Why when we use primers with an opposite orientation, in the end we get a DNA amplified fragment, which in the middle are the unknown DNA sequences and in the terminals are the targets of the primers?
Best regards
the idea of inverse pcr is that the primers face away from each other. So when your primer sequences are in circular dna as in after you have ligated the ends of your unknown sequence the primers are now facing towards each other if they can extend round the circle of dna they will eventually extend over the other primer and then the usual amplification takes place just as if the template was linear and the primers facing towards each other. In circles things that face away from each other are actually facing towards each other
the idea of inverse pcr is that the primers face away from each other. So when your primer sequences are in circular dna as in after you have ligated the ends of your unknown sequence the primers are now facing towards each other if they can extend round the circle of dna they will eventually extend over the other primer and then the usual amplification takes place just as if the template was linear and the primers facing towards each other. In circles things that face away from each other are actually facing towards each other
You can find some nice figure on Google (see below, the Nested PCR step is optionnal).
The tricky thing with inverse PCR is that you have to find the correct DNA dilution before ligation : if it's not sufficiently diluted, you'll have a lot of religation of your restricted fragments instead of circular self-ligations. If the dilution is too strong, you'll have to (i) scale up your dilution reaction, (ii) concentrate your sample before the PCR step and/or perform the optionnal nested-pcr step (implying to design some specific primers).
When I was younger (doh >_< !), we didn't have these fast and processive polymerases like the Phusion (or equivalent...) and we needed to try few restriction enzymes to ensure to have a restriction fragment with a nice size (not too long so that the taq could amplify, not too short so that the resulting sequences could be usefull).
I wonder how these polymerase improvements make the inverse PCR easier (or not ?) nowadays.
https://framapic.org/DLJBHzFsRM7t/IkaXaVDJtSKF.jpg
great question, generally inverse PCR is carried out to amplify sections which lies between the circular DNA, hence even if it is unknown it gets amplified, see all cloning plasmid diagram for expample
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