Methods of DNA Isolation

Isolation of DNA is a very important technique which is the foundation for many types of techniques such as the diagnosis of many genetic diseases as well as fingerprinting DNA. How much amount and purity required, the DNA type is what makes the difference for the different methods for DNA isolation. There were three different E. coli cultures the aim was the analyse the DNA of the E. coli.

Multiple amounts of techniques were used to manipulate and isolate the DNA from E. coli. We start of by Isolating the plasmid DNA from the 3 cultures using alkaline lysis. Alkaline lysis is an extraction method used to isolate plasmid DNA from bacteria. Next the DNA which has been isolated restriction enzymes is used for digestion. Restriction enzymes are able to cleave DNA and make them into fragments and this is within the molecule at sites called restriction sites. Bacterial transformation was also done. Finally, is the analysis of the E. coli which is transformed and this is achieved by using agarose gel electrophoresis.

Practical 1

Three E. coli cultures A, B and C were provided which has been grown overnight shaken at 37°C. The bacterial pellet is dissolved in 100 ml of solution 1. Solution 1 contains 50Mm glucose the glucose aims to provide an osmotic balance between the cell and the solution and this prevents the cells from bursting at this stage. Solution 1 also contains 25Mm Tris (pH 8.0) this is used to stabilise the ph in the solution. EDTA 10Mm is also a chemical which is necessary to allow DNA degrading enzymes. The main purpose for EDTA is to bind to magnesium and calcium and this stops the DNA from degrading. The EDTA is also able to stabilise the DNA phosphate backbone as well as the cell wall.

Next solution II is added, solution II is 0.2M NaOH and 1% (w/v) SDS. This strong alkaline solution is able to disturb the cell membranes and allows to come in contact and denature the plasmid and chromosomal DNA. The cell contents made contact with the extracellular chemicals which allowed EDTA to chelate with the metal ions in the cells. SDS precipitate with proteins in the cell contents and form insoluble complex. As a result, precipitates were observed in the solution. Chromosomal DNA and plasmid DNA were denatured by the high pH in the solution. The process is known as denaturation as it

Solution 3 is added next which is 3M potassium acetate pH 4.8. The potassium acetate is able to decrease the alkalinity of the solution so it able to renature the plasmid DNA but does not renature the chromosomal DNA. The ssDNA can re nature the dsDNA because the hydrogen bonds between the single stranded DNA is re-established. Through hydrophobic relations a white precipitate is formed by the SDS, denatured cellular proteins and the single stranded genomic DNA all sticking together whereas the double stranded plasmid dissolves in the solution.

At this point most of the cells debris is separated from the plasmid DNA but in the solution there is the debris the salts, Rnase and EDTA so the solution has to be cleaned up and the plasmid DNA concentrated. 70% ethanol is added next and it is able to change the DNA’s structure as they aggregate and precipitate from the solution. Using centrifugation, the DNA which is precipitated can be separated.

Practical 2

From the E. coli which had been isolated next we begin to degrade the DNA using restriction nucleases. Restriction enzymes cut DNA molecules in specific areas to cut them into smaller fragments. Different kind of DNA sequences are cut and recognised by different restriction enzymes. These restriction enzymes also need a buffer which is suitable this includes magnesium as a co factor. A certain concentration and a Tris to buffer the ph. For different kinds of enzymes there are different optimum salt concentrations. Samples B and C are isolated with 10 units of enzyme.

There are 2 tubes called tube BR and tube CR. Tube BR contains DNA B, 10 x EcoR1 buffer, EcoR1 enzyme and water, Tube CR contains the same but instead of DNA B it contains DNA C. There is a specific order in which these are added. Firstly, water is added then it is the buffer, the DNA and then finally it is the enzyme. The reason for this order is because a suitable environment has to be created before the enzyme is added. Eco R1 is basically a restriction enzyme isolated from E. coli, which at particular locations cuts DNA double helix at specific restriction site. EcoR1 is able to make cuts in the backbone of both of the strands, and this allows there to be two sticky ends at the cutting site of the DNA.

There is a specific sequence which the EcoR1 can recognise this sequence is GAATTC and the enzyme cuts in between the G and A on the strand which is complimentary. To start the solution is added orderly with water, buffer and the enzyme. The water is used to dilute the buffer because the manufacturer concentrates the buffer. The EcoR1 buffer is there as it is the optimal buffer used for the enzyme performance. When the conditions are finally suitable for the enzyme it is added. This is when it opens up or fragmentise the plasmid DNA. Once the both the tubes had been completed they were incubated.

Transformation of plasmid

Now a technique known as bacterial transformation is used and two tubes B and B are diluted in a Tris buffer at pH 7.6 and this makes up 40 folds of the final volume of the mixture, as they have been diluted they are labelled diluted B and diluted BR. The purpose of bacterial transformation is to introduce DNA into bacterial cells. There are many techniques used to achieve this but the reliable technique is a heat shock technique. When the DNA has been taken up it has to either join with a host genome or autonomously replicate. Circular forms of DNA are the only DNA which are going to be able to replicate, the linear form which use restriction enzymes will not be able to transform. The circular form when introduced to E. coli will be able to replicate.

The heat shock technique uses calcium chloride which creates a calcium rich environment, between the plasmid DNA and the bacterial cellular membrane the rich calcium environment cancels the electrostatic repulsion between them. In the bacteria pores are created as there was been a sudden increase in the temperature so this allows the entry of plasmid DNA to the bacterial cell. When the cell takes up the DNA it establishes itself to create a steady tranformant. In the practical the unknown strain of E.coli cells were added with calcium chloride and pre cooled in ice. The procedure is repeated twice and kept in ice. At the same time tube 1 which contains no plasmid DNA is prepared, tube 2 containing diluted plasmid B is prepared and tube 3 containing diluted plasmid BR is prepared.

