Sense and antisense direction - final explanation - Let solve this finally! (Oct/02/2007 )

I would be pleased if someone with experience can help me in solving the problem regarding direction of reading of plasmid in order to get sense and antisense RNA probe for in situ RNA hybridization.

I am confused as I got two completely opposite answers from two molecular biologists. So, question is: how do I know which sequence I use for sense and which for antisense production. Take this example: I know orientation of my insert and I will use polymerase (T7, Sp6, or whatever, depending of plasmid) in the direction in which is exactly sense strand of DNA (+/+). Do I get senseRNA or antisenseRNA probe? Two mol. biologists confused me: one told me that plasmid DNA is recognized as any other DNA, in other words, if polymerase reads template (not sense) strand, than I would expect that reading in sense DNA direction produces senseRNA. However, I got information that in this commercial plasmids it is just opposite: one should understand that polymerase reads exactly the cloned strand (not template), so sense strand produces antisenseRNA. To make things even more complicated, one biologists told me that DNA during cloning can make flip/flop movement, so, sense and antisense can exchange places "laterally". (how is that possible? DNA should know which is 5 and which is 3 end?!). Theoretically, one could test that only by sequencing (as restriction enzymes cut both strands and this do not change orientation of cloned DNA).

Thank you very much!

well i think both are quite right, even if i don't figured it out completely.
The plasmid point of view tells you what is sense and antisense, according to gene nomenclature.

In commercial kit, i think it's mentionned antisense to figure that the probe you construct is perfectly annealing with the RNA you want to probe. So in this point of view, your probe is an antisense probe.

Well, let's start the with RNA rather than the DNA. Sense RNA is RNA which can be translated into protein by ribosomes. It has a 5' AUG codon and a 3' UAA, UGA, or UAG stop codon. There is no ambiguity about this.

Confusion starts with the DNA. A promoter makes a sense piece of RNA by copying a DNA strand which is its reverse complement. This is sometimes called the template strand. But if you are starting at the promoter and reading left to right, this template strand will the reverse complement of the sequence you normally read. I don't care what you call the two strands of DNA, but the one you normally read starting at the promoter is the same (except for the T->U difference) as the RNA strand. Now this strand is not the one which is actually used to produce the RNA (that is, it is not the template) but it is the one which is normally thought about. It will include a start codon ATG at the 5' end, and a stop codon TAA, TGA or TAG at the 3' end.

Thank you for answers, but one can still be confused. Lets take an example: sequence which I want to detect using in situ RNA hybridization is ATC TAG. This is, of course, ++ strand of the gene. I clone this in the plasmid, check orientation and now I know that on the "left" side of it I have T3 polymerase and on the "right" T7. In other words, T3 will go from A to G in my sequence and T7 goes in an opposite direction. BUT! Does T3 polymerase read "ATC TAG", making TAG ATC or it reads template of it, making "ATC TAG" (as in "normal", eucariotic cells). In the first case, I will get "antisense riboprobe" and in another "sense riboprobe"!
So, which polymerase gives sense and which antisense in this very example?

???Any idea???

So, here is your plasmid:
5' <T3 promoter ----> ATCTAG <---- reverse complement of T7 promoter> 3'

The T3 promoter will make the RNA molecule 5' AUCUAG 3'. The T7 promoter will make the molecule 5' CUAGAU 3'. To detect the RNA sequence 5' AUCUAG 3' which I gather is what you want to do, you need the molecule 5' CUAGAU 3'. made from the T7 promoter, since this is the one which will hybridize with your desired target.