Restriction Enzyme Digest of Genomic DNA - How to get complete digestion? (Jul/22/2009 )

I am trying to digest (human) genomic DNA, but Im having problems with incomplete digestion (I really need it to be very close to complete).

Right now Im doing my digests in 10ul with 10-20ng DNA, BSA and 4U Enzyme for 16h.

The DNA im testing the system on is GenomiPhi-amplified (unmethylated) and diluted in water (no EDTA etc.)

Any tips on improving efficiency?

Substance on Jul 23 2009, 04:56 AM said:

Do you have any gel images for us? They will help a great deal. Do you run undigested gDNA as well as the cut DNA?
As a first thought, I'd add more enzyme and digest in a larger volume.

What volume of enzyme are you adding to add 4U? Your enzyme volume should not exceed 10% of your final digestion volume, because most enzymes are stored in 50% glycerol, and greater than 5% glycerol inhibits digestion.

swanny on Jul 23 2009, 03:36 AM said:

Gel images (I assume you mean of raw digested gDNA) would be great yes, but as Im only digesting 10-20ng i can't really run it on a gel (can I?) and expect to see anything. The reason for using small volumes is that i want to use the entrie digest (or half of it in the 20ul case) product as template for a PCR. Right now Im doing my tests in duplicate (+/- enzyme), so when I run my PCR I expect to get product in the -enzyme reaction and no product (or at least substantially less product) in the +enzyme one.

But band intensities (250bp fragment, 2%Agarose gel, EtBr) after PCR are pretty much the same for the +/-RE incubations (the PCR is not saturated).

Should I do a large scale test gDNA digest (500ng+a ton of enzyme) and compare with undigested gDNA? All my enzymes are six-cutters with exclusively G's and C's in the recognition sequence (rare cutting enzymes that is), does that matter? How will it show on a 0,8% agarose gel?

The problem with using a larger volume is that I then decrease the DNA concentration, which I've read can also be a problem! Because I intend to screen the methylation status of primary tumor DNA (a limited resource) the system has to work with as little input DNA as possible, preferably I would like to use 10-20ng input gDNA/digest. Is that too little?

When it comes to the other question of what volume of restriction enzyme I use the answer is 10% of the total volume, 1ul in a 10ul digest. Is that pushing it? To mee it feels like pretty much standard.

Has anyone here got experience with Spermidine? I read some articles where they mentioned that it enhances digestion efficiency, so I ordered a gram out of shear desperation. If someone has experience then what concentrations do you use (the article said 1mM)? Anything I should know about Spermidine and how to use it apart from concentrations?

How are you assessing the completeness of digestion if not by an agaose gel? Also, I'm clearly missing something here -- why must your DNA be digested to do PCR on it?

HomeBrew on Jul 23 2009, 07:05 PM said:

Ok so Im trying to look at the methylation status of a gene that I suspect is silenced through promoter methylation in cancer.

I do that by using methylation sensitive restriction enzymes couppled with PCR.

I have designed 4 primer pairs that cover selected regions of the CpG island in the promoter region of my gene. In the region between the forward and reverse primer (of each pair) there are sites for 2-3 different methylation sensitive restriction enzymes (in total I can probe the methylation status of 10 individual CpGs in the island).

So when I digest normal (gene is expressed-->promoter unmethylated) gDNA with a RE theoretically all the template strands are broken and the subsequent PCR has no intact template (or atleast so little of it that it doesnt reach the detection limit of my PCR)---> no product.

However if the gene is silenced through promoter methylation, the RE cant cut--> template for my PCR--> Product

The degree of methylation is (rough estimate) determined by comparing the PCR-product intensities of RE-treated gDNA to a no-RE gDNA control.

Right now Im trying to get my control for digestion to work (100% unmethylated DNA should be clevaed completely), but because I dont get good enough digestion I still have so much template left in the digestion product that a band always shows up in the PCR.

Basically I do asess the completeness of my digestion on agarose gel, but only after PCR amplification. And since I can see almost equal amounts of PCR product in the samples amplified from RE treated gDNA and untreated gDNA i can assume that the digestion efficiency wasnt all that great.

In every digest-round I do I also through in a positive control (200+ng of PCR amplified fragment that has the RE-site in it) and it has always been cleaved so its not that the enzyme isnt doing anything, just not doing enough.

If you do a normal PCR with 30-35 cycles, the amount of final product will be almost independent of the amount of template which is present. To do anything quantitative, you'll need to determine which cycle produces significant product, using either a QPCR cycler, or by sampling your cycling reaction every 3-4 cycles and running those samples on a gel.

phage434 on Jul 25 2009, 05:23 AM said:

I know, but as I said above the PCR is NOT the problem!

Optimizing my PCR to get into a (semi) quantitative range was the first thing I did, before any of this restriction enzyme crap.

I know pretty much precisely how many cycles i need for any given amount of input DNA (and primer pair) to stay above the detection limit but well below saturation.

The question is not so much PCR-related!

I only asked for tips on improving digestion efficiency!

So, then I assume you are running parallel PCR reactions with 10x and 100x dilutions of your DNA sample, to make sure your semi-quantitative detection is working? A 90% cutting efficiency (which is pretty good) will give only about 3 cycles of difference in when the amplification occurs. How are you purifying your DNA following genomiphi amplification? How are you quantifying it?

How big is the piece of tempate DNA? Maybe you could enhance efficiency by first cutting the chromosome with an initial enzyme that would have your target DNA on a reasonably sized piece. If you first were to recover that, and digest it with your methylation sensitive enzymes, you could get complete digestion and clearer results.

Or, do two rounds of digestion on your full sized chromosome with your methylation sensitive enzymes -- do the first one the way your doing it, then do a second round of digestion in a final volume double the first volume (add 1 ul of enzyme, 2 ul of 10X buffer, and 7 ul of water to your first 10 ul digestion, and incubate at 37C again).