How to prevent melting of short DNA duplexes

Steve S.H at l.u.c
Fri Mar 27 10:36:53 EST 1998


In article <3517F8B5.1AA2 at chugaibio.com>, dspinella at chugaibio.com,
@chugaibio.com wrote:

> Dear BioNetters:
> Does anyone know of a simple way to enhance the stability of short
> double-stranded (say 10-12 base pairs) DNA fragments in a complex
> mixture?  I'm trying to find a way to prevent even AT-rich sequences of
> this size from melting apart in solution that won't interfere with
> subsequent enzymatic manipulation of the fragments (particularly
> ligation).  Suppose for example you were trying to make a library of 12
> bp fragments.  How could you clone the fragments into a vector under
> conditions that wouldn't select against the AT-rich fragments because
> they melted apart before encountering the vector?  Obviously, I could
> increase the salt concentration, and/or decrease the temperature of the
> solution (with attendant adverse affects on the subsequent ligation
> reaction).  How about TMAC? Polyamines? Any suggestions would be
> appreciated.  Thanks.
> --Dom Spinella

I had exactly (well, more or less) the same problem a few days ago. I
solved it by first phosphorylating the oligos (T4 PNK) then assuming that
the single-stranded linkers would ligate first to the double stranded
vector, and only then would they anneal for a final ligation.

E.g: 8 pb phosphorylated linker, with an EcorV site:


   5'-XXXXXXXXXXXX     +    5'-XXGATATC
      XXXXXXXXXXXXXX-5' 

                      AND

                         5'-XXXXXXXXXXX
        CTATAGXX-5'    +      XXXXXXXXX-5'

   
                        |
                        |
                   first ligation
                     1h at RT
                        |
                       \|/
                        |


    5'-XXXXXXXXXXXXXXGATATC        5'-XXXXXXXXXXX
       XXXXXXXXXXXXXX-5'        CTATAGXXXXXXXXXXX-5'


                        |
                        |
                  second ligation
                     O/N at 16°C
                        |
                       \|/
                        |

    5'-XXXXXXXXXXXXXXGATATCXXXXXXXXXXX
       XXXXXXXXXXXXXXCTATAGXXXXXXXXXXX-5'

And there you are!

The only important thing, is that your oligos need to be previously
phosphorylated, otherwise it will never work that way...

I hope it helped



More information about the Methods mailing list