Isolation of PCV-1 and Confirmation by Restriction Digestion


AIM 

Aim of this experiment is the isolation of pCV1 plasmid DNA from E.coli bacterial culture grown overnight in LB medium with a suitable antibiotic such as tetracycline which selects the cells having plasmid and so tetracycline resistance gene. Also, aim is to cut the pCV1 plasmid by PstI restriction enzyme for controlling that isolated plasmid is actually pCV1 or not via Nanodrop measurements and also agarose gel electrophoresis. 

INTRODUCTION 

Plasmid is an extra chromosomal, supercoiled, circular double stranded DNA molecule which found in bacteria and some eukaryotes. Plasmids have major three elements including an origin of replication, an antibiotic resistance gene like resistance to ampicillin and also a multiple cloning site. Moreover, they have selectable markers, promoter region and primer binding site. Plasmids provide the adaptation to the host cells under the unavailable environmental conditions via their included genes [1]. Plasmids can be important and essential for many cellular processes in molecular biology such as determination of the gene function, because they are easy to work due to their stabilities and capable of independent replication [2]. pCV1 plasmid derived from pCV is a mammalian expression vector which includes  the hybrid regulatory sequences of HIV and also tetracycline resistance gene. This plasmid can be cut by PstI restriction enzyme which forms two fragments like 3.3 kb and 7.0 kb [3]. 

Isolation of Plasmid DNA  

Plasmid DNA isolation is a process in which plasmid DNA is obtained by separating from other cellular components via applying physical or chemical methods. In the plasmid isolation, there are 4 basic stages. Firstly, a bacterial culture is grown overnight in a LB medium with a suitable antibiotic. Generally, ampicillin and kanamycin are used for selection. After that, obtained bacterial culture containing plasmid of interest is harvested by spinning and lysate by physical or chemical methods such as using of the chemical agents that break down the cell integrity. Lysozyme, EDTA, SDS and strong alkali reagent are examples of these chemical agents. Lysozyme breaks down the cell wall polymeric compounds which provide the integrity. EDTA damages the cell envelope structure by removal of magnesium ions from the cell membrane. SDS is a detergent which removes the lipid molecules from cell membrane in the lysis stage. Also, strong alkali reagents denature the host cell chromosomal DNA without any damaging the plasmid DNA due to its compact structure. After the lysis of the cells, all components are removed except the plasmid DNA and finally, plasmid DNA is purified [4]. For the plasmid DNA isolation, many methods are used including the Miniprep Kit and Alkaline Lysis methods. ALS is a method which isolates the plasmid DNA in the presence of three buffer solutions with different chemicals which are glucose, Tris-HCl and EDTA for Alkaline Lysis Buffer I; NaOH and SDS for Alkaline Lysis Buffer II; potassium acetate and glacial acetic acid for Alkaline Lysis Buffer III.  

Miniprep method also defined as minipreparation of plasmid DNA depends on the small-scale for the isolation of plasmid DNA from bacteria by usage of a spin-column and also different types of chemicals. 

DNA Concentration Determination 

In the molecular biology, nucleic acid quantitation is important that how much DNA or RNA is present in the prepared solutions for performing the experiments about the genes. So, DNA concentration in a sample is determined by UV absorbance spectrophotometry at 260 nm. The UV absorbance spectrophotometry can also assess the purity of a DNA sample based on the ratio of absorbance at 260 and 280 nm. For the purity of a DNA sample, the A260/A280 ratio is accepted as 1.8. However; if this ratio become less than 1.8, this means that there is any protein or phenol contamination, because proteins can give absorbance at 280 nm. If the ratio become above 1.9, this means that there is RNA contamination, because RNA can give absorbance at 260 nm. Moreover, A260/A230 ratio is used for determination of purity which indicates the alcohol contamination of any solutions, when the ratio becomes less than the 1.8 [5]. 

Restriction Enzyme 

Restriction enzymes also called restriction endonucleases are DNA-cutting enzymes in the bacteria which cleave DNA at the specific recognition sequences, palindromic sequences, by recognizing them for producing the 5’ phosphates and 3’ hydroxyls. A palindromic sequence is the same nucleic acid sequence when it read from 5’ to 3’ or 3’ to 5’. Restriction enzymes naturally can be found in bacteria types. Bacteria use them for protection themselves against the phage DNA and they have ability to prevent them cut by their restriction enzymes. They can change the recognition sequence of the enzyme and so, the enzyme does not work. Also, restriction enzymes form 2 types of ends including sticky-cohesive ends or blunt ends. Sticky-cohesive ends include 5’ or 3’ overhangs due to asymmetrically cutting of the enzyme such as Bam HI (5’ overhangs) and Kpn I (3’ overhangs). In contrast, the blunt ends occur without any overhangs, when the enzyme like Sma I cut the both strand of DNA in the middle of the recognition sequence [6]. 

