Sat May 27 22:54:16 EST 1995
hdang at cns.neusc.bcm.tmc.edu (Hong Dang) wrote:
> In article <95144.215132MAYL at QUCDN.QueensU.CA>, <MAYL at QUCDN.QueensU.CA> writes:
> |> We usually do this in the following manner:
> |> Design 4 primers,
> |> 1 5' to your mutation (a)
> |> 1 3' to your mutation (d)
> |> 2 at your mutation, going in opposite 'directions' (b and c)
> |> For this to work, the primers at the 5' and 3' sites have
> |> to contain unique restriction sites, and the mutation has
> |> to create a new restriction site...
> |> a b
> |> ------> ----->
> |> ===================x==================
> |> <----- <------
> |> c d
> |> PCR out two fragements, one using Primers a and c, the other
> |> using primers b and d. Cut the products with the appropriate
> |> enzymes, and ligate into a vector (3 part ligations are *fun*)
> |> then sequence to check for errors. Or subclone each fragment,
> |> sequence, then cut and ligate.
> |> Good luck:)
> |> Lorraine
> We used 3 primers, a, b, and d. Do PCR with b and d, then a second PCR
> with a and the product from the 1st PCR, with Pfu polymerase for the
> 1st reaction (works well at least under 500 bp), and 5% DMSO. Then subclone
> the final product, works quite well for us.
There is another PCR-based approach that applies to either mutagenesis
or fusing heterologous DNA fragments, WITHOUT RESTRICTION ENZYMES.
It is called Splicing Overlap Extension (Horton et al, BioTechniques
Vol. 8, No. 5, p. 528, 1990). As you'll see in the reference, this is
a very flexible approach to mutagenesis, without much of the annoying
three-way ligations necessary with more standard approaches.
More information about the Methods