2 replies to this topic

[semperfi]

Hi there

Is it possible to have a primer that extends from the end of one exon to the begining of the next exon..Thoretically , you can  align this  in a blast search in a NM_sequence of any sort, but in terms of the reaction biochemically will the intron areas be a problem, before splicing? thanks in advance for any assistance.

[pito]

I do not understand what you want.
You want to create a primer that extends an entire exon and an intron (with the start of the next exon included?).


You can make a primer as you like it to be and let it extend as long as you want in theory... (in practise their are limitations, but you could do it).

(keep in mind: you always need 2 primers if you want to generate a certain piece of DNA , copy it.)


And what about the splicing? I do not see where the splicing come froms when doing a PCR reaction?



PS. you are a marine?

[Trof]

semperfi, on Jun 12 2010, 04:02 PM, said:

Hi there

Is it possible to have a primer that extends from the end of one exon to the begining of the next exon..Thoretically , you can  align this  in a blast search in a NM_sequence of any sort, but in terms of the reaction biochemically will the intron areas be a problem, before splicing? thanks in advance for any assistance.

What you described is called intron spanning. qPCR primers are often designed this way (one primer or probe on the exon-exon boundary), because they don't amplify gDNA, only cDNA. mRNA used for reverse transcription is already spliced, so you don't have problems with that.