HPLC protocol for methylation quantification - (Nov/12/2005 )

Cheers HN!

I will give it a go and then let you know how it turns out.

Thanks again
Dave

I've tried out your method HN, but it isn't quite what I was hoping for! I've had better results using DNAse I prior to the P1 step, so I'll now combine the both and see how it goes.

HN: When you said 8ul of DNA, what concentration was this?

The only other thing that I can think of is possibly different kinds of alkaline phosphatase, but I used the appropriate amount of units.

Anyone else got some ideas?

I think if you use HPLC you have to use more DNA, at least 1ug. Since I am using LC-MS I need much less DNA. The graph I had is 200fmol DNA standard. For my sample I always use less than 100ng for one injection. the alkaline phosphatase is from Sigma, cat# p-5521

Good luck!

Thanks hn, that may explain my results.

I've been using 10ug of DNA with slightly more P1, when really I should be using closer to 10 times the amount! I will need to optimise as p1 isn't cheap...

Thank you once again!
Dave

I do think I used a lot of P1 but it is easier. The other protocol I tried first with less P1 also worked. Here is what I did. (probably even less than this still work)

300ng (3ul) DNA + 0.75ul ZnSO4 (10mM) + 1.25ul P1(200u/ml) at 37C for 16hrs.

Resuspend P1 in H2O first to get 1u/ul, then diluted 5 times in 30mM NaAcetate to get 200u/ml.

Oh, I use 2.5M (NH4)2SO4 to dilute alkaline phosphatase.

Was that P1 from US Biologicals?

The data sheet (from US Bio) says that it is supplied in lyophilized powder in 20mM sodium Acetate buffer, 5mM Zinc chloride and 50mM sodium chloride so I haven't been adding any extra.

In case you haven't worked out the kinks in your digests yet you may want to read through this paper....
J Neurosci Methods. 2004 Jun 15;136(1):69-76.
Single extraction protocol for the analysis of 8-hydroxy-2'-deoxyguanosine (oxo8dG) and the associated activity of 8-oxoguanine DNA glycosylase.
Bolin C, Stedeford T, Cardozo-Pelaez F.

I have used this technique to digest DNA for detection by both electrochemical and diode array (HPLC). The most important part of this protocol is denaturing the DNA and getting the nuclease P1 in before reannealing as it cannot work on dsDNA. Also we use enzymes in excess to see the best results.

Trust me if you perform this protocol correctly you will see beautiful separation of individual bases.

Hey all HPLC'ers

I just wanted to give my 2 cents to this discussion, as I worked on genomic DNA methylation analysis via HPLC
for 6 months for my master thesis.

Two things I can say:

1.) All that has been said here about the digest is right: for HPLC use at least 10 µg - I used 15 -20 µg
Be aware to denature properly, and always add excess of DNaseI and Nuclease P1.

2.) THIS has not been mentioned here:
I found that the isolation method for the genomic DNA makes a huge difference on the purity of the genomic DNA.
I started out using phenol/chloroform (the "old" standard) and Promega Wizard genomic DNA kit (works by precipitation).
The results with these isolations were strongly contaminated with unknown peaks.
I then used column based isolations (I used Qiagen, but I am sure any silica-gel based or ion-exchange based column will work just as well)
These isolations were great !!! (unfortunately by the time I realized this my time for MSc thesis research was up )

So: Use Column-based genomic DNA isolation methods if you are going to analyze with HPLC !!!

good luck all,

PFA-Goofy