Principle : site-directed mutation is introduced through the following protocol. 
1) Insert target DNA into multicloning site of pUC vectors. Choose one of MUT Primers to destroy a restriction site based on the direction of R1 Primer (primer for introducing a mutation) and the restriction site used for DNA insertion. Perform the first PCR by the combination of R1 Primer and M13 Primer RV (or M13 Primer M4), MUT Primer and M13 Primer M4 (or M13 Primer RV) separately in two tubes.
2) After the elimination of the excess amount of the primers, amplified products are mixed, heat denatured and annealed.
3) Add TaKaRa LA Taq to complete heterogeneous double strand.
4) Perform the second PCR by using M13 Primer M4 and M13 Primer RV, which will result in two types of the amplified products (a) and (b).
5) Digest the amplified products with two restriction enzymes, one of which should recognize the site (X) that had been destroyed by MUT Primer and the other should recognize the appropriate site (Y) within the multicloning site.
6) Reclone the digested fragment into the vector digested with the same two restriction enzymes. Only the fragment (a) which contains the mutation introduced by R1 Primer sequence will be recloned.
Methods
두 개의 PCR tube에 반응액1 과 반응액2를 준비한다.
1) Insert target DNA into multicloning site of pUC vectors. Choose one of MUT Primers to destroy a restriction site based on the direction of R1 Primer (primer for introducing a mutation) and the restriction site used for DNA insertion. Perform the first PCR by the combination of R1 Primer and M13 Primer RV (or M13 Primer M4), MUT Primer and M13 Primer M4 (or M13 Primer RV) separately in two tubes.
2) After the elimination of the excess amount of the primers, amplified products are mixed, heat denatured and annealed.
3) Add TaKaRa LA Taq to complete heterogeneous double strand.
4) Perform the second PCR by using M13 Primer M4 and M13 Primer RV, which will result in two types of the amplified products (a) and (b).
5) Digest the amplified products with two restriction enzymes, one of which should recognize the site (X) that had been destroyed by MUT Primer and the other should recognize the appropriate site (Y) within the multicloning site.
6) Reclone the digested fragment into the vector digested with the same two restriction enzymes. Only the fragment (a) which contains the mutation introduced by R1 Primer sequence will be recloned.
Methods
두 개의 PCR tube에 반응액1 과 반응액2를 준비한다.
반응액1
10x reaction buffer 5 μl
dNTP mix 1 μl
Template DNA 1 μl
Primer P1과 P2 각각 0.5 μl
H2O 41 μl
Taq polymerase 1 μl
반응액2
Primer P1 과 P2 대신에 primer P3 와 P4 를 사용하고 그 외는 반응액1 과 동일하다.
2. 1차 PCR을 아래와 같이 시행한다.
94°C에서 5분 1회,
94°C 30초, 55°C 30초, 72°C 1분(template 길이에 따라 적당히 조절한다.), 25회,
72°C에서 10분
3. 반응액1과 반응액2를 이용하여 2차 PCR을 시행한다.
10x reaction buffer 5 μl
dNTP mix 1 μl
반응액1과 반응액2 각각 1 μl
Primer P1과 P4 각각 0.5 μl
H2O 40 μl
Taq polymerase 1 μl
94°C에서 5분 1회,
94°C 30초, 55°C 30초, 72°C 1분(template 길이에 따라 적당히 조절한다.), 25회,
72°C에서 10분
