The expostulation of this practical is to magnify a section of actin cistron from cauliflower genome DNA ( gDNA ) . Practical conducted consisted of extraction of Brassica oleracea botrytis gDNA and elaboration of the partial I?-actin cistron from extracted gDNA utilizing two cauliflower-specific primers, BRAS-6F and BRAS-6R. Chicken liver DNA is used as mark Deoxyribonucleic acid in this prac to see if the particular designed primers can magnify the same part of the lily-livered I?-actin cistron. Finally, the PCR samples were run through gel cataphoresis.
Polymerase Chain Reaction ( PCR ) is a powerful method developed by Kary Mullis in 1983. PCR has been widely applied in molecular biological science, genetic sciences and forensic biological science ( Bartlett and Stirling, 2003 ) Using the ability ofA DNA polymeraseA to synthesise new strand of DNA complementary to the templet, it enable research workers to magnify specific familial sequences from a low concentration of sample.
The PCR reaction is based onA thermic cycling. One typical PCR rhythm involves with three stairss ( McPherson and Moller, 2006 ) :
( 1 ) Denaturation. The two strands of the parent DNA molecule are separated into single-stranded Deoxyribonucleic acid by heating the solution to 94-96° C.
( 2 ) Annealing. The solution is rapidly cooled to 50-65°C to allow the primers anneal to a Deoxyribonucleic acid strand. One primer anneals to the 3 ‘ terminal of the mark strand while the other primer anneals to the 3 ‘ terminal of the complementary mark strand. Then each transcript will be the templet in the following rhythm. This primer tempering depends on the thaw temp ( Tm ) of the primer.
( 3 ) Extension. The solution is so heated to 72°C which is the optimum temperature forA TaqA DNA polymerase, a sort of DNA polymerase working at high temperature. This polymerase elongates both primers at 3 ‘ terminals, in the way of the mark. DNA synthesis continues until the temperature rises and the double-stranded DNA is denatured once more.
In this practical, we amplified a fragment ( 193bp ) of the ?-actin cistron from the Brassica oleracea botrytis genome which had been extracted in earlier pattern. Actin is a constitutively expressed protein which is rather conserved across beings. The primers BRAS-6F and BRAS-6R were specifically designed based on the sequence of the Brassica oleracea botrytis actin cistron. We besides tested the primers on poulet liver Deoxyribonucleic acid to see if they can magnify the same part of the lily-livered ?-actin cistron.
Materials and Methods
Target DNA Preparation
Cauliflower DNA was extracted from cauliflower blossomings. Tissues were homogenized in CTAB buffer and heated in 65A°C H2O bath for 10min to do DNA dissolved wholly. Chloroform and isoamylalcohol ( 24:1 ) mixture was added to the homogenate and the homogenate was centrifuged for 10 min at maximal velocity ( 1300rpm ) in a benchtop microfuge, so that the DNA solution and other substances were separated. The upper aqueous stage which contained extracted DNA was transportation to a clean Falcon tubing and re-extracted with chloroform/isoamylalcohol and centrifugation. Then the Deoxyribonucleic acid was precipitated with isopropyl alcohol and re-dissolved in 3mL of TE buffer. Purified DNA solutions were stored at 4A°C. Chicken liver DNA was provided by the coach. The concluding reaction contain 200ng of Deoxyribonucleic acid 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/I?m. In the concluding reaction, 5 units of Taq polymerase was added to each group. This thermophilic DNA polymerase is thermally stabled and has an optimal temperature of 72A°C.
Primer Design and Analysis
A brace of primer sequences BRAS-6F ( 5′-GCCGAGCGGGAAATTGTAA-3 ‘ ) and BRAS-6R ( 5’-CCACAAACGATGGCTGGAACA-3 ‘ ) were available from the prac coach. Each primer was composed of 10-11 ( G+C ) and 9-10 ( A+T ) bases. The Tm value was calculated as: Tm = 4A- ( G+C ) +2A- ( A+T ) . Thus the Tm of BRAS-6F and BRAS-6R is 58A°C and 64A°C severally.
Three groups of samples were amplified in PCR system utilizing 0.5mL microfuge tubings. The templets in each group were cauliflower gDNA, chicken liver gDNA and dH2O ( negative control ) severally. All four dNTPs were at 200I?M in each group. 40 rhythms were used and each rhythm was as follows: denaturation at 94A°C for 30 sec, tempering at 58A°C for 30 sec and so extension at 72A°C for 45 sec. The procedure included an initial 2-min clasp at 94A°C and a concluding 5-min clasp at 72A°C.
Analysis of PCR Merchandises
Three groups of PCR reactions were analysed by cataphoresis on a 1.5 % agarose gel incorporating ethidium bromide. 10I?L of each PCR reaction was assorted with 2I?L of 6X gel lading buffer and loade into the gel. The gel was electrophoresed at 100V for 30 proceedingss.
The partial I?-actin cistron from cauliflower gDNA was amplified. An agarose gel cataphoresis was carried out utilizing the three groups of PCR reaction. Fig 1 illustrates the consequences of the gel cataphoresis. Fig 2 shows the standard curve of molecular weight log10 against the distance the sets migrated from the well.
Fig Agarose gel cataphoresis of PCR elaboration from Brassica oleracea botrytis. Lane 1 & A ; 5, PCR elaboration from cauliflower Deoxyribonucleic acid ; Lane 2 & A ; 6, PCR elaboration from chicken liver DNA ; Lane 3 & A ; 7, negative control ; Lane 4, Molucular weight marker ( I¦174 cut with HaeIII ) . Lane 1-3 were conducted by H-J Cheng ; Lane 5-7 were conducted by X. Chen.
Fig 2 The criterion curve for Fig 1 Lane 4 MW marker, constructed utilizing the log10of molecular weight in bp against the
distance migrated by Deoxyribonucleic acid in the gel
Table 1 The Data from the gel incorporating MW marker ( Fig 1 Lane 4 )
Distance ( millimeter )
Molecular Weight log10 ( bp )
In this experiment, we have used cauliflower-specific primers ( BRAS-6F and BRAS-6R ) to magnify a fragment of cauliflower I?-actin cistron with the size of 193bp.
In Lane 1 and Lane 5 of Fig 1, the presence of a individual distinguishable set somewhat below the 234bp set of MW marker indicates that a big sum of DNA fragment was produced from cauliflower gDNA. Comparing the set with the MW marker, we can find that the molecular weight of PCR merchandises is about 200bp.
In Lane 2 and Lane 6 of Fig 1, dimer and dispersed sets was presented, which means really few DNA were amplified. This consequence indicates that the cauliflower-specific primers ( BRAS-6F and BRAS-6R ) does non fit I?-actin cistron from chicken liver DNA good.
In Lane 3 and Lane 7 of Fig 1, the negative control shows no sets as expected, because there was no Deoxyribonucleic acid in the samples ; this consequences besides shows that there was no possible taint which could hold affected the PCR consequences obtained.
To further place the size of PCR merchandises from cauliflower gDNA, I produced a standard curve utilizing the MW marker ( Fig 2 ) . The graph shows that the relationship of log10 of DNA molecular weight and the distance of the set migrated from the well is about additive. The distance of the set in Lane 1 from the well is 31.5 millimeter. From Fig 2 we can acquire log10 of MW is 2.34. Therefore, the size of PCR merchandises in Lane 1 is 102.34 = 219 bp.
In decision, we have successfully amplified a section of I?-actin from cauliflower Deoxyribonucleic acid utilizing primers BRAS-6F and BRAS-6R and the agarose gel cataphoresis have shown a set of dependable consequences.