Star Activity 

Under the extreme reaction condition in terms of elevated pH or low ionic strength, restriction enzymes can cleave similar, non-identical sequences and this condition can change the specificity resulting in the star effect or star activity [7]. 

Agarose Gel Electrophoresis 

Agarose gel electrophoresis is a method that separates the molecules based on their molecular weights through a gel by applying an electric field. The movement of molecules is from the cathode (-) towards the anode (+) due to negative charge of phosphate group in the double-stranded DNA molecules at neutral pH. The supercoiled DNA moves faster than the linear form of the same plasmid and linear DNA moves faster than the circular nicked form of the same plasmid. Moreover, the movement of the DNA molecules can be affected by other factors including the conformation of nucleic acids, buffer composition, agarose concentration, strength of electric field and excess amount of intercalating dye such as ethidium bromide. In the agarose gel electrophoresis procedure, the samples are loaded into the wells by mixing DNA samples with a loading dye which includes glycerol and bromophenol blue. In this case, glycerol provides the samples to sink into the wells and bromophenol blue provides to visualize the DNA migration through the agarose gel. Also ethidium bromide provides to visualize the DNA bands in the agarose gel under the UV light [8]. 

MATERIALS&METHODS  

 pCV-1 plasmid                               –Ethidium bromide                          –Eppendorf tubes 

–Nanodrop spectrophotometry         –Restriction enzyme PstI                 –Pipette and tips 

–LB medium                                     –6X Gel loading dye                        –Glove 

–Tetracycline                                    –Miniprep Kit                                   —E.coli bacteria 

–TAE buffer                                     –Agarose gel electrophoresis apparatus 

–Alkaline Lysis Buffer I                  –Alkaline Lysis Buffer III 

–Alkaline Lysis Buffer II                 –Ethanol (100%-70%) 

ALS METHOD 

Firstly, overnight E.coli culture including the pCV-1 plasmid and TAT gene was centrifuged at ≥12000 x g for 1 min and supernatant was discarded. Then, bacterial culture was added into the remained pellet and centrifuged at ≥12000 x g for 1 min again for the precipitation of bacterial cells and the supernatant was discarded again. After that, 100 µl of ice-cold Alkaline Lysis Solution I is added into the remained pellet and gently vortexed. Then, 200 µl of Alkaline Lysis Solution II is added into the mixed solution without vortexing for preventing of plasmid and then 150 µl of Alkaline Lysis Solution III was added into the solution and waited on the ice for 3 minutes. After 3 min, the bacterial lysate was centrifuged at maximum speed for 5 minutes at +4 oC. After the centrifugation, the supernatant was transferred into a new Eppendorf tube and then 900 µl of 100% ethanol was added into the solution to precipitate the nucleic acids and vortexed and waited for 2 minutes at room temperature. Then, precipitated nucleic acids were centrifuged at maximum speed for 5 minutes at +4 oC in a microfuge for the collection of nucleic acids. After centrifugation, the supernatant was discarded and remained pellet was solved with 1 ml of 70% ethanol by inverting of the closed tube several times and again centrifuged at maximum speed for 2 minutes at +4 oC in a microfuge for recovering of the DNA. After centrifugation, the supernatant was discarded gently and the open tube was waited for evaporation of all liquid. Finally, the plasmid DNA was collected in the eluate and the amount of DNA was measured by Nanodrop spectrophotometry. 

Restriction reaction for ALS; 

After the Nanodrop measurements, the required amounts of buffer O (10X), plasmid DNA and water were calculated as 2 µl, 0,95 ~ 1 µl and 16 µl, respectively for the restriction reaction. Firstly, water and buffer were mixed and then plasmid DNA was added into the reaction mixture and finally, alredy calculated 1 µl of PstI enzyme was added into the mixture and mixture was incubated at 37 oC for 1 hour. 

