TA Cloning of TAT


AIM 

Aim of this experiment was to amplify TAT gene by PCR technique and to purify the obtained TAT gene fragments by applying the gel extraction method such as Thermo Scientific GeneJET PCR Purification Kit for the TA-cloning of TAT.  

INTRODUCTION 

Polymerase Chain Reaction 

PCR (polymerase chain reaction) is a common laboratory technique which provides the amplification of a specific segment of DNA sequence from a mixture of DNA molecules. The polymerase chain reaction is performed into three stages including the denaturation, annealing and elongation or extension which are also called PCR cycle. In the denaturation stage, applying the heat (more than 90 oC) separates the double-stranded DNA into two single-stranded DNA by breaking down the weak hydrogen bonds between the bases. However; the stronger bonds between deoxyribose and phosphates do not break. In the annealing stage, the DNA mixture is cooled (40 oC and 65 oC) allowing the primer binding and annealing to target complementary sequence in the template DNA. In the elongation stage, the temperature is increased to 72 oC which is optimal temperature for DNA polymerase activity allowing the DNA polymerase to synthesize new strand of DNA by also adding the nucleotides (dNTPs) onto the end of the extending primers. Synthesis of new strand always starts at 3’ end of the primer and continues in the 5’ to 3’ direction. As a result, two new DNA strands identical to original target can be amplified. The PCR cycle is repeated many times (30-40) and by this way, the synthesized products in a given cycle can serve as templates for the next cycles and so the number of target DNA copies become about double in every cycle [1]. 

The PCR reaction is set up with essential components including the a template DNA, a suitable DNA polymerase enzyme like Taq DNA polymerase, a buffer solution for the enzyme, magnesium chloride, dNTP mix, forward and reverse primers and water. By combining of all these components, a reaction mixture is prepared for performing the PCR. In this case, a template DNA also known as obtained DNA from samples includes target sequence of interest which will be amplified.  The DNA polymerase is an enzyme which synthesizes a new strand of DNA also via sequential addition of nucleotides [2]. Taq DNA polymerase is a highly processive thermostable enzyme which synthesized a new strand of DNA by addition of dNTPs onto the end of an annealed primer. For its activity, it needs to magnesium ions in which magnesium chloride. Taq DNA polymerase also adds the single deoxyadenosine (A) to 3’ end of the PCR product which is ligated with 3’ T overhangs of the cloning vector for the TA cloning process [3]. Buffer is a salt-solution which provides the optimal pH and salt condition for DNA polymerase and other reaction components. Primers including the forward and reverse primers are small segments of single stranded DNA (15-30 bases long) which provide annealing to their complementary sequence on the template strand. They provide initiation sites for DNA synthesis by binding to end of the target DNA sequence. In the PCR technique, magnesium as a cofactor is used in the magnesium chloride form and it provides the optimum activity for DNA polymerase enzyme. Lastly, dNTPs are four nucleotides which extend an annealed primer by acting of DNA polymerase [2]. 

For the PCR optimization, each required component must be mixed with appropriate concentrations of components during the PCR reaction. Buffer concentration including the pH and salt concentration must be in an appropriate concentration in which high salt conditions damage the DNA by denaturation resulting in the failure of the reaction and also low pH conditions affect the enzymatic activity (not worked). Also primer concentration must be appropriate at which high primer concentration causes mispriming and accumulation of nonspecific products resulting in decreasing the specificity and at which low concentration causes the completing reaction early resulting in the less yield of desired product. Moreover, high enzyme concentration causes the amplification of non-specific annealed primers resulting in the decreasing the specificity and low enzyme concentration causes low amplification of annealed primers resulting in the less yield of desired product. High DNA concentration causes the false priming resulting in the less DNA synthesis and low DNA concentration causes the less yield of desired product. The last important factor in PCR optimization is water concentration which arranges the optimal pH and salt concentration by dilution of buffer like from 10X to 1X [4].    

Gel Extraction 

Gel extraction is a method allowing the high purity of DNA which is used to isolate and purify the desired fragment of DNA from other bigger and smaller DNA (non-specific) based on their size by cut out of the visualized band on the agarose gel with performing the specific gel extraction processes especially spin-column kit procedure [5][6]. 

 

 

MATERIALS&METHODS 

PCR Reaction for TAT Gene 

For the PCR reaction, the isolated pCV-1 plasmid including the TAT gene from previous experiment was used as a template. The reaction mixture was prepared as 4 reactions for preventing losing of materials in the presence of any problems. Thereby, required amounts of all PCR components waited on ice were mixed (except the DNA) for the four reactions master mixture in order as shown below table:  

Table 1: The required amounts of PCR components for one reaction and four reactions to set up PCR reaction mixtures 

Component  Volume (µl)  Master Mix Volume (4 rxn) 
Nuclease-free dH2O  13.5 µl  54 µl 
10X Taq Buffer  2.5 µl  10 µl 
dNTP mix, 10 mM each  1 µl  4 µl 
25 mM MgCl2  2.5 µl  10 µl 
Forward primer (2 pmol)  2 µl  8 µl 
Reverse Primer (2 pmol)  2 µl  8 µl 
Taq Polymerase (5u/µl)  0.5 µl  2 µl 
DNA ng/µl  1 µl  Total 24 µl for each tube 

+ 1 µl of DNA 

TOTAL  25 µl 

 

After set up of master mix, 24 µl of mixture without the DNA sample was transferred into 2 PCR tubes for each group and then 1 µl of DNA was added into the each tube as the total volume of reaction mixture has been reached to 25 µl. The PCR tubes holded on ice was gently flicked to mix the solution and briefly centrifuged. Finally, the PCR tubes were placed into PCR machine and PCR cycles were performed under certain conditions as below: 

 

  • Initial Denaturation: 94 oC for 3 min 
  • Denaturation: 94 oC for 30 sec 
  • Annealing: 53 oC for 30 sec            30 cycle 
  • Extension: 72 oC for 45 sec 
  • Final Extension: 72 oC for 5 min 

Agarose Gel Electrophoresis 

After performing the PCR reaction, the PCR products including the amplified TAT gene and other molecules were loaded into an agarose gel. Before loading the samples, 0.8% agarose gel was prepared previously. In this case, to prepare 8% agarose gel, 1.2 g agarose was weighed and it was solved into 150 ml TAE Buffer by applying the heat and then cooled for addition of intercalating agent ethidium bromide. After preparation of agarose gel, all of 25 µl of PCR products were loaded into three gel wells with mixing 5 µl of 6X loading dye as the last concentration of loading dye comes to 1X which calculation for it is shown as below: 

Stock concentration of loading dye = 6X 

Final concentration of loading dye = 1X     So; 

M× V1 = M× V2 

1X × (25 µl +?) = ? × 6X                    

? = 5 µl of loading dye required 

The gel was run by 100 V electric current for 30 min. Finally, the gel slice including the desired DNA fragments were removed from the agarose gel by cut out of them under the UV light and the removed gel slices (2 gel slices for each group) were weighed for the next processes as gel extraction. 

Gel Extraction  

Firstly, the weight of cut out gel slices containing the fragment were determined as 0.2157 g ~ 216 mg for our group and 0.1666 g ~ 167 mg for other subgroup. The gel extraction processes were performed by Thermo Scientific GeneJET PCR Purification Kit. Then 1:1 volume of Binding Buffer, 216 µl of Binding Buffer was added into the gel slice and the gel mixture was incubated at 60 oC for 10 min until the gel slice was completely dissolved. In this stage, the tube with gel slice was mixed by inversion at certain intervals to facilitate the melting of gel slice. On the other hand, during dissolving, the color of solution was checked to control pH level for DNA binding. The color of our solution was yellow which indicates an optimal pH for DNA binding. But, if the color of solution was orange or violet, 10 µl of 3 M sodium acetate, pH 5.2 solution will be added into the gel mixture for obtaining the yellow color. After the gel slice completely dissolved, a 1:2 volume of 100% isopropanol, 216 µl of 100% isopropanol was added into solubilized gel solution and it was mixed. Then, up to 800 µl of the solubilized gel solution was transferred to the GeneJET purification column and centrifuged for 1 min. After the centrifugation, the supernatant was removed and the column was placed back into the same collection tube. After that, 700 µl of Wash Buffer added into the GeneJET purification column and centrifuged for 1 min and again the supernatant was removed. The empty GeneJET purification column was centrifuged for an additional 1 min to completely removal of residual wash buffer. Then, the GeneJET purification column of one subgroup was transferred into a clean 1.5 ml microcentrifuge tube and 45 µl of Elution Buffer was added to the center of the purification column membrane and centrifuged for 1 min. After centrifugation, the same Elution Buffer (45 µl) was used for our group’s GeneJET purification column and again centrifuged for 1 min. Finally, the purified DNA of each subgroup of our group was collected into the same tube by discarding the GeneJET purification columns and some of purified DNA was stored at -20 oC and some (2 µl) was used for Nanodrop measurements. 

Nanodrop Measurements 

For the Nanodrop measurements, firstly, 2 µl of dH2O was put into the pit of Nanodrop machine for cleaning the pit and it was dried. Then 2 µl of solvent of DNA (TE/ dH2O) was put into the pit as a blank in order to calibrate and pit was dried again. 2 µl of sample (DNA in TE/dH2O) was put into the pit and absorbance values including the A260/A280 and A260/A230 and also total DNA amount (ng/ µl) were determined and the results were recorded. 

 

RESULTS 

By performing the polymerase chain reaction we’ve amplified the TAT gene and also we’ve visualized the TAT gene fragments for each group by applying the agarose gel electrophoresis method and so, we’ve examined the PCR products on the agarose gel under the UV light which shown in below figure; 

   

Figure 1: The agarose gel image of amplified TAT genes results of the performing the PCR reaction in the presence of a ladder with 1 kb length bond size. TAT gene has a 263 bp length and so, the bands at approximately 300 bp indicate the amplified TAT genes. In addition to this, G1-1, G1-2 and G1-3 DNA bands indicate that the 1st group’s amplified PCR products including TAT gene with approximately 300 bp. G2-1, G2-2 and G2-3 DNA bands indicate that the 2nd group’s amplified PCR products including TAT gene with approximately 300 bp. G3-1, G3-2 and G3-3 DNA bands indicate that the 3rd group’s amplified PCR products including TAT gene with approximately 300 bp which not work the PCR correctly. Because, the bands are not very clear on the gel image especially the indicated band of G3-3 as shown above figure. G4-1, G4-2 and G4-3 DNA bands indicate that the 4th group’s amplified PCR products including TAT gene with approximately 300 bp. G5-1, G5-2 and G5-3 DNA bands indicate that the 5th group’s amplified PCR products which not worked PCR and so we’ve not observed any bands for TAT gene with approximately 300 bp in the 5th group’s gel results. 

After the agarose gel electrophoresis each group has cut out the own gel slice from the agarose gel under the UV light and performed the gel extraction method to isolate and purify the desired fragment of DNA from other bigger and smaller DNA molecules (non-specific). Thereby, at the end of the gel extraction processes, we’ve measured the absorbance values including the A260/A280 and A260/A230 ratios for checking the purities of extracted DNA molecules and also total DNA amount (ng/ µl) by using the Nanodrop Spectrophotometry and recorded the datas which shown below table: 

 

Table 2: The measured results of purified DNA molecules including the TAT gene with absorbance ratios and also total amount after by performing the gel extraction processes  

  Gel Extraction Method 
A260/A280  1.88 
A260/A230  0.05 
Total DNA (ng/µl)  20.7 ng/µl 

 

Based on the Nanodrop results, we’ve checked the purities of collected DNA molecules for our group and observed that the extracted DNA molecules were pure when we’ve examined the A260/A280 ratio, whereas when we’ve look at the A260/A230 ratio, we’ve observed that the alcohol contamination. Also, we’ve determined the total DNA amount as 20.7 ng/ µl which was unexpected result. We’ve used this data for the calculation of required DNA amount which will be used for the ligation reaction. 

DISCUSSION 

In this lab session, we’ve aimed the amplification of TAT gene by performing the polymerase chain reaction in the presence of required reaction components and we’ve aimed the controlling of amplified TAT genes by agarose gel electrophoresis method followed by the cut out of the gel slice with desired DNA fragment, we’ve aimed to purify the DNA molecules with amplified TAT gene fragments by applying the gel extraction method such Thermo Scientific GeneJET PCR Purification. For this purposes, at first we’ve performed the polymerase chain reaction by preparation of master mix including four PCR reaction mixtures for preventing losing of materials in the presence of any problem such as in pipetting, mixing, etc. In the PCR reaction, we’ve used the isolated pCV-1 plasmid from previous experiment as a template which includes target sequence of interest as TAT gene. Moreover, we’ve used the essential amounts of other PCR components such as MgCl2, dNTP mix, forward and reverse primers, Taq DNA polymerase enzyme and buffer for Taq DNA polymerase as shown in table 1. We’ve used the Taq DNA polymerase for synthesizing the new strand of DNA with also sequential addition of nucleotides like dNTPs. Also we’ve used it for production of PCR product with 3’ end which is necessary for TA cloning processes. The Taq DNA polymerase activity depends on the magnesium ions and buffer conditions. Magnesium ions interact with negatively charged molecules due to its positive charge in the reaction such as template DNA, primers and also dNTPs [2]. Moreover, we’ve used the buffer solution for optimal pH and salt condition for DNA polymerase activity and stabilization of other components. Also we’ve used the forward and reverse primers extended by dNTPs for annealing to their complementary sequences on the template strand. When we’ve set up the PCR reaction, we’ve mixed the PCR components in an order as water, buffer, dNTP mix, magnesium chloride, forward and reverse primers, Taq DNA polymerase and lastly template DNA. We’ve mixed the water and buffer at first because the water decreases the harmful effect of buffer solution on enzyme activity and other components by diluting the buffer. We’ve added the enzyme and DNA lastly for preventing the any damage to enzyme or starting reaction from nonspecific regions at which primers bind randomly. At the end of this stage, we’ve total 25 µl of reaction mixture and performed the PCR cycles under the suitable PCR conditions. After performing the PCR reaction, we’ve prepared the 0.8% agarose gel which 1.2 g of agarose was solved in 150 ml TAE buffer by applying the heat and then the solution cooled before addition of ethidium bromide intercalating agent for preventing the its harmful effect. We’ve prepared the 0.8% agarose gel for the separation of large DNA fragments. So, we’ve loaded the all of PCR products into three gel wells with mixing 6X loading dye as the last concentration of loading dye as 1X. We’ve used the loading dye including the glycerol and bromophenol blue to sinking of the samples into the well by glycerol and visualization of DNA migration by bromophenol blue. Also we’ve used the ethidium bromide intercalating agent for visualization of DNA bands under the UV light. After the agarose gel electrophoresis, we’ve cut out the gel slices (two gel slices for each group) for the gel extraction processes. Based on the agarose gel image shown in figure 1, we’ve expected to observe bands at 263 bp (~ 300 bp) length for amplified TAT genes. In addition to this, we’ve observed the approximately 300 bp bands for the G1-1, G1-2 and G1-3 DNA bands which indicate the 1st group’s amplified PCR products and this result was expected. We’ve observed the approximately 300 bp bands for the G2-1, G2-2 and G2-3 DNA bands which indicate the 2nd group’s amplified PCR products. But the band in G2-1 was clearer than G2-2 and G2-3. The reason of these less clear bands can be excess amount of enzyme and long extension time [8]. We’ve observed the approximately 300 bp bands for the G3-1, G3-2 and G3-3 DNA bands which indicate the 3rd group’s amplified PCR products. But the band in G3-2 was clearer than G3-1 and G3-3. The reason of these less clear bands can be excess amount of enzyme and long extension time [8]. We’ve observed the approximately 300 bp bands for the G4-1, G4-2 and G4-3 DNA bands which indicate the 4th group’s amplified PCR products and this result was expected. Moreover, we’ve not observed any bands for G5-1, G5-2 and G5-3 DNA bands which indicate that the 5th group’s amplified PCR products. This means that the PCR cycles have not performed correctly or the agarose gel electrophoresis has not performed correctly even TAT gene fragments have amplified. This result was unexpected. The reason of this can be too short extension time, very high annealing temperature, and inappropriate buffer causing the enzyme or DNA denaturation, low primer concentration, long denaturation time and inappropriate amount of magnesium ions [7]. After the agarose gel electrophoresis, we’ve performed the gel extraction method by using the Thermo Scientific GeneJET PCR Purification Kit. We’ve performed the gel extraction method because, the PCR mixture was not used directly in cloning and in addition to this and to remove the other bigger and smaller nonspecific DNA molecules and we’ve isolated and purified the desired fragment of DNA. For this purposes, at first we’ve determined the weights of the gel slices containing the fragment as 0.2157 g ~ 216 mg for our group and 0.1666 g ~ 167 mg for other subgroup. Based on these weights we’ve performed other processes. We’ve used Binding Buffer for dissolving the gel slice by incubation at 60 oC. In this case, the concentration of gel slice was depending on the buffer, heat and incubation time. On the other hand, during dissolving, we’ve checked the color of solution to control pH level for DNA binding. The color of our solution was yellow which indicates an optimal pH for DNA binding. But, if the color of solution was orange or violet, 10 µl of 3 M sodium acetate, pH 5.2 solution will be added into the gel mixture for obtaining the yellow color. Also, we’ve used the 100% isopropanol for precipitation of DNA and Wash Buffer for removal of undesired components. Until the elution stage, high salt condition can be provided the attachment of DNA molecules to the GeneJET purification column, but in the elution stage low salt condition can be provided the collection of DNA molecules by Elution Buffer solution for downstream applications. At the end of the gel extraction method, we’ve stored some of purified DNA at -20 oC and used some (2 µl) for Nanodrop measurements. We’ve measured the absorbance values including the A260/A280 and A260/A230 ratios for checking the purities of extracted DNA molecules and also total DNA amount (ng/ µl) by using the Nanodrop Spectrophotometry and recorded the datas which shown table 2. Based on the Nanodrop results, we’ve checked the purities of collected DNA molecules for our group and observed that the extracted DNA molecules were pure when we’ve examined the A260/A280 ratio, because if the A260/A280 ratio is about 1.8, this means the DNA sample is pure, so we’ve not observed any protein or phenol contamination. However; when we’ve look at the A260/A230 ratio, we’ve observed that the alcohol contamination. Because if the is less than 1.8, it can be said that there is alcohol contamination. The reason of this situation can be presence of organic contaminants such as phenol or other organic compounds. Because many organic compounds can show the strong absorbances at about 225 nm causing interfere with downstream applications [8]. Also, we’ve determined the total DNA amount some less as 20.7 ng/ µl than expected result. The reason of this can be incomplete solubilization of the gel slice, inefficient DNA binding, membrane wash or DNA elution. At the end of this experiment, we’ve used this data for the calculation of required DNA amount which will be used for the ligation reaction in TA cloning processes. 

REFERENCES 

  1. PCR: How We Copy DNA 

http://molecular.roche.com/pcr/Pages/Process.aspx 

  1. Polymerase Chain Reaction (PCR) 

http://vlab.amrita.edu/?sub=3&brch=186&sim=321&cnt=1 

  1. Innis, M.A., Myambo, K.B., Gelfand, D.H. and Brow, M.A.D. (1988) Proc. Natl. Acad. Sci. USA 85, 9436. 

Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216. 

Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 6008. 

Chou, Q., Russel, M., Birch, D., Raymond, J., Bloch, W. (1992) Nucl. Acids Res., 20, 1717. 

Sharkey, D.J., Scalice, E.R., Christy, K.G., Atwood, S.M., Daiss, J.L. (1994) BioTechnology, 12, 506. 

  1. Apte A., Daniel S. (2009) PCR primer design. Cold Spring Harb. Protoc. doi:10.1101/pdb.ip65. 

Newton C.R., Graham A. (1994) Introduction to biotechniques (Bios Scientific, Oxford, UK). 

  1. Purifying DNA from an Agarose Gel 

https://www.addgene.org/plasmid-protocols/gel-purification/ 

  1. DNA extraction from agarose gels (basic method) 

http://www.methodbook.net/dna/gelextrc.html 

  1. PCR Troubleshooting Guide 

https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide 

  1. Interpreting Nanodrop (Spectrophotometric) Results 

http://www.u.arizona.edu/~gwatts/azcc/InterpretingSpec.pdf 

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