How to define CpG islands and what to do if they aren't found? - (Feb/03/2010 )

As far as I can tell there is yet no well-defined definition of what exactly a CpG island is. Generally said it is an area rich in CG content and preferably in the promotor region about 200bp long (correct me if I'm wrong).

I have been trying to develop MSP primers for some genes, my question is the following:
what if software doesn't find any CpG islands? (I have used cpgislands2, methyl primer express and MSPPrimer)
From certain articles I read they suggest a nested approach is best:
first 2 pair of primers, 1 for methylated and 1 for unmethylated.
Then a second pair of nested primers that can be used for both methylated as unmethylated.

If I use methyl primer express and I tinker with the settings a bit I can get 2 CpG islands. However, there are CG's spread all over the 1000bp long gene... it is almost impossible to find methylated and unmethylated primers with high enough Tm's, let alone for the nested primers.

I know BSP primers are an alternative, but they ask of me to develop MSP primers. I am at a bit of a loss here. I only used lightcycler probe design before which made everything for me. So needless to say, this is a bit much to deal with especially because the gene in question isn't really suitable it seems.

My reference article is:

Methods Mol Biol. 2009;507:305-23.
Methylation-specific PCR.
Licchesi JD, Herman JG.
Cancer Biology Program, Sidney Kimmel Comprehensive Cancer Center Johns Hopkins, Baltimore, MD, USA.

>>As far as I can tell there is yet no well-defined definition of what exactly a CpG island is. Generally said it is an area rich in CG content and preferably in the promotor region about 200bp long (correct me if I'm wrong).

You are right.

>>what if software doesn't find any CpG islands?
My thought is if there is no CpG island or CpG rich region (regions that don't meet the classic criteria, but still CpG rich) associated with the promoter of a gene, DNA methylation may not play a role in gene expression, or there is no methylation at all.

>>From certain articles I read they suggest a nested approach is best:
first 2 pair of primers, 1 for methylated and 1 for unmethylated.
Then a second pair of nested primers that can be used for both methylated as unmethylated.

The first pair should be specific for bisulfite modified DNA with no bias toward methylated or unmethylated DNA, ie, the primers should not contain any CpGs.


>>If I use methyl primer express and I tinker with the settings a bit I can get 2 CpG islands. However, there are CG's spread all over the 1000bp long gene... it is almost impossible to find methylated and unmethylated primers with high enough Tm's, let alone for the nested primers.

In this case, you have no choice but to design primers whereever possible regardless of Tm values. That's why bisulfite PCR is challenging.

>>I know BSP primers are an alternative, but they ask of me to develop MSP primers. I am at a bit of a loss here. I only used lightcycler probe design before which made everything for me. So needless to say, this is a bit much to deal with especially because the gene in question isn't really suitable it seems.

If you have lots of samples to study and just want to know whether methylation status in those samples, such as clincial samples, MSP probably is the best choice. If you study cell lines and want to understand how methylation affects gene expression, BSP is the choice.

Thank you PCRman for your response.
At the moment we have stepped down from our target gene for a while and are trying to get a succesfull nested amplification done on some genes that have already been studied well and that have a "well-defined" CpG-island.

At the moment our work consists of the following workflow:

- gDNA extraction with either ZYMO plant/seed DNA kit or the Qiagen plant DNA kit
- gDNA cleanup with ZYMO DNA clean/concentrator kit
- gDNA bisulfite treatment using ZYMO methylgold kit
- BSP primers developed with methprimer with a Tm of about 60°C and amplifying a part between 450-400bp long
- I have ordered Red Hot Taq Polymerase to increase specificity and to get less primer dimers (at least thats what it says it should do)
- I will use protocols I found on this forum

Is there anything else I should take into account or is there comments on one of the kits I use? Suggestion are always welcome be it positive or negative:)

Thank You

I have now tested the methyleasy xceed kit and used the supplied control samples. Both of them are negative after the PCR.
Can anyone tell me if Red Hot Taq polymerase from thermoscientific is good for bisulfite PCR?
I am getting bands on the other samples funnily enough just not on my controls. I did everything exactly as they said and no bands at all.
Any tips please?

Cheers