MINIPREP KIT METHOD 

All stages in the kit were performed at room temperature. Firstly, overnight E.coli culture including the pCV-1 plasmid and TAT gene was centrifuged at ≥12000 x g for 1 min for harvesting of the cells. After the centrifugation, supernatant was removed and 1 ml of cells were added into the pellet in the microcentrifuge tube and then again it was centrifuged at ≥12000 x g for 1 min and after the centrifugation the supernatant was removed. After that, the remained bacterial pellet was resuspended with 200 µl of the Resuspension Solution including the RNase A by applying the pipetting until completely resuspension. Then, 200 µl of Lysis Buffer was added into the resuspended cells for the lysis of bacterial cells and waited for 5 minutes. After 5 minutes, 350 µl of Neutralization/Binding Buffer was added into the cells by inverting it 4-6 times and then the solution was centrifuged to precipitate the cell debris at max speed for 10 min. At the same time, column preparation was made by adding the 500 µl of Column Preparation Solution into a GenElute HP Miniprep Binding Column and applied the vacuum until the whole solution passes through the column. After that, the lysate formed in neutralization stage was transferred into the column and centrifuged at ≥12000 x g for 1 min and the passed lysate through the column was removed. Then, column was washed with 500 µl of Wash Solution 1 and again the passed solution through the column was removed and again column was washed with 750 µl of diluted Wash Solution 2 and passed solution through the column was removed. After this stage, the column was transferred into a new microcentrifuge tube (2 ml) and centrifuged at ≥12000 x g for 1 min. Then 30 µl of Elution Solution was added into the column for obtaining only plasmid DNA. Finally, the plasmid DNA was collected in the eluate and the amount of DNA was measured by Nanodrop spectrophotometry. 

Restriction reaction for Miniprep; 

After the Nanodrop measurements, the required amounts of buffer O (10X), plasmid DNA and water were calculated as 2 µl, 4.36 ~ 4.5 µl and 12.5 µl, respectively for the restriction reaction. Firstly, water and buffer were mixed and then plasmid DNA was added into the reaction mixture and finally, alredy calculated 1 µl of PstI enzyme was added into the mixture and mixture was incubated at 37 oC for 1 hour. 

RESULTS 

Results of Nanodrop Spectrophometry 

At the end of the plasmid DNA isolation processes, we’ve used the plasmid DNAs eluted from both Miniprep Kit and ALS methods for measurement of the total DNA amount in the samples which also required for the restriction reactions and also determining the ratio of A260/A280 and A260/A230 to check the purities of the obtained DNA samples. Thereby, we’ve placed the 2 µl of DNA samples into the Nanodrop spectrophotometry and measured the each value which given below table 1: 

Table 1: indicates that Nanodrop spectrophotometry results for plasmid DNA samples obtained by ALS and Miniprep Kit method 

  Miniprep Kit Method  ALS Method 
A260/A280  1.77  1.68 
A260/A230  1.81  1.83 
Total DNA (ng/µl)  229.2  5248.9 

 

Based on the determined total DNA amount for both Miniprep Kit and ALS method, we’ve calculated the required DNA amount for the restriction reactions as the total volume of reaction mixture has been reached to 20 µl. So, we’ve calculated the required DNA amount as 4.36 µl ~ 4.5 µl for restriction reaction of Miniprep Kit and as 0.19 µl ~ 0.2 µl for restriction reaction of ALS method which are shown below: 

Calculations for Miniprep Kit:           1µl             229.2 ng DNA  

x               1000 ng 

x = 1000/ 229.2 = 4.36 µl plasmid DNA 

We’ve calculated the required amount of DNA as 4.36 µl by using above equation but, we’ve used 4.5 µl of DNA in the restriction reaction and for the complete reaction we’ve calculated the amount of buffer and water which will be used in restriction reaction as shown below: 

M1 × V1 = M2 × V2                                So,       4.5+2+1 = 7.5 µl  

10X × V1 = 1X × 20 µl                                      20 µl – 7.5 µl = 12.5 µl of water 

V= 2 µl of buffer                                             (1 µl is the amount of PstI enzyme) 

 

Calculations for ALS:                       1µl              5248.9 ng DNA  

x               5000 ng 

x = 5000/5248.9 = 0.95 µl plasmid DNA 

We’ve calculated the required amount of DNA as 0.95 µl by using above equation but, we’ve used 1 µl of DNA in the restriction reaction and for the complete reaction we’ve calculated the amount of buffer and water which will be used in restriction reaction as shown below: 

M1 × V1 = M2 × V2                                            So,     1+2+1 = 4 µl  

10X × V1 = 1X × 20 µl                                      20 µl – 4 µl = 16 µl of water 

V= 2 µl of buffer                                             (1 µl is the amount of PstI enzyme) 

 

