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Protocol for Preparation of Covalently Closed, Double-stranded Linear DNA Molecules (with Nb.Bpu10I)

Covalently closed, double-stranded, linear DNA molecules can be used as minimalistic transfection vectors in pharmaceutical applications. As an example, MIDGE^® (Minimalistic Immunologically Defined Vectors for Gene Expression) are covalently closed DNA transfection vectors used for gene therapy or genetic vaccination*. MIDGE^® minimalistic transfection vectors combine the advantages of viral vectors (cell specificity and high expression levels) with those of plasmid vectors (no immunogenicity or danger of viral recombination and relatively low costs). Such vectors contain only an expression cassette (promoter, coding sequence, and terminator/poly-A-site) of the gene of interest.

I. Design of plasmid

1. The plasmid used for MIDGE^® construction should have the desired insert DNA fragment flanked by two inverted Bpu10I recognition sequences on either side. An interrupted palindrome spacer sequence between the two inverted Bpu10I recognition sites should be included, which after a nicking reaction forms a hairpin self - complementary structure between the top and bottom (5' and 3') DNA strands.

Note 
Cleavage efficiency of Bpu10I nickase depends on the recognition sequence and flanking regions.
 

II. Protocol for production of linear, double-stranded, covalently closed DNA molecules

1. Add the following components to a reaction tube:

   Purified MIDGE® production plasmid (50 µg)	50-170 µl
   10X R reaction buffer	20 µl
   Nb.Bpu10I (5 u/µl)	10 µl
   Water, nuclease-free	to 200 µl

2. Vortex the tube and spin down in a microcentrifuge for 3-5 s.
3. Incubate the mixture at 37°C for 1 hour.
4. Incubate the reaction tube at 95ºC for 5 min.
5. Wait for several minutes for reaction mixture to cool to room temperature and add the following components:

   ATP (10 mM)	20 µl
   T4 DNA Ligase (30 u/µl)	1 µl**
   10X Buffer R	2 µl

6. Vortex the tube and spin down for 3-5 s.
7. Incubate the mixture at 22ºC for 1 hour.
8. Stop the reaction by heating at 70ºC for 10 min.
9. Select a restriction enzyme that has no recognition sites in the gene of interest, but has recognition site(s) in the vector backbone and is active in R reaction buffer. To the reaction tube from step 8, add the following components:

   Water, nuclease-free	275 µl
   10X Buffer R	33 µl
   selected restriction enzyme (10 u/µl)	25 µl**

10. Mix the tube contents and incubate 1 hour at 37ºC.
11. Heat-inactivate the restriction enzyme (optional).
12. Add 5 µl** of T7 DNA Polymerase (10 u/µl) and incubate at 37ºC for 1 hour.
13. Stop the reaction by heating at 70ºC for 10 min. This solution should contain linear, double-stranded, covalently closed DNA.
14. Purify the reaction mix (for example by phenol/chloroform extraction).
15. Quantify the DNA and use for gene-transfer experiments (e.g. transfection with polymeric transfection reagents, ballistic gene-transfer, microinjection, etc.).

*  The MIDGE^® Vector design and production process are covered by German patents DE19753182 and DE19781276D and corresponding foreign counterparts issued to Mologen Holdings GmbH. Licenses for commercial and/or clinical use may be obtained from Mologen Holdings GmbH, license for research use only.

** Quantities of T4 DNA ligase, restriction enzyme and T7 DNA polymerase could be decreased (≈ 10 times) if incubation time of each reaction is prolonged to overnight incubation.

Ordering Information
Nb.Bpu10I	#ER1681	100 u
Exonuclease III	#EN0191	4000 u (200 u/µl)
T7 DNA Polymerase	#EP0081	300 u (10 u/µl)
Water, nuclease-free	#R0581 #R0582	5x1 ml 30 ml

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Updated gegužės 10, 2007 13:00