely. Chloroform and isoamylalcohol (24:1) mixture was added to the homogenate and the homogenate was centrifuged for 10 min at maximum speed (1300rpm) in a benchtop microfuge, so that the DNA solution and other substances were separated. The upper aqueous phase which contained extracted DNA was transfer to a clean Falcon tube and re-extracted with chloroform/isoamylalcohol and centrifugation. Then the DNA was precipitated with isopropanol and re-dissolved in 3mL of TE buffer. Purified DNA solutions were stored at 4°C. Chicken liver DNA was provided by the tutor. The final reaction contain 200ng of DNA in each of the two groups. Besides, a dH2O control sample was used as a negative control.
Taq DNA polymerase
The concentration of Taq DNA polymerase used in this prac was 5 units/Î¼m. In the final reaction, 5 units of Taq polymerase was added to each group. This thermophilic DNA polymerase is thermally stabled and has an optimum temperature of 72°C.
Primer Design and Analysis
A pair of primer sequences BRAS-6F (5'-GCCGAGCGGGAAATTGTAA-3') and BRAS-6R (5'-CCACAAACGATGGCTGGAACA-3') were available from the prac tutor. Each primer was composed of 10-11 (G+C) and 9-10 (A+T) bases. The Tm value was calculated as: Tm = 4Ã-(G+C)+2Ã-(A+T). Thus the Tm of BRAS-6F and BRAS-6R is 58°C and 64°C respectively.
Three groups of samples were amplified in PCR system using 0.5mL microfuge tubes. The templates in each group were cauliflower gDNA, chicken liver gDNA and dH2O (negative control) respectively. All four dNTPs were at 200Î¼M in each group. 40 cycles were used and each cycle was as follows: denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec and then extension at 72°C for 45 sec. The process included an initial 2-min hold at 94°C and a final 5-min hold at 72°C.
Analysis of PCR Products
Three groups of PCR reactions were analysed by electrophoresis on a 1.5% agarose gel containing ethidium bromide. 10Î¼L of each PCR reaction was mixed with 2Î¼L of 6X gel loading buffer and loade into the gel. The gel was electrophoresed at 100V for 30 minutes.
The partial Î²-actin gene from cauliflower gDNA was amplified. An agarose gel electrophoresis was carried out using the three groups of PCR reaction. Fig 1 illustrates the results of the gel electrophoresis. Fig 2 shows the standard curve of molecular weight log10 against the distance the bands migrated from the well.
Fig Agarose gel electrophoresis of PCR amplification from cauliflower. Lane 1&5, PCR amplification from cauliflower DNA; Lane 2&6, PCR amplification from chicken liver DNA; Lane 3&7, negative control; Lane 4, Molucular weight marker(Î¦174 cut with HaeIII). Lane 1-3 were conducted by H-J Cheng; Lane 5-7 were conducted by X. Chen.
Fig 2 The standard curve for Fig 1 Lane 4 MW marker, constructed using the log10of molecular weight in bp against the
distance migrated by DNA in the gel
Table 1 The Data from the gel containing MW marker (Fig 1 Lane 4)
Molecular Weight log10(bp)
In this experiment, we have used cauliflower-specific primers (BRAS-6F and BRAS-6R) to amplify a fragment of cauliflower Î²-actin gene with the size of 193bp.
In Lane 1 and Lane 5 of Fig 1, the presence of a single distinct band slightly below the 234bp band of MW marker indicates that a large amount of DNA fragment was produced from cauliflower gDNA. Comparing the band with the MW marker, we can determine that the molecular weight of PCR products is approximately 200bp.
In Lane 2 and Lane 6 of Fig 1, dimer and dispersed bands was presented, which means very few DNA were amplified. This result indicates that the cauliflower-specific primers (BRAS-6F and BRAS-6R) does not match Î²-actin gene from chicken liver DNA well.
In Lane 3 and Lane 7 of Fig 1, the negative control shows no bands as expected, because there was no DNA in the samples; this results also shows that there was no potential contamination which could have affected the PCR results obtained.
To further identify the size of PCR products from cauliflower gDNA, I produced a standard curve using the MW marker (Fig 2). The graph shows that the relationship of log10 of DNA molecular weight and the distance of the band migrated from the well is approximately linear. The distance of the band in Lane 1 from the well is 31.5 mm. From Fig 2 we can get log10 of MW is 2.34. Thus, the size of PCR products in Lane 1 is 102.34 = 219 bp.
In conclusion, we have successfully amplified a segment of Î²-actin from cauliflower DNA using primers BRAS-6F and BRAS-6R and the agarose gel electrophoresis have shown a set of reliable results.