Replies

• Dorothy Clyde

  06 March 2008 | 10:29

  Hi Farasha,

  Thank you for posting your question. I’d say that your options largely depend on the size of your DNA fragment (is it small enough to allow PCR?) and the purpose of the experiment (is some extra sequence allowed e.g. by adding restriction enzyme sites).

  There is a short discussion on overlap extension PCR here. However, I’m not sure this technique can be applied to splicing identical fragments; you would not be able to design primers that could distinguish the individual fragments from the spliced ones for the final PCR step.

  Perhaps you could add restriction enzyme sites to the ends of your fragments (e.g. by PCR)? If you want to be sure that fragment inserts in the right orientation, you could perhaps design a scheme where you used different restriction sites at the 5’ and 3’ ends – but which produce compatible cohesive ends. When such sites are ligated, they produce a hybrid recognition site that can no longer be cleaved by either of the two enzymes. Commonly-used examples of such enzymes are: EcoRI and MfeI; and BamHI and BglII. The Promega or NEB websites and catalogues are very useful sources of relevant information on this and other cloning-related topics.

  So, using EcoRI and MfeI as an example (assuming that the vector has an EcoRI site and the fragment is engineered to have an MfeI site at it’s 5’ end and an EcoRI site at it’s 3’ end) an outline of the cloning process might be:

  Step 1: Cut the vector with EcoRI. Double digest the insert with EcoRI and MfeI and ligate to the cut vector. After ligation, the 5’ vector/insert junction will have a hybrid EcoRI/MfeI site that can no longer be cut by either enzyme. The 3’ junction will have a functional EcoRI site.

  Step 2: Repeat step 1 using the “vector+insert” created by the first ligation and the same insert preparation. After ligation, a hybrid site will be created between the junctions of the 2 inserts, and a new EcoRI site will be formed between the junction of the second insert and the vector.

  This stepwise procedure could be used to add as many MfeI/EcoRI fragments as needed.

  Remember: if the cloned product is to be used to express a protein, you will need to take into account the possible effects of adding restriction enzyme sites (e.g. frameshift, additional codons etc.). In this case, you may prefer to investigate the GATEWAY cloning system, which is based on site-specific recombination rather than ligation.

  I hope this helps – and good luck!

  Best wishes,
  Dot

• Farasha Almadani

  26 March 2008 | 22:43

  Hi Dot

  Thanks so much for your detailed reply. Actually just before I saw your reply I had already ordered primers for PCR by overlap extension ! but as you said every time in the second PCR I got amplification of the original individual fragments although I managed to get a band indicative of two spliced fragments but following digestion I was not able to get any DNA probably because of the low concentration (I purified by gel extraction).

  Now I will definitely revert to the enzyme method but I will still try to precipitate the DNA and ligate in the hope that I will get results.

  Thanks again

  Farasha

• Martha Toy

  12 May 2008 | 08:39

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