Which MSP & BGS primer design tool do you "swear" by? - (Oct/12/2005 )

Hi All,

I'm new to Epigenetics and there's LOTS of things I'm not sure. Hope to have some advice from you guys.

I looked up the web, and found several MSP and BGS design tools, like MethPrimer, BiSearch, PerlPrimer, Primo, etc. Which one, in your opinion, do you think works the best? Also, what's the advantages and disadvantages?

Also, I heard from my seniors that the type of primer purification (i.e. Cartridge purified primers works better than just PCR grade desalted primers) also affect the methylation PCR. Is that true? Even if it affect the PCR, by how much?

So sorry for "bombarding" you guys with so many questions. I'm ..... Thanks so much in advance for your kind help!!!

Darthvader

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Frankly speaking, I don't think there is a best program among the list in terms of the quality of primers picked by those programs because the extreme sequence composition after bisulfite modification makes any program hard to pick good primers. Certainly there are subtle differences in algorithms used by them.


I agree. If you can afford it, use PAGE purified primers.

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I agree with pcrman, I won't swear by any computer program and will pick primers by eye only.

For MSP especially the purity of your primer synthesis is very important.

good luck

Nick

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Hey thanks pcrman and Nick!! My boss have already helped me designed the primers by eye, and I've sent them for synthesis using cartridge purification. Apparently, the local primer synthesis company have 3 types of purification: Cartridge, HPLC and PAGE, which gives 75-85%, 95% and 98% purity respectively. And PAGE is so expensive!! According to my boss, Cartridge purification should work well for me. Keeping my fingers crossed now...

Anyway, thanks so much for all the advises in this forum. I really learned a lot for the past one day. I'm so glad I found this forum. Thanks again guys!

Vader

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glad to help....

in my experience for BSP primers I have not needed to have any purification done on them except desalting.

good luck with it, hope you bisulfite well!

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Well I am happy to swear by Methprimer for BSP, they always yeild a product (sometimes quite alot of optimisation is required) and no programme is going to be able to account for possible non-specific products. As for the primers, I've never found a need for HPLC or PAGE purification.
The 3 other tips that I can give are...

Set up all your reactions on ice

Use a kit based bisulphite treatment kit

Most importantly, use the best hot start enzyme you can afford.
I switched to BD Clontech's Titanium TAQ a couple of years ago and it's made a world of difference.

Hope that helps

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My MSP primers ordered from Biosource. It is called Econopure, since it is not HPLC or PAGE purified, I guess it is cartridge? They worked fine for me.

I used Chemicon Fast kit for bisulfite reaction. And Jumpstart RedTaq ReadyMix from SIGMA for PCR. Thanks to the folks here, I followed their suggestions and never tried other enzymes since it works well.

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Also I used Methprimer to design my MSP primers, so far three genes I tried looks primers work OK!

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Thanks for all the replies guys!!

Just received my purified primers today. Going to test them out tomorrow morning. Hopefully I'll be able to get them to work this time. Else I will use Methprimer to design them to try them out.

By the way, some of my friends working in related fields told me that normal de-salted MSP and BGS primers tend to "lose their effectiveness" after just one or two freeze-thaw cycles. Do you guys get that sort of problems to with normal primers?

Vader

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with BSP primers, no

I order mine from invitrogen, they come lyophilised and I will resuspend them in a stock concentration of 250uM. I will then aliquot a working stock at 10uM concentration. This minimises the freeze thaw cycles of the stock primers and also reduces the liklihood of cross contamination

Good luck! May the methylation gods be with you.......always

Nick

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