Candidate gene methylation analysis - Some advice needed! (Aug/18/2004 )
Hi
I am about to start a project looking at the methylation status of some candidate genes. It would be good to get some quantitative value of methylation, but failing this a 'yes' / 'no' answer would be OK initially. I have some questions which someone may be able to help me with!
- quantity / quality of DNA : how important is this? some of our samples are quite precious and so DNA has to be used efficiently. Also, some of the DNA has been obtained using buccal swabs - will this be OK (our extration methods are good and produce good yields of cleanish DNA)
- can someone reccommend a good bisulphate treatment method. I can only find 2 on the market, neither seem to be available from distributors in the UK. I like the look of methyleasy as this seems to be effective even on small quantities of starting DNA. Is this in reality the case?
- following this treatment, what is the best way of detecting methylation status. I seem to have uncovered about 10,000 variations in methods! Methylation-specific PCR seems fairly straightforward. Can sequencing give quantitative data? And also, we have a DHPLC in our lab, I have seen somewhere that this can be used - how easy/reproducible is data using this method??
Thanks in advance for any help!!
Jon
Hi Jon,
- quantity / quality of DNA : how important is this? some of our samples are quite precious and so DNA has to be used efficiently. Also, some of the DNA has been obtained using buccal swabs - will this be OK (our extration methods are good and produce good yields of cleanish DNA)
A: DNA quantity is not a problem. Even pico grams of DNA can be successfully amplified. Actually the theoretical limit for bisulfite based methods is ONE cell because there is an amplification step. However, DNA quality is important. If DNA is not pure, you may end up with incomplete bisulfite conversion.
- can someone reccommend a good bisulphate treatment method. I can only find 2 on the market, neither seem to be available from distributors in the UK. I like the look of methyleasy as this seems to be effective even on small quantities of starting DNA. Is this in reality the case?
A: Yes, a kit can save you some pain. I think most kits should be able to handle small quantities of DNA. I have done bisulfite PCR on DNA from a few microdissected cells without using a kit some time ago. The most important parameter to evaluate a kit is the purification method it uses since DNA loss during purification is a big concern.
- following this treatment, what is the best way of detecting methylation status. I seem to have uncovered about 10,000 variations in methods! Methylation-specific PCR seems fairly straightforward. Can sequencing give quantitative data? And also, we have a DHPLC in our lab, I have seen somewhere that this can be used - how easy/reproducible is data using this method??
A: Based on you needs (a few candidate genes, quantatitive results, small quantities of DNA), I would recommend realtime MSP. Althogh regular MSP is one of the simplest methods but not quantatitive. Realtime MSP is increasingly used by researchers for screening large number of clinical samples across multiple genes. A good strategy for working with precious DNA is to design a pair of universial primers outside your MSP target which amplifies only bisulfite modified DNA and makes no discrimination against methylated or unmethylated by excluding any CpG sites in the primer sequence. After a first round PCR using the universial primers, the resulted products can be used for MSP using primers specific for methylated or unmethylated template. In this way, you won't be worried about running out of your DNA.
Hope those helps.
Many thanks for your comments and advice.
I came across another method yesterday that seems ideal for our purposes - basically quantitative MSP using fluorescent primer extension reactions (e.g. SNaPshot). I was just wondering if anyone had any experience of using this procedure to quantify methylation?
Jon