Dear Pooja, 

Methylation sensitive/depended restriction Enzymes could do the job or COBRA (http://en.wikipedia.org/wiki/Combined_bisulfite_restriction_analysis).

Dear Pooja,

Ms-SNuPE http://www.ncbi.nlm.nih.gov/pubmed/12095270 is a good way to analyze single CpG sites. COBRA would be perfect - as mentioned by Oliver - if your CpG is located in a sequence context where restriction enzymes are available for. If COBRA does not work then clone sequencing is a method which is relatively expensive but does not require a lot of experience.

Good luck,

Dimo

Dear Pooja,

For validating single CpG site, we found the MethylQuant method useful. This is a real-time PCR (SYBR Green) aproach, here is the link to the paper:  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC535695/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC535695/

COBRA has the limitation of restriction enzyme substrate sequence. A rather simple and cost-effective sequencing-based way for this purpose is Pyrosequencing.

Thank you for your valuable suggestions Olivier, Dimo, Eran and Omid.

Hi Pooja,

I would suggest you two ways to validate your result:

1) check whether the CpG site constitutes a restriction site of any enzyme, preferentially methyl-sensitive one. if so and if it is a methyl-sensitive you can amplify by PCR a fragment including your CpG (400-500 pb should be enough),  digest it using your enzyme and then run a agarose 2% gel. the result you will get depend on the methylation status: if gain of methylation the enzyme would not cut otherwise it will cut. if the CpG is restriction site of non methyl-sensitive enzyme, do a COBRA instead. DNA bisulfiting, PCR amplification of a fragment including the CpG, digestion with your enzyme then run a 2% agarose gel.

2) bisulfite treatment, PCR amplification and then sequencing after cloning. this technique is laborious time consuming and high risqué of contamination. but it still feasible since you have only 1 patients and to circumvent the contamination risk check whether you have a SNP that you can embed into your PCR fragment so that you can differentiate between the 2 alleles.

Good luck          

Forget the first part of the point one because when you PCR amplify you will loose methylation so your methyl-sensitive enzyme will cut anyway. the COBRA is the best.

Apologies for the confusion!

Thank you all

When you analyze your sequence for whether it is suitable for COBRA, remember that bisulfite conversion causes the two opposite strands of DNA to be non-complementary--and thus the PCR products derived from them have different sequences.  Thus you may find that one strand provides no suitable restriction enzyme site for detecting the difference between the methylated/unmethylated state, but there still may be one on the opposite strand.  (I was pleasantly surprised to encounter exactly this situation recently for a CpG site in a promoter I was analyzing.)  If this is the case, then you need to be careful to select your PCR primers to amplify the correct bisulfite-converted strand.  

Bisulfite pyrosequencing is the gold standard for validation of specific sequences. In the best case, both strands should be analyzed.

you can Bisultphite treat your DNA and then use a LM-PCR design for that SPECIFIC C residue that if remains unmethylated will get converted and thereby detected otherwise. This can be both quantitative quick and can be highthroughput and cost-effective since it involves a quick PCR

Thank you Subhojit

I had a similar doubt. but instead of pyro if I am opting for COBRA or MSP, can anyone tell me which is better of the two, MSP or COBRA?

This course might be of interest to you!

Nature Events: Epigenetics Workshop - DNA Methylation Data Analysis

http://www.nature.com/natureevents/science/events/35473-Epigenetics_Workshop_DNA_Methylation_Data_Analysis

Shyamashree - COBRA can only be used if the "specific C residue" you need to analyse falls under a restriction site. MSP on the other hand, can analyse single or multiple Cs as long as t hey fall under the primer annealing site. however the latter can be challenging if the difference is only for a single C methylation. The LM-PCR design of bisulphite treated DNA i suggested circumvents problems of the above two procedures, when designed appropriately. However if you are querying multiple Cs the MSP is easier to design, and if your C's of interest are part of an RE site - you can use COBRA as well which is easier to do.