about the methylation content (MSP) - (Apr/19/2006 )
QUOTE (purplefetus @ Apr 28 2006, 10:56 AM)
hmmmm, so, overall if I just did direct sequencing of my BSP products would it be highly criticized? Would those findings not be valid in the scientific community unless cloning and sequencing was performed?
not at all purple fetus, in fact there are many papers that show direct sequencing results in many of the high ranked journals. I suppose, you have to consider that the question you are asking first and look to see if direct sequencing or cloning and sequencing is appropriate.
Take for example you are looking at an imprinted region where you have one allele fully methylated and the other fully unmethylated. Only by cloning and sequencing you would see in the normal situation, 50% of your clones un methylated and the other 50% fully methylated at all CpG sites. With direct sequencing you will only get one chromatogram representing the whole population of templates whereby at every CpG site you will see 50% methylation/50% unmethylated, you can not however tell from this, if you have templates where all CpG's were unmethylated or was it just that there was variagated methylation across all templates.
There are pros and cons for both techniques, direct sequencing is certainly much cheaper to perform than cloning and sequencing as in direct, only one sequencing reaction is performed, while you would have to sequence at least 1o clones for each loci to get a representative result.
Phew, ...relieved. Thanks Nick!