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about semi-quantity of PCR products on agarose gel


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#1 cneagle66

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Posted 07 October 2003 - 12:59 AM

Hi, dear everyone,

I'm investigating methylation status of DNA samples. After DNA digestion by enzymes Msp I and Hpa II, I use special primers

#2 cneagle66

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Posted 07 October 2003 - 01:46 AM

Hi, dear everyone,

I'm investigating methylation status of DNA samples. After DNA digestion by enzymes Msp I and Hpa II, I use special primers, which span the sequence recognized by the two enzymes above, to amplify DNA samples. PCR products were run on agarose gel. No PCR product after Msp I digestion (as negitive control). No PCR product using DNA digested by Hpa II if no DNA methylation. PCR of non digestion DNA as positive control.

Till now, I haven't seen any complete non-methylation (which means no PCR product using DNA digested by Hpa II as template) within the region in 24 of all my 90 samples (a few groups treated in different way). I guess there is just different extent in CpG methylation status in them. Then I want to semi-quantify the extent of methylation. But how I can do it?

It's impossible that I run all the PCR samples in one gel, also this method is not accurate (I think) to compare the density of bands in different gel or even in one gel. There are many things which can influence the compared result, such as sample loading, photo taking, and the EB amount difference between first half and second half after a long time running.

My friend gave me advice about it: comparing the ratio of band density. For example, I can compare the ratio between HpaII/MspI and positive control/MspI. Maybe it's a good idea?

Or else, can I use control such as PCR of Actin simultaneously to adjust ? Can I use a DNA sample whose amount is known to run in same gels as a quantity control?

Can you give me any idea for it?

Many thanks!

cneagle

#3 il0postino

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Posted 09 October 2003 - 09:03 PM

Maybe it is better to use this method with bisulphate?
You treat your starting DNA chemically to change non metylated C's into A's. The methylated residues are protected against this treatment (as far as i recall, but you can look this up). Then you PCR using carefully designed primers (they should work both on modified and non-modified DNA). And then you just sequence the PCR product. This technique is strand specific.
You might want to look in to this method to see if it is any use to you.

Alternatively you could try your method in conjuction with real time PCR if you have the equipment. You should be able to calculate how much DNA you started with. If you do not have the equipment for this you might be able to use the syber green technique and measure every few cycles to see if you can come up with something consistent.
Syber green intercalates only with double stranded DNA and then becomes fluorescent. So after every PCR cycle you have more double stranded DNA (your DNA fragment) (you can not have any background bands for this technique).

good luck




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