Whole cell PCR - DNA purification without using proteinase K? (Mar/18/2009 )

Hi guys,
I have been learning a lot from your expertise in this web site and I have some questions that might have good solutions from you.

We have been working on developing a whole cell PCR without using any column or centrifuge machine.
We can trap mammalian cells easily in our PCR chamber under microscope and lyse them successfully with our laser.
We can see that the cell is breaking apart with every laser shot in the 10's nanoliter volume chamber.
As a proof of principle, we tried to run the 18s assay with the lysed cell with the Taqman pcr.
But it didn't work out.
My best guess is that the genomic dna was not accessible by the primers or probes because the histone was not digested in my experiment even though the chromosome was exposed.
The problem is I cannot add or mix the cell lysate with another reagent once I started, so I cannot use the proteinase K here because it will digest the polymerase which was already mixed in the sample.
Basically, I make a PCR mix with live cells in the first place and I lyse the cell with the laser and try to run PCR with cell lysates as my template right away.
My question is if anybody knows any method that can separate the genomic dna from the histone other than using proteinases (such as proteinase K) so that the method does not inhibit the downstream pcr reaction.
If you can share any good reference that does whole cell PCR (mammalian), it would be greatly appreciated too.
Thank you in advance.
Good luck with all your lab projects.

Have you tried just adding the cells to the PCR mix and starting the amplification run by heating the tube to 94C for 5-10 minutes? That will not only lyse all cells, but also denature all non-thermostable proteins. Then you just run the PCR as normal.

swanny on Mar 18 2009, 02:46 PM said:

I believe that worked out well with the bacterial cells but it didn't work out with the mammalian cells in our lab...
Thank you for the suggestion though...^^

Have you tried varying the amount of cells being used in the PCR reaction? Try using less (or more) cells.

Have you tried different formulation of PCR buffer?

Youp on Mar 19 2009, 09:49 AM said:

Ok, fair enough, what about a simple osmotic shock? Spin down the cells, resuspend in a small volume of a hypotonic solution, then use that as the template, with a long denaturation step at the start.

Youp on Mar 18 2009, 11:49 PM said:

For me this worked quite well with mammalian cells and a hot-start polymerase. I easily got DNA fragments up to 4 kb amplified.

Maybe you have too little cells or you destroy them with the laser.

Frija on Mar 19 2009, 01:26 AM said:

That's very encouraging.
Could you share your protocol with me or any reference I can follow?
For us, we are not doing an end point PCR but we are doing real-time PCR from ABI.
Unfortunately, my probes seem to be aggregate together with some cell debris as soon as they are mixed with cells.
So, I'm trying to use SYBR green master mix and actually waiting for them.^^
Thank you.

swanny on Mar 18 2009, 06:32 PM said:

Our problem is right there.
Once we prepare for the sample in our chamber, the only weapons we can use are optical methods such as lasers.
And we want each cell live until we lyse them with the laser.
Thank you.

Frija on Mar 19 2009, 02:26 AM said:

I have had the same issue with whole cell PCR. What I did was heating the lymphoma cells in PCR mix at 95 degree C for 10 min for hot start. I could not get products even after 40 cycles. When observing under microscope, I found lots of cells remain intact after heating. Could you share your protocol with me and what cell line you are working with? Many thanks.