DISCUSSION 

In this experiment, at first, we’ve aimed the isolation of plasmid DNA including TAT gene from overnight E.coli bacterial culture in the LB medium including the tetracycline and so we’ve eliminated some of bacteria which are sensitive against tetracycline antibiotic, in other words, not have plasmid. Because, the pCV1 plasmid contains the tetracycline resistance gene and so we’ve selected the bacterial cells without plasmid by this way. Also, we’ve aimed to check that isolated plasmids are actually pCV1 or not in the presence of PstI restriction enzyme. For the isolation of plasmid DNA, we’ve used the 2 different methods including Miniprep Kit and ALS methods. In the Alkaline Lysis method, we’ve used some chemicals in which Alkaline Lysis Buffer I, II and III. Firstly, we’ve used the Alkaline Lysis Buffer I containing the glucose, EDTA and Tris-HCl agents for resuspension stage of isolation procedure. Glucose maintains the osmotic pressure for preventing the cells to burst. EDTA is chelating agent that discards the Mg ions which important for cell envelope structure and also prevents the activity of DNases which degrade the DNA structure and makes cells more permeable to SDS agent for lysis processes. Therefore, we’ve aimed to preserve the plasmid DNA structure. Also Tris-HCl arranges the pH level of the environment. Secondly, we’ve used the Alkaline Lysis Buffer II containing NaOH and SDS for cell lysis stage of isolation procedure. In this case, NaOH helps to break down the cell wall and also breaks down the hydrogen bonding between the DNA bases, in other words, it makes from double-stranded DNA molecule to single-stranded DNA molecule. SDS is a detergent which provides the solubilization of cell membranes by removal of the lipid molecules and so, it provides the chromosomal DNA and plasmid DNA to pass into the solution. Lastly, we’ve used the Alkaline Lysis Buffer III containing the potassium acetate, glacial acetic acid and water for neutralization and reducing of the pH processes. Potassium acetate provides the reducing the pH level by precipitation of SDS with its lipids and proteins and also glacial acetic acid brings the pH to neutral and so, DNA strands can be renatured. Moreover, at the end of the Alkaline Lysis method, we’ve also used the 100% ethanol to prevent the dissolving of DNA molecule and also to precipitate the other salts or anything and before measurement by Nanodrop, we’ve used 70% ethanol for precipitation of plasmid DNA. After additional of ethanol chemicals, we’ve waited for evaporation of entire alcohol from the DNA sample to obtain accurate measurements by Nanodrop spectrophotometry. Thereby, after evaporation,  we’ve measured the amount of DNA and also A260/A280  and A260/A230 ratios by Nanodrop spectrophotometry. Other method which we’ve used was Miniprep Kit method. Actually, when we’ve compared the two plasmid DNA isolation methods, we’ve realized that Alkaline Lysis is a manual method which used basic three buffers with different components and also alcohol for the precipitation of DNA and so it’s also known as alcohol precipitation method, however; although the Miniprep Kit method is similar to ALS method, it is applied based on the small-scale commercial kit procedures including a spin-column and also different types of solutions and buffers which also show the similar effects with buffer components in ALS method. Also, we can obtain more plasmid DNA molecule by Alkaline Lysis method compared to Miniprep Kit. So, we’ve used the some chemicals including the Resuspension Solution containing RNase, Lysis Buffer, Neutralization/Binding Buffer, a GenElute HP Miniprep Binding Column, Column Preparation Solution, Wash Solution 1 and Wash Solution 2 and Elution Solution during the Miniprep Kit procedure. We’ve used the Resuspension Solution for resuspending the cells like ALS I buffer and in this method it contains the RNase, unlike in the ALS method, there is not RNase. This means, the RNA contamination can observe in the Nanodrop results for ALS method. Also, we’ve used Lysis Buffer for cell lysis stage like ALS II buffer, Neutralization/Binding Buffer for neutralization like ALS III buffer, a GenElute HP Miniprep Binding Column, Column Preparation Solution, Wash Solution 1 and Wash Solution 2 for removal of other components like salts and Elution Solution for elution of plasmid DNA molecules together. At the end of the Miniprep Kit procedure, we’ve measured the amount of DNA and also A260/A280 and A260/A230 ratios by Nanodrop spectrophotometry. According to Nanodrop results, we’ve not observed any alcohol contamination for both ALS and Miniprep Kit. Because, A260/A230 ratio checks the alcohol contamination and we’ve measured the ratio as 1.81 for Miniprep Kit and 1.83 for ALS method. If these values could be less than 1.8, it could be said that alcohol contamination occurred in these procedures. This result was expected. Also we’ve observed protein contamination for both ALS and Miniprep Kit. Because, A260/A280 ratio