The pre cold competent cells were added to the tubes 1 2 and 3 and mixed gently. As the cells are fragile, it is important to avoid using the vortex. After 15 minutes the tubes were shocked with hot water at 42°C. At this stage the cell membrane becomes thinner and plasmid DNA can enter the cell body. After 2 minutes the tubes were set in ice for 5 minutes to allow the cell membrane to recover. L broth is added to each tube and water bathed at 37°C for at least 20 minutes. After that cells in each tube were transferred LB amp plate spread and incubated overnight.

Practical 3

Agarose Gel electrophoresis is a common used method for analysing the size, purity, quantity and the sequence of DNA molecules and plasmid DNA molecules. Agarose is a polysaccharide it is a one of the components to agar and is extracted from red seaweed. It is also made up of anhydrous-galactose units. There are many reasons why the agarose gel is beneficial for gel electrophoresis. Between the polysaccharide unit’s non-covalent bonds are formed by the agarose gel. A sol state is formed as non-covalent bonds hold the structure of the agarose gel so it undergoes a phase transition at high temperature, When the running buffer and the agarose powder is mixed it creates the gel with later arrangement of the sol state at a higher temperature and also arranged cooling.

To start an agarose gel is prepared by the molten agarose being poured in the former. Wells are formed in the DNA sample for the DNA to be loaded by combs, they are then left for approximately 20 minutes to set. Once the gel has been set TBE buffer is used to carry a current and provide ions it is also able to maintain the Ph. As we know DNA is negatively charged, so when the electric field is applied for the period of electrophoresis there will be movement of the DNA towards the anode which is the positive pole. The sample loading wells be towards the negative pole which is the cathode, so when the gel is placed in the electrophoresis tank it is orientated.

A loading buffer solution is used to treat the plasmid sample before it is loaded on the gel. The density is increased of the sample as the loading buffer contains glycerol. DNA is able to travel towards the positive electrode as the larger fragments are slowed down in compare to the smaller fragments which is why they do not travel far. A band is also formed as all the fragments gather at a point and travel at more or less the same rate. So now when all the fragments have travelled and separated the different sized fragments, there is a dye known as SYBER-SAFE and this is used to visualise the DNA. When the dye is hit by a UV radiation there is an orange coloured fluorescent light. Finally, the UV trans illumination photographs the gel which contains the stained DNA molecules. A loading buffer completes the circuit as well as balances the pH in the gel.

Results

The transformed E. coli from tube 1, 2 and 3 were grown in the agar plate. Tube contained 0 colonies, tube 2 contained 300 colonies and tube 3 had contained 5 colonies. Tube 1 contained 0 colonies as it only contained buffer. Tube 2 had contained 300 colonies as it contained circular plasmid DNA. Tube 3 had only contained 5 colonies as it only contained linear DNA so the only way it may have contained colonies may have been contamination or mutation. From the results Tube C shows it did not travel far as the molecules were large or had a low molecular charge so they were not able pass through the gel network. Incomplete precipitation of the chromosomal DNA could be a possible error.

It is known that tube A contains no plasmid but tubes B and C contain plasmid. It is because the results show that there are 2 DNA bands on both tubes B and C but there are no DNA bands in tube A. The plasmid DNA which is within E. coli is in a circular form. The bands on the top of tubes B and C are known as nick DNA they were linearized by alkaline solutions and are not able to renature. Circular DNA is able to travel further as its shape causes less friction and it is smaller in size. The nick DNA does not travel far as it is in a linear form which causes more friction. And it has a larger molecular size.

Plasmid DNA in tube C is cleaved into two fragments by the restriction enzyme because it contains two recognition sequences. The band of the two fragments can travel further than the band of plasmid DNA C because they experience less resistance. Therefore, the band of the linear plasmid DNA C has lower position than circular plasmid DNA C. Whereas plasmid in tube B cleaves at only one recognition site the circular plasmid opens up forming a nick DNA. Which experience higher friction than the circular plasmid DNA. Therefore, circular plasmid DNA C can travel faster than the nick plasmid DNA C forming a band at a low position.

The marker X contains linear double stranded DNA with no molecular weight. When it is loaded in electrophoresis, the DNA with different molecular weight runs to different position and forms a DNA ladder. By comparing the bands of linear plasmid DNA from B and C with the ladder the molecular weight of the plasmids is known. Linear plasmid DNA of E. Coli B has a molecular weight of 3kbp. The linear plasmid DNA fragments have molecular weight of 4.7 and 5.3 kbp therefore the molecular weight of plasmid DNA C is 10 kbp

Discussion and Conclusion

To start most of the macro molecules such as the proteins, chromosomal DNA and high molecular weight RNA were all removed by the process of Alkaline Lysis. But from the results it has shown there was some macromolecules which had remained in tube C. The possible reason for the Tube C containing the macromolecules could be Tube B+ had the RNA degraded using the RNase. When the alkaline lysis experiment was performed there was no single stranded linear DNA when denaturing was done, and this has shown that there was control on the pH. 5 colonies of antibiotic resistant were grown on plate 3 by linearized plasmid B. There was linearized plasmid in tube BR but at low points the reason for this is there was no circular plasmid DNA seen in electrophoresis. Post inspection should be done on the colonies formed on the L-amp plate. When bacterial transformation had been carried out there may have been a chance some of the plasmid B which is linearized has merged with the chromosomal DNA.