Isolation of Deinococcus radiodurans' genomic DNA

 A Summary of DNA Isolation

February 25th, 2022

Overview

The process of DNA isolation is one with many steps, and its uses are numerous, one of which is carrying out PCR type reactions. This week's laboratory procedure involves isolating genomic DNA from a broth culture of Deinococcus radiodurans with an average OD600 reading of 5.30. The purpose of this DNA isolation is to gather genomic DNA for use in the future in PCR type reactions that amplify fragments of the zwf gene iconic for the stress-resilience properties of D. radiodurans under oxidative stress. The zwf gene in D. radiodurans is known to code for the enzyme glucose-6-phosphate 1-dehydrogenase (G6PD), a key enzyme in the first step of the Pentose Phosphate Pathway (PPP). Primers chosen from the two ends of the zwf gene will be ordered in the future to amplify the gene using PCR. Subsequently, this gene is to be deleted and replaced with the gene which codes for kanamycin resistance isolated from Escherichia coli through a process called Gibson Assembly. 

Procedure

For this procedure, a protocol for the DNeasy UltraClean Microbial Kit is being used. The steps of DNA isolation can be summarized in the following bullet points:

• About 1.8 mL of  D. radiodurans broth culture samples (4 samples) is added to a Microcentrifuge Tube and centrifuged for 1 minute to collect a pellet of cells after discarding the supernatant.
• These cells are then resuspended in 300 microliters of PowerBead Solution containing a buffer and salts to help homogenize them.
• 50 microliters of Solution SL is added to the mixture which was transferred to a PowerBead Tube and vortexed for 10 minutes. Cell lysis takes place during this step. A centrifugation step for 1 minute follows to get rid of cell debris on the bottom of the tube. 
• After transferring the resulting supernatant to another Microcentrifuge Tube, 100 microliters of Solution IRS is added and the mixture is then incubated at 4 degree Celsius for 5 minutes followed by a centrifugation step. This ensures that non-desired cell debris and proteins precipitate to the bottom of the tube. The supernatant is then transferred to a clean Microcentrifuge Tube without disturbing the pellet of left over cell materials.  
• 900 microliters of Solution SB is then added to help the existing DNA bind to the MB spin Column membrane to which the resulting mixture is then transferred. This is done in 700 microliter batches to fit the spin column. A centrifugation step for 1 minute follows each transfer. DNA is now bound to the membrane of the spin column. 
• 300 microliters of Solution CB is added to the MB Spin Column to wash the DNA that's bound to the membrane from residue salts. The flow-through of the wash is discarded followed by centrifugation. 
• The MB Spin Column is transferred to a clean Microcentrifuge Tube and 50 microliters of Solution EB is added to the center of the membrane of the spin column. This releases the bound DNA into the Microcentrifuge Tube.
• DNA is now ready for different applications. It is then stored as a frozen stock solution. 

After 4 samples of 50 microliters of DNA are isolated, the OD260 of each sample was measured. The absorbance of the double stranded DNA was as follows:

Sample 1: 14.5 

Sample 2: 235.7

Sample 3: 128.9

Sample 4: 190.9

Conclusion

As shown above, the assay of purifying DNA is a complex process with many chances of error, so the resulting amount of DNA depends on the amount of bacterial cells used in the first step. DNA is an organic compound that needs to be handled carefully, thus numerous buffer solutions were used to guide the behavior of this compound. For example, the highly concentrated salts Solution SB ensures that DNA binds to the membrane silica of the MB Spin Column while Solution EB contains no salts and is slightly basic (PH 8) to unbind the DNA from the membrane and release it into the collection tube. One experimental error that occurred during the isolation procedure was that a different spin column was used for sample 1, so a sufficient concentration of DNA was not collected, and this is evident in the OD260 reading of sample 1 (14.5). This absorbance is extremely low, so this sample is not reliable for carrying out PCR type reactions. Thus, this isolated DNA sample will be discarded. 

All in all, DNA purification is an important stepping stone for many molecular applications, and the process has countable steps to minimize error during the production of a highly pure DNA solution.