Directed mismatch allows you to create or remove restriction sites on the current DNA sequence or its variants by incorporating single or double mismatches at a site near the mutation. Using this function you can create or destroy restriction sites in order to distinguish the wild type allele from a common mutant allele.
1.Choose the Restriction | Directed Mismatch command to open the Directed Mismatch dialog box.
2.Select a restriction enzyme file and the Cutter option. “restrict.enz” is the default enzyme file in DNAMAN.
3.Type a number in the Mutation at box to select the mutant position.
4.Type A, C, G or T code in the for box to define the mutation.
6.Click the Site button to confirm the sequence and mutation.
7.Select one of the Accept Mutation options:
Non option searches for restriction sites without any mismatch.
<= 1” option searches for restriction sites that can accept a single mismatch.
<= 2 option searches for restriction sites that can accept double mismatches.
Example:
1 TTTGACTGCCACTTCCTCGATGAAGGTTTTACTGCCAAGG
-------------------C--------------------
Mutation at position 20
Maximum Mismatch = 2
W.T. indicates the wild type sequence
'*' indicates mutation position
'^' indicates mismatch
AflII
Mutant:CTGAAG
Enzyme:CTTAAG
* ^
A single mismatch “T” near the mutant allele “C” creates the enzyme site of AflII in the mutant sequence, but not in the wild type sequence.
Eco57I
Mutant: CTGAAG
Enzyme: CTGAAG
*
The enzyme site of Eco57I exists in the mutant without incorporating any mismatch.
XbaI
W.T. : CCTCGA
Enzyme: TCTAGA
^ ^ *
Double mismatches “T” and “A” near the wild type allele “A” creates the enzyme site of XbaI in the wild type sequence, but not in the mutant.