pros and cons of Blunt vs sticky ligation - (Nov/05/2007 )
Hi readers,
I am attempting to reconstruct a protocol from scratch using two papers as reference. Both touch on similar application of a method (Nimblegen's sequence capture arrays, published in Nature Methods Oct).
In one paper, the authors used blunt-end ligation of double-stranded universal adaptors to the library of DNA fragments (with polished ends).
In the other paper, 3'-A overhangs in the DNA (the 3'- A are introduced by Taq Pol) are ligated to adaptors with complementary 5'-T overhangs.
In your experience, would you prefer to do blunt-end or sticky-end ligation and why?
Thanks in advance for any useful advice!
jenny
I am attempting to reconstruct a protocol from scratch using two papers as reference. Both touch on similar application of a method (Nimblegen's sequence capture arrays, published in Nature Methods Oct).
In one paper, the authors used blunt-end ligation of double-stranded universal adaptors to the library of DNA fragments (with polished ends).
In the other paper, 3'-A overhangs in the DNA (the 3'- A are introduced by Taq Pol) are ligated to adaptors with complementary 5'-T overhangs.
In your experience, would you prefer to do blunt-end or sticky-end ligation and why?
Thanks in advance for any useful advice!
jenny
I always prefer "sticky end ligation". But that is my personal opinion. Ppl around are happy with the adapter ligation as well, so I don'T think there would be a huge difference since there are a kits available which are good and easy to handle.
Btw do u mean by adapter ligation that u add an adapter which will be cut by a specific restriction enzyme afterwards and then be cloned into a vector cutted by the same re? If u would end up in a sticky end ligation as well.
I prefer the sticky version because I have the idea that nuclotides always need a partner to bind and form a hydrogen bond between each other (which would be 2 between A and T but better than none). With the blunt end ligation there is only the chance that the double strands ligate by the phosphodiester bond from the 5′-phosphate to the 3′-hydroxyl group.
So its ur choice!
In general
sticky end cloning.
Pro: Able to orientate fragment, able to perform multiway ligation.
Con: Must know sequence. May need to buy primers.
Blunt end cloning
Con: Difficult. Do not touch with a barge pole. Easier to encounter concept of 'eternal failure' and taste the finges of Hades/Hell/damnation. (Cloning is hard enough as it is. No need to go looking for trouble)
Pro: Able to encounter early experiences of Hades/Hell/damnation. (What doesn't kill you only makes you stronger/stranger)
The only real pro for blunt end cloning, is that you don't need to know your insert's sequence and be able to engineer unique restriction site.
But this is where TA cloning (vector has 3'T and insert has 3'A) comes in. So there is no need for such blunt end horrors. Depending on what you are doing, you may use TA cloning kits or engineer your own TA vector.