Hairpin template in a PCR

[Peter Ellis]

Peter Ellis wrote:
> TR wrote:
>>
>> Well,
>> after the initial panic, I have been reading and came up with an
>> alternative solution. And again I would like to know your opinion
>> about this plan B:
>
> I don't see why you can't make the two halves of the hairpin
> separately, with compatible sticky ends, and just ligate them
> together.
> Thus:
>
>> --------amplicon---------|
>> --------amplicon---------|
>
> Amplify with an extended primer to introduce a restriction site at
> one end
>> --------amplicon---------|---RRRR---
>> --------amplicon---------|---RRRR---
>
> Cut to leave a sticky end
>
>> --------amplicon---------|---RRRR
>> --------amplicon---------|---
>
> Ligate this back to itself
>
>> --------amplicon---------|---RRRR---|---------nocilmpa--------|
>> --------amplicon---------|---RRRR---|---------nocilmpa--------|


Note that doing this leaves you with the "spacer" section also forming an 
inverted repeat - the hairpin continues all the way to the restriction site. 
However, if you need the spacer loop to *not* form an inverted repeat, you 
can just do two initial PCRs, thus:


|--------amplicon---------|
|--------amplicon---------|

Do two different PCR amplifications with different extended primer to 
introduce a restriction site at one end

|--------amplicon---------|abcdeRRRR---
|--------amplicon---------|abcdeRRRR---

|--------amplicon---------|zyxwvutsRRRR---
|--------amplicon---------|zyxwvutsRRRR---


Cut these to leave a sticky end

|--------amplicon---------|abcdeRRRR
|--------amplicon---------|abcde

|--------amplicon---------|zyxwvutsRRRR
|--------amplicon---------|zyxwvuts



Ligate these together, you'll get three products.

1)
|--------amplicon---------|abcdeRRRRedcba|---------nocilmpa--------|
|--------amplicon---------|abcdeRRRRedcba|---------nocilmpa--------|

2)
|--------amplicon---------|zyxwvutsRRRRstuvwxyz|---------nocilmpa--------|
|--------amplicon---------|zyxwvutsRRRRstuvwxyz|---------nocilmpa--------|

3)
|--------amplicon---------|abcdeRRRRstuvwxyz|---------nocilmpa--------|
|--------amplicon---------|abcdeRRRRstuvwxyz|---------nocilmpa--------|

The third one is the one you want.  If you design your initial primers 
properly so they're different lengths, you can then gel-purify the correct 
one.

Peter