Evaluating Methods of DNA Isolation for High Molecular Weight Segments

 Introduction: 

    In a previously successful attempt to genetically sequence the genome of Deinococcus aquaticus, which mapped 95% of the genome, the DNA isolation procedure utilized was a methodology that provided high concentrations of DNA, but lacked in molecular weight. Larger segment sizes of DNA from isolation is ideal for more complete genomic sequencing, and a goal for future sequencing runs of other types of bacteria. The average base pair length of the previous run was around two to three thousand base pairs, whereas the goal moving forward is potentially ten thousand base pair segments of DNA for genomic analysis. The protocol for DNA isolation used was a secondary method to a QIAGEN kit that specializes in producing high molecular weight samples for testing. The QIAGEN kit had been tested to evaluate potential samples for genetic sequencing, but each trial was unsuccessful, all attempts not producing significant amounts of DNA. It was determined that the protocol may not be suitable for Deinococcus species, and may need to be supplemented with a mechanical lysing step which is not originally included, presumed to prevent DNA breakage. But Deinococcus aquaticus as well as others in the genus, are notoriously difficult to lyse and it is suspected that incubation with lysozyme, as used in the protocol, was not effective enough to lyse the cells necessary, which is why little to no DNA was evident in the resulting sample. The methods of mechanical manipulation and adjustments to time and methods was evaluated this week to increase DNA yield from this protocol, and hopefully maintaining a high molecular weight of approximately ten thousand base pairs. 

Methods; 

 Deinococcus aquaticus was inoculated in ten milliliters of TGY broth media and allowed 48 hours for growth in an agitation incubator at thirty degrees Celsius. An OD600 reading was taken and the culture diluted with TGY broth in a separate tube for a total of one ml volume, and a secondary OD readings taken to ensure it was within protocol standards (0.05 to 0.3). 

The QIAGEN protocol was followed for steps two through four. The one ml sample was equally aliquoted into four separate tubes, and then five microliters of lysozyme was added to each tube and 11.25 microliters of proteinase K. Tube A was inverted by hand continuously for 60 seconds prior to incubation, and Tube B was put in the bead beater on  1m/s for five seconds prior to incubation. All four tubes were incubated for 45 minutes, rather than 30 minutes as the protocol calls for. Post incubation, Tube C was treated mechanically identical to Tube A and Tube D was mechanically treated identical to Tube B. The samples were tested for dsDNA concentrations.  

Another trial was accomplished identically except the time were adjusted for each tube. The initial OD600 reading was also kept within 0.001 between the two trials. The inverting time of tubes A and C was decreased to 30 seconds, and tubes B and D were put in the bead beater for two seconds. Tubes A and B were still pre incubation and C and D post incubation. The samples were tested for dsDNA concentrations. 

A 0.5% agarose gel was run with the second set of samples, using a HindIII ladder and 1x TAE solution. The samples were prepared by the same QIAGEN protocol, which called for adding 0.7 volumes of isopropanol to each sample, which was 140 microliters for each, and after allowing a pellet to form washing the pellet with 70% ethanol (220 microliters), centrifuging, and then pouring off the supernatant. The sample was then resuspended in 20 microliters of TE with a pH of 8.0. The gel was ran at 85 mA and for approximately thirty minutes before analysis was conducted. 

Results:

    The first round of samples are shown in Figure 1, and had a DNA concentration between 210 ng/ul and 301 ng/ul. The 260/280 and 260/230 values were low but disregarded at this stage of the protocol. The second set of samples are shown in Figure 2, with three of the four samples being between 37 ng/ul and 39 ng/ul and the tube D being the outlier at 98.9 ng/ul. The gel ran on these samples produced no bands or smears for any of the samples. 

Tube	ug/ul	260/280	260/230
A	290.4	0.49	0.19
B	284.6	0.48	0.18
C	301.0	0.49	0.19
D	210.7	0.61	0.08

Figure 1

Tube	ug/ul	260/280	260/230
A	39.2	0.91	-0.75
B	38.6	0.95	-0.95
C	37.2	1.00	-0.73
D	98.9	0.68	-0.69

Figure 2

Conclusions:

    The difference of values seen in set one and set two are substantial. The samples in set one were not satisfactory because they indicated too much mechanical disruption had been implemented, and the goal is to find the most minimal amount of mechanical force necessary to produce a sample with approximately 100 ng/ul after the lysing step of the protocol, because it is presumed that that would be enough to compensate any losses and result in a sample for genomic sequencing with enough DNA at the highest possible molecular weight. Any additional mechanical disruption more than necessary will potentially break the DNA fragments. Decreasing the time of mechanical manipulation by approximately half produced the samples in set two, which have significantly lower concentrations, besides tube D. Further trials should be conducted to evaluate the correct amount of time for each method that is needed to find the correct balance between sufficient cell lysis and high molecular weight DNA. The data does potentially indicate there is not much of a difference in DNA yield between pre and post incubation methods, and maybe for bead beating and inverting as well. A successful gel in the future will be able to indicate if the two methods have any effect on the size of the DNA fragments, but the gel was unsuccessful this time so no indication was given. The gel was likely a failure due to misunderstanding or mistakes in the cleaning process for each sample since the directions were very vague. There is no complete certainty the cleaning process was accomplished correctly, which would explain why no smears were seen of any kind.