Evaluating Methods of High Molecular Weight DNA Extraction for the Deinococcus genus

 Introduction:

    Due to the importance of preparing a protocol for future genetic sequences of members of the Deinococcus genus, further testing was done to evaluate methods of DNA extraction for high molecular weight. The primary focus this week was to utilize the kit that gave us a successful sequence of D. aquaticus and modify that method for the future paper that does not require as high molecular weight in samples as does when evaluating isolates. D. aquaticus and D. radiodurans are effectively being used as standards in preparation for many different types within the genus. D. sonorensis was also briefly evaluated this week due to it's strange plaque like structures that it forms. It is still hopeful that a chemical process, can be used without mechanical processing, in the Zymo kit. Above all else, it is imperative to accurately determine if results can be reproduced effectively using whatever protocol is ultimately decided upon. 


Methods: 

    The below protocol was followed identically as seen with the exception of the bead beating time. Bead beating was done for 30 seconds, 1 minutes, 2 minutes, and 3 minutes for a sample of Deinococcus radiodurans and Deinococcus aquaticus. For the second set of data collected this week one more sample from each type of Deinococcus was done with bead beating for 30 seconds, to evaluate repeatability. One sample of D. radiodurans and D. aquaticus were done identically except with a bead beating tube with glass beads instead of the one provided. A total of 0.15 g of two sizes of beads was added to each empty tube for this step. D. sonorensis was also evaluated with both methods, 30 seconds of bead beating with a standard tube and a tube with glass beads. 


D. radiodurans and D. aqauticus also tested for the protocol shown below, in which a Zymo kit was utilized for the lysis portion of the procedure and a DNA cleanup kit done to evaluate the results. 



DNA cleanup procedure

All samples were tested using a Nanodrop One and run on agarose gel of 1% with 1X TAE buffer with 10 microliters of sample loaded into each well. 

Results:

    


Tube

ug/ul

260/280

260/230

Aqua30

74.2

1.93

1.98

Aqua1

68.9

1.93

1.88

Aqua2

112.1

1.94

2.11

Aqua3

71.9

1.92

1.62

 Exp 1 (Tuesday) 

 

Tube

ug/ul

260/280

260/230

Radio30

47.3

1.90

1.50

Radio1

117.5

1.93

1.95

Radio2

79.6

1.92

2.04

Radio3

135.4

1.93

2.02

Exp 1 (Tuesday) 


Tube

ug/ul

260/280

260/230

Aqua Glass

72.7

1.96

1.42

Aqua 30

93.5

1.96

2.00

Aqua Zymo

1.1

1.36

0.33

Son30

1.9

1.05

0.40

 Exp 2(Thursday) 

 

Tube

ug/ul

260/280

260/230

Radio Glass

170.4

1.94

1.97

Radio 30

119.6

1.93

2.02

Radio Zymo

1.5

1.85

0.08

Son Glass

1.8

1.75

1.40

Exp 2 (Thursday) 

                                                      Gel 1 (left to right) D. radiodurans 3 minutes, 2 minutes, 1 minute, 30 seconds. D. aquaticus 3 minutes, 2 minutes, 1 minute, 30 seconds. Indy III ladder. 

                                                        Gel 2 (left to right) D. aquaticus 30 seconds Thursday sample, glass beads, 30 seconds Tuesday. D. radiodurans 30 seconds Thursday sample, glass beads, 30 seconds Tuesday sample. Indy III ladder. 

                    

Conclusions:

    The samples that did not have sufficient DNA to run on a gel, including the D. sonorensis samples and the Zymo samples, show that chemical lysis is likely not possible at this point and that D. sonorensis will likely need special accommodations. It is decided further testing will be done with this type, and a special mechanical step, not bead beating, will be used to break down the plaques for sufficient testing and hopefully DNA extraction. The ng/ul of each sample that had a value above 2 was roughly identical for D. aquaticus across the board, but D. radiodurans had more variability, The results on the gel show that the separate samples made Tuesday and Thursday are nearly identical in molecular weight and ng/ul which is a solid representation of reproducibility. The samples using glass beads, particularly the D. radiodurans, shows higher molecular weight and may indicate that this method should be utilized going forward for optimal sequencing results.  

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