10 replies to this topic

[sri2010]

Hi everyone.. I just joined a lab, and this my first time ever doing bench work other than lab component part of my undergrad courses. I'm totally lost! My PI asked me to design a bisulfite sequencing for the promoter region of a gene. Apparently its a new assay in the lab, and no one else in my lab has any experience with it neither does my PI.

Anyway, so I read up on this, and I understand the basics, essentially to find out "whats methylated and whats not". I ordered the Bisulfite sequencing modification kit from Sigma Aldrich, so I'm essentially taking samples from DNA patients and treating with the kit.

I ordered primers based on the MethylPrimer design. I'm assuming I don't use the primers with the kit?

What next? (in very basic language) How do I find out whats methylated and whats not? What are my next steps?

I'm so confused!

Thank you for looking..

[methylnick]

best thing for you to do is to find an epigenetics text book and read up on the assays you can use to measure them.

[sri2010]

methylnick, on 13 October 2010 - 04:22 PM, said:

best thing for you to do is to find an epigenetics text book and read up on the assays you can use to measure them.

thanks, is there one you could specifically suggest? from reading the posts, theres PCR involved, how do I go about developing the cycles for the PCR? would it be in the epigenetics textbook?

[epigenius]

How interesting, I didn't know that they made Epigenetics textbooks already.

Anyway, PCR is involved only if you choose to do PCR based sequencing. I recommend MSP (methylation specific PCR), and not a direct PCR sequencing method which is alot more cumbersome in my opinion. If you want to go the easy way, there are several commercial MS-PCR kits out there that you can use and just follow the instructions for programming your cycles, etc. Alternatively you can use microarrays although this may be beyond the scope of what you need to do.

I also encourage you look at some publications which used bisulfite sequencing and learn from their protocols.

Edited by epigenius, 14 October 2010 - 12:08 PM.

[sri2010]

epigenius, on 14 October 2010 - 12:05 PM, said:

How interesting, I didn't know that they made Epigenetics textbooks already.

Anyway, PCR is involved only if you choose to do PCR based sequencing. I recommend MSP (methylation specific PCR), and not a direct PCR sequencing method which is alot more cumbersome in my opinion. If you want to go the easy way, there are several commercial MS-PCR kits out there that you can use and just follow the instructions for programming your cycles, etc. Alternatively you can use microarrays although this may be beyond the scope of what you need to do.

I also encourage you look at some publications which used bisulfite sequencing and learn from their protocols.

Epigenius, thank you for your reply.

So basically,

1) Bisulfite DNA Modification kit
2) MS-PCR kit (using the modified DNA). Is there a kit you suggest specifically? I'm assuming I can use the primers I got from the program online(meth primer)

3) After steps 1 -2 , how do I analyze the result? I'm still left "with liquid in the PCR tube"....now what? Do I need special instrumentation? Do I send for "sequencing" Do I run a gel to check the product?

Sorry I'm being blunt about everything, but I'm confused...


Also, are there kits for microarrays? I read the microarrays are for "genome wide analysis" i guess which wouldnt be very applicable in my situation since im looking at the promoter region of a single gene...?

Edited by sri2010, 14 October 2010 - 01:29 PM.

[epigenius]

For microarray, you need the right platform such as from Illumina or Agilent.

Also, scratch what I said about MSP. It is actually more cumbersome in general (although I prefer MSP for quantitative analysis) -- but regardless there are still post-conversion kits out there for use with regular PCR.

[epigenius]

I'm not sure what you mean by "after steps 1-2" -- do you mean after the bisulfite conversion process and the PCR program cycle, or after the first two steps of your bisulfite kit's protocol?

[epigenius]

sri2010, on 14 October 2010 - 01:10 PM, said:

I read the microarrays are for "genome wide analysis" i guess which wouldnt be very applicable in my situation since im looking at the promoter region of a single gene...?

Yes correct.

[sri2010]

epigenius, on 14 October 2010 - 01:41 PM, said:

I'm not sure what you mean by "after steps 1-2" -- do you mean after the bisulfite conversion process and the PCR program cycle, or after the first two steps of your bisulfite kit's protocol?

Yes- sorry, I mean after the Bilsulfite conversion and running the PCR....I'm left with "liquid".

Now what?

Should I run it in a gel to make sure I have product? Are there specific requirements to "prep the product" when running it in a gel?

I saw chromotograms as a method of analysis...can I use this?

Again, thank you so much for your help.

[epigenius]

sri2010, on 14 October 2010 - 01:56 PM, said:

epigenius, on 14 October 2010 - 01:41 PM, said:

I'm not sure what you mean by "after steps 1-2" -- do you mean after the bisulfite conversion process and the PCR program cycle, or after the first two steps of your bisulfite kit's protocol?

Yes- sorry, I mean after the Bilsulfite conversion and running the PCR....I'm left with "liquid".

Now what?

Should I run it in a gel to make sure I have product? Are there specific requirements to "prep the product" when running it in a gel?

I saw chromotograms as a method of analysis...can I use this?

Again, thank you so much for your help.

Here I'll break it down for you:

First do the bisulfite conversion of your DNA. This converts cytosine to uracil while 5-methylcytosine does not get converted. Next do the PCR amplification and get your PCR product. Now you have to check if the PCR generated the anticipated DNA fragment -- you can make a gel and compare it with a DNA ladder (kind of like a measuring ruler). Next you can perform a gel purification to isolate your desired fragment of DNA. Now you can continue to do subcloning and ultimately Dye-Primer sequencing. The result from the sequencing is then compared to the original sequences of which you can then determine any methylated/unmethylated cytosines. Methylated C's stay as C's while unmethylated C's are now T's.

I hope this helps.

[sri2010]

Now you have to check if the PCR generated the anticipated DNA fragment -- you can make a gel and compare it with a DNA ladder (kind of like a measuring ruler).

>>Okay, how would I prepare the samples for the gel? Is it the same as any other gel? What kind of loading dye should I use?

Next you can perform a gel purification to isolate your desired fragment of DNA.

>>Is there a kit for this?

Now you can continue to do subcloning and ultimately Dye-Primer sequencing.

>>How do I go about doing the "subloning" and the "Dye-Primer sequencing"? Is there a kit for this too?

You have been an invaluable source, sorry, I might sound "lazy" with the kits, but I feel since its the first time I'm doing this, I thought the kits would make me "less lost".

Again, thank you so much for your patience and advice.