In a cloning process, there are two major steps: generating an Insert and a Vector fragments and ligating them to create a new clone. DNAMAN mimics this process to produce a new cloned sequence. This function can help you design a cloning strategy prior to laboratory works.
Insert and Vector fragments can be obtained from the same or different source sequences.
1) Perform restriction analysis with the source DNA, check the Results: Draw restriction pattern option. The analysis results are shown in a restriction pattern graph.
2) Move the cursor to the desired fragment, then click the left mouse button to display a popup menu. If the enzyme cutting sites are available, this menu allows you to load the fragment as Vector or Insert.
3) Select the fragment as the Vector or the Insert that will be stored in the memory.
Repeat the process to load the other source of DNA for cloning if necessary. The Restriction | Cloning menu becomes activated when the Vector is loaded into memory.
Choose the Restriction | Cloning command to perform ligation. A Cloning dialog box appears to display the information of the Insert and Vector. Check the Vector and Insert information to make sure that no error occurs. Otherwise, you must back to restriction analysis and load the correct fragment into the memory.
There are some conventions for DNA fragments in the Cloning table:
1) The direction of a fragment is defined from the 5’ end to the 3’ end. The ligation is performed between the 5’ end of the insert and the 3’ end of the vector, and between the 3’ end of the insert and the 5’ end of the vector.
2) For each fragment, the blunt end is Blunt. The symbols ‘+’ and ‘-’ are related to the upper strand.
5’ end +: 5’ protruding termini; 5’ end -: 5’ recessed termini
3’ end +: 3’ protruding termini; 3’ end -: 3’ recessed termini
Example:
5’-AGCTGCGCGCGCTTTATTATAGCTGTAGCTA-3’
3’- CGCGCGCGAAATAATATCGACATCG -5’
The ends are: 5’ end +AGCT 3’ end +TA
5’-AGCTGCGCGCGCTTTATTATAGCTGTA -3’
3’- CGCGCGCGAAATAATATCGACATCG -5’
The ends are: 5’ end +AGCT 3’ end - CG
3) Fragment end may be modified by clicking the button behind the position box. There are three options: Keep, Fill in and Delete.
4) Insert sequence may be reversed by checking the Reverse insert orientation option.
5) If you use linkers in the cloning construction, the linker sequences are related to the upper strand. The linker Vector-Insert is between the 3’ end of the Vector and the 5’ end of the Insert, and the linker Insert-Vector is between the 3’ end of the Insert and the 5’ end of the Vector.
6) You may perform Vector self-ligation by checking the Vector recircularization option.
If the ends of the insert and vector are not compatible, a message box will appear to indicate the incompatibility.