checks the purity of DNA preparation as 1.8 value and so, we’ve measured both value as less than 1.8 and there can be protein contamination for both procedure. Because, proteins give absorbance at 280 nm and so accumulation of proteins in the samples or not completely removal of them can be caused thid situation. In other words, in the presence of high concentrations of proteins, proteins can contribute to the 260 and 280 absorbance. This result was unexpected. However; when we’ve compared the total DNA amount of both ALS and Miniprep Kit methods, the results were expected meaning that more DNA can obtain by ALS method instead Miniprep Kit method. After the Nanodrop measurements, we’ve prepared the restriction reaction mixture by using the calculated amounts of buffer O (10X), obtained plasmid DNA, PstI restriction enzyme and distilled water. When we’ve prepared the reaction mixture, we’ve take car about the order of materials which will be added and so, we’ve mixed at first distilled water, buffer and plasmid and at last we’ve added the enzyme into the mixture for preventing the enzyme activity before formation of the best media and also preventing the denaturation of enzyme. On the other hand, for the visualizing the uncut and digested DNA molecules obtained by ALS and Miniprep Kit method, we’ve performed the agarose gel electrophoresis method by using the Ethidium bromide for observation of DNA bands under the UV light and loading dye including glycerol providing the samples to sink into the wells and bromophenol blue for visualization of DNA migration through agarose gel. For this process, 1% agarose was prepared in the TAE buffer and samples were run on 100 volt for 30 minutes. For this purpose, the ladder with 10.0 kb in length was used and at the end, the samples were loaded into the wells of gel in order to, first ladder, uncut ALS isolated plasmid (1 µl of plasmid), digested ALS isolated plasmid (5 µg of plasmid), uncut Miniprep isolated plasmid (1 µl of plasmid) and then digested Miniprep isolated plasmid (1µg of plasmid). Thereby, the gel image was obtained. The used pCV1 plasmid is 10.3 kb in length and so when it was digested by enzyme, the digest fragments should be 7.0 and 3.3 kb in length. In other words, we should observe one band at only about 10 kb for uncut ALS and Miniprep isolated plasmid and also two bands at about 7.0 kb and 3.0 kb for digest ALS and Miniprep isolated plasmid. So, depending on the agarose gel image, the results for the digested ALS and Miniprep isolated plasmid were expected. Because, two bands formed at about 7.0 kb and 3.0 kb, but also a smear formed in the lane of digested ALS isolated plasmid which not expected result. The reason of this can be contamination of DNA with protein meaning that can be accumulation of protein or not completely removal of the protein from the samples and also some RNA contamination due to absence of RNase enzyme in the ALS procedure. Also the reason can be excess salts which accumulate by deficiency of ethanol precipitation. Moreover, the DNA can be degraded by DNases due to error in the EDTA agent. However; when we’ve examined the results of uncut ALS and Miniprep isolated plasmids, we’ve observed that more than one band as a smear between 6.0 kb and 10.0 kb for uncut ALS plasmid with also a smear which can be any protein or RNA contamination and also one band at about 8.0 kb and one band at about more than 10 kb for uncut Miniprep plasmid. Actually, we should observe one band for these uncut samples, however; the reason of this can be supercoiled DNA and the reason of other unexpected results can be related to E.coli cells or not completely elution of plasmid. As a result, when we’ve examined the whole gel image, we can also realize that the supercoiled (uncut) DNA migrates faster than linear (digested) form of the same plasmid which also expected result. 

REFERENCES 

  1. Bacterial Plasmids 

https://www.csun.edu/~hcbio027/biotechnology/lec2/PL/pl.htm 

  1. What is a Plasmid? 

https://www.addgene.org/mol-bio-reference/plasmid-background/ 

  1. Arya SK., Guo C, Josephs SF, Wong-Staal, F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science 229:69-73, 1985. 
  1. Sabine Ehrt and Dirk Schnappinger, Isolation of Plasmids from E. coli by alkaline Lysis, Methods in Molecular Biology, Pages 79-82. 

Craig Winstanley and Ralph Rapley, Extraction and Purification of Plasmid DNA, The Nucleic Acid Protocols Handbook (2000),Pages 327-331 

  1. How do I determine the concentration, yield and purity of a DNA sample? 

https://worldwide.promega.com/resources/pubhub/enotes/how-do-i-determine-the-concentration-yield-and-purity-of-a-dna-sample/ 

  1. REBase: Database from New England Biolabs with detailed information on restriction endonucleases. 
  1. Nasri, M. and Thomas, D. (1986) Nucleic Acids Res. 14, 811. PMID: 3003698 
  1. Gel Electrophoresis 

http://www.phschool.com/science/biology_place/labbench/lab6/gelelect.html 

 

 

 

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