7 replies to this topic

[Lala]

Hello, I would really appreciate if someone could explain or point me in the right direction , to the question of:

why does methylation information on the cytosine base, get erased when you do PCR?

i know in order to conserve the methylation information you have to use bisulphite conversion. but i dont understand why PCR erases methylation information.

thank you so much for your help, i really appreciate it!

[pcrman]

DNA polymerase used in PCR cannot differentiate 5'methyl cytosine from cytosine, thus during PCR, 5' methyl cytosines are copied as cytosine.

[Lala]

pcrman, on Jun 17 2009, 01:11 PM, said:

DNA polymerase used in PCR cannot differentiate 5'methyl cytosine from cytosine, thus during PCR, 5' methyl cytosines are copied as cytosine.

awesome pcrman. that's a really good answer, thank you so much!!!   that makes so much sense now

Edited by Lala, 18 June 2009 - 06:20 AM.

[mdfenko]

to expand on pcrman's answer, methylation is a post translational modification. first the dna is assembled then it would get methylated if the mechanism to methylate is present and active (as in an intact cell).

pcr is only the assembly mechanism.

[Lala]

mdfenko, on Jun 18 2009, 08:07 AM, said:

to expand on pcrman's answer, methylation is a post translational modification. first the dna is assembled then it would get methylated if the mechanism to methylate is present and active (as in an intact cell).

pcr is only the assembly mechanism.

thanks mdfenko!!! thats a really good explanation.    thats a good answer.
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[Lala]

can i ask another question please  

okay so during methyLight reaction, primers and probes are used. you have 1 set of primers and probes for the GENE of interest, and then you have a primer and probe for a REFERENCE gene. like a housekeeping gene

some use the gene COL2A1 (which encodes for the alpha 1 chain of type II collagen found in cartilage for eg and part of the eye aswell i think)

and the reason you use a reference gene, is to normalise for input DNA.

my question is. how does that actually work. how does using a reference gene, normalise for the input DNA     

is the reference gene normally NOT methylated?

thanks so much in advance for your help, i really appreciate it  

[pcrman]

Your guess is right. The reference genes are supposed to be not methylated. The signal from the reference gene is thus only determined by the amount of template DNA and can be used to normalize signal from GOI.

[Lala]

brilliant pcrman!! thanks so much !! you've been a great help thats a great answer, very well explained.

its finally beginning to make sense now.  

i've spent so long trying to figure out , and you guys have helped me so much in a day!

i gave you guys both 5 stars in your profile

i have written down all your answers in my notebook

Edited by Lala, 18 June 2009 - 02:47 PM.