BSP,MSP primer design? - (Apr/21/2009 )
I am newbile in methylation, I have an easy question in bsp and msp primer design, how can i design primer for methylation? can i use software methprimer or methyl primer express? which is better in designing primer? please tell me how to design primer in detail, pcrman,please writedown your detailed procedure because there are so many novice here , thank you
Before you start your experiments you should read some of the original papers which describe the invention of the bisulfite modification based PCR methods such as bisulfite geneomic sequencing, MSP, etc. Only after you have gained a solid understanding of the methodology, you will be able to do it right.
For DNA methylation analysis, there are many different methods available. Which method to choose from depends on the purpose of your experiments, number of samples, and personal preference, etc. For example, if you want to study how a gene's transcription is regulated by methylation, you can choose BSP which give you results with high resolution and wide coverage of CpGs. Further after BSP, you have to decide whether you want to drectly sequence your PCR products or sequence plasmid after cloning the products into plasmid.
If you have known that some particular CpG sites affect gene transcription and you want to map the methylation status of those sites in many clinicial samples, then MSP probably is the first choice, because absesne or presense of amplification can directly tell you whether a sample has methylation at the primer binding sites.
As for primer design, first you need to choose the right sequence to pick primers on. This is critical. The sequence you should focus on is the proxima promoter seuqnece and the promoter should contain CpG island. I think it is meaningful only to analyze methylation for promoters that contain CpG islands. About 60% of human genes have it. Sometimes the CpG island extend into the first exon and intron.
Once you have decide which sequence region to examine DNA methylation, you can input the sequence into different online programs to let the software to help you choose primers. These programs should do a better job than you do it mauanlly because they calculate many parameters for any possible primer selection and return the best ones to you. This FAQ may help you understand how such program choose MSP or BSP primers.
After you have designed and ordered your primers, the actually bisulfite modification procedure and the PCR reaction are also challenging. That is why there are many different bisulfite modfication kits on the market.
Hope that helps. If you have more specific questions, you can post them here.