Possible to sequence directly from cDNA? - (Aug/23/2008 )
So, my partner has a question... which as far as I know should be possible, but may be difficult...
Is it possible to sequence directly from cDNA? If not, why not?
The problem is that she works in an metatherian mammal species that hasn't got much published sequence. The gene she works on is NBC and she has fully cloned it but the full clone was made using a primer derived from a consensus sequence, so may not be the actual sequence from this species. The lab now wants to express this gene in a cell culture system and if the sequence is wrong, may get erroneous results, so they need to check the first 20 bases or so at the 5' end of the gene. Rather than perform another RACE and go through the cloning again if they don't need to, why not sequence directly from the cDNA using a single primer pointing in the 5' direction?
Any thoughts will be appreciated.
This is unlikely to work. The specific cDNA is likely in very low concentration. The reverse transcriptase does not amplify the RNA, so you get only the same number or cDNA molecules as you had RNA molecules. The sequencing reaction also does not amplify, except linearly with the number of cycles. You will need much more DNA than a typical cDNA reactions produces.