I performed a single digest (XbaI) to cut my vector and insert and I have done the ligation and transformation experiments. I would like to determine the orientation of my insert using PCR. I used overlapping PCR to insert a frameshift mutation into the insert and I understand there is also a way to utilize these primers in order to determine insert orientation. Can anyone please explain this process? I would like to understand the protocol and the logic/reasoning behind the steps (especially the choice of primers) in the protocol. Thanks!
P.S. I understand orientation can also be determined using digestion, feel free to expound on this if you are familiar with it.
Hi there,
The usual PCR technique for fragment orientation involves one primer specific of the vector (upstream or downstream of the cloning site) and on primer within the cloning sequence. If the primer set is correctly oriented, PCR will give a unique band with a specific size according to the distance between primers. If not properly oriented, there will be no amplification. For digestion, principle is the same : using one restriction enzyme specific of the vector (a unique site close to the cloning site) and one restriciton specific of the insert but not lying right in the middle of it (it may be the same restriciton enzyme specific of the vector). You then may calculated the distance between the two cleavage sites according to insert orientation relative to the vector. As the site lying within the insert is not right in the middle of it, digest is generating two different fragment sizes according to insert orientation.
Hi there,
The usual PCR technique for fragment orientation involves one primer specific of the vector (upstream or downstream of the cloning site) and on primer within the cloning sequence. If the primer set is correctly oriented, PCR will give a unique band with a specific size according to the distance between primers. If not properly oriented, there will be no amplification. For digestion, principle is the same : using one restriction enzyme specific of the vector (a unique site close to the cloning site) and one restriciton specific of the insert but not lying right in the middle of it (it may be the same restriciton enzyme specific of the vector). You then may calculated the distance between the two cleavage sites according to insert orientation relative to the vector. As the site lying within the insert is not right in the middle of it, digest is generating two different fragment sizes according to insert orientation.
Hi Erome,
In order to determine insert orientation, one of your primers has to be within the vector. If the primers you have used for making the frameshift are all entirely internal to the insert, it is theoretically impossible to use them to determine insert orientation.
If, on the other hand, you have a vector-specific primer available (e.g. a sequencing primer), orientation is easy. Run 2 PCRs - one with sequencing primer + an insert-specific forward primer, one with seq. primer and insert-spec. reverse primer.
For restriction digestion, which is what I have used, the simplest way is to use an enzyme that cuts once in the insert and once in the vector. As you have been using PCR, I assume you know your insert sequence, and the vector sequence will be available on ENTREZ nucleotide. There are several programs that will allow you to search a sequence for enzymes that cut only once and will give you their positions. Try REMAP or the New England Biolabs website.
Given that information, together with the position of the XBaI site in the vector, it's simply a matter of adding fragment sizes and comparing them to what you actually get on a gel. Drawing a circular diagram of the two possible plasmid restriction maps is a good idea.
Hope this helps. Good luck.
And try to find a copy of 'Molecular Cloning, A Laboratory Manual'. I like the first edition (first author Maniatis), but the others (eg Sambrook et al) are also great. No molecular biology lab is complete without one.
Sequence with primers that align in the vector regions. (Each vector use to have standard sequencing primers)
Thank you all! Andrew, I especially appreciate your recommendation on a good molecular biology manual. I have been looking for one and I will most certainly follow up on your suggestion. As far as my cloning is concerned, I have realized that insert has a promoter, rbs and terminator within its sequence. From what I understand, the outcome of this is that the orientation does not affect the transcription of the inserted gene; regardless of its orientation it will be expressed. Is this correct?
Hi Erome, sometimes the orientation does not affect (if the inserted gene has its own promoter and terminator in the same direction). Sometimes the orientation does make a difference. Assume you have a 5'-promoter-(3'-gene-5')-terminator-3'.
Re the promoter - rbs - terminator. Yes, your insert will be expressed regardless of its orientation - but how much will depend on the transcriptional environment. A strong promoter in tandem will increase expression; a strong promoter in opposition will suppress it. If your vector has a lac promoter (they usually do), then as long as you don't induce it with IPTG, you can ignore it.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques