https://www.researchgate.net/post/How_to_process_hydrolysed_genomic_DNA_for_running_in_HPLC_MS

Dear Dr. Chung

I suggest increasing the DNA degradase to 10-20 µl to ensure complete digestion of DNA.

Hi Seied,

Have you tried using 10-20 ul of degradase to digest genomic DNA samples?

Thanks,

Lyra

According to the Zymo Research Corporation's protocol, it is suggested to increase the enzyme concentration to 5-10 times to ensure complete digestion of DNA.

Hi All,

Yesterday I have set up an experiment to track the genomic DNA degradation by LC-MS. I used 0.5ug of genomic DNA and digested with 1ul, 2ul or 3ul of DNA degradase plus. I quench the reaction after 0, 2, 4, 6 hrs and overnight by 87.5ul of 0.1% formic acid. The samples were injected into the LC-MS.

I have attached my data for the 1ul here (adding more enzyme do not help at all - I will attach the files tomorrow). It seems that comparing to the pure 5mdC and dG sample, there are some chunk of DNA that is not degraded overnight.

I am contacting the Zymo Research tech support right now.

Any suggestion for protocol optimization will be deeply appreciated!!

Best,

Lyra

Hi All,

Here are the data of adding 2ul and 3ul of DNA degradase plus to degrade 0.5ug of genomic DNA.

Today I am trying two different modifications:

(1) Mix 0.5ug of genomic DNA and ddH2O, heat the sample at 100 degree for 5 mins and put the sample on ice for 2 mins. Afterward, add 1ul of DNA degradase plus and buffer. Incubate at 37 degree. Quench the degradation at 0, 2, 4 hr by 87.5ul of 0.1% formic acid.

(2) Mix 0.5ug of genomic DNA and ddH2O, Add 0.9ul of RNaseA (20mg/ml stock), incubate for 10 mins. Afterward, add 1ul of DNA degradase plus and buffer. Incubate at 37 degree. Quench the degradation at 0, 2, 4 hr by 87.5ul of 0.1% formic acid.

Hopefully I can see some differences! I suspect that there are some structures with the methylated part of genomic DNA that is preventing the degradase activity.

We will see. I will update my results here.

Best,

Lyra

Hi

Did you contact the Zymo Research Co., what was their recommendation?

Following is their reply:

One thing that we do notice is that the signal for your actual samples are very low in comparison to the Pure 5mdC and dG. Since your actual samples signal is low, it will be more prone to noticing the background. If you look at the pure 5mdC and dG, the intensity of the signal is much higher, so it will minimize any background. Usually the lower intensity may indicate that you may need to increase your sample input in order to get more distinguishing peaks then compared to the background. In addition, to confirm that your DNA concentration is correct, we would recommend using a dsDNA -specific quantification methods such as Qubit or Picogreen to quantify your DNA.

I know you said that the kit contains RNAse in the protocol, but sometimes the RNase may just degrade the containing RNA, but it isn’t removed – it may just be lower molecular weight nucleic acid. Using Nanodrop will inflate your actual DNA concentration because it cannot distinguish DNA and RNA. Therefore, you will actually be putting less DNA into the system then you think, causing the lower signals. If you can double check your DNA concentration by using a dsDNA-specificmethod, it would be very helpful in diagnosing the situation. We have found that accurately quantifying the dsDNA helps with the consistency in the LC/MS results on our end.

In addition, several of the peaks that you notice are not actually an accumulation of 5mdC. 5mdC signal is very specific (see below). There are many derivatives of cytosines, so several of the peak that you may be noticing may also be due to 5hmdC, 5mdC, and dC.

As for your 0 hour start control, how do you generate that? Do you add the DNA directly to the reaction and immediately followed by the solvent, or did you try to inactivate the enzyme before that? Although 37C is the optimal temperature, the enzyme is very robust and start digesting the sample at room temperature.

Some update:

1. Heat denaturation help a bit, but not much.

2. RNase doesn’t help at all.

3. I am going to switch to using Nuclease P1, phosphodiesterase 1, and alkaline phosphatase treatment. It’s better since at least I know the identy of enzymes I am working with and it’s easier to optimize.

Update:

1. I found that the DNA degradase (Zymo Research) is contaminated by some nucleosides (or other small molecules) that give me those extra peaks. I just mixed 98ul 1% formic acid + 2 ul of degradase plus (inject 20ul) and I saw those extra peaks without adding any genomic DNA (see the attached file).

2. I quantified my DNA with Qubit and the concentration is quite similar between using nanodrop and the Qubit dsDNA BS kit. So the DNA concentration is not the issue.

I think I just have to give up on the DNA degradase Plus for my assay. I am going to optimize my assay based on the attached paper and use Nuclease P1 (Wako), phosphodiesterase 1 (Worthington), and alkaline phosphatase (CIAP) (NEB).

I made following solutions and inject into LC-MS:

(1) 1ul of Nuclease P1 (1U/ul) + 109 ul 0.1% formic acid

(2) 1ul of Phosphodiesterase I (12.5 mU/ul) + 109 ul of 0.1% formic acid

(3) 1ul of CIAP (10U/ul) + 109 ul 0.1% formic acid

At least this time no peaks were found close to where 5mdC or dG peaks are. We are going to try optimizing the protocol. I will let you guys know how it goes.

What was the final